Method for producing nicotinamide mononucleotide

Abstract

The present invention pertains to a method for producing nicotinamide mononucleotide. Provided is a method for producing nicotinamide mononucleotide that comprises a step for reacting a microorganism, which is capable of producing nicotinamide mononucleotide, in a solution at pH 4-10.

Description

Technical Field

[0001] The present invention relates to a method for producing nicotinamide mononucleotide, which includes a step of reacting a microorganism having nicotinamide mononucleotide-producing ability in a solution with a pH of 4 to 10 (for example, a buffer solution or the like).

Background Art

[0002] In recent years, aging and aging-related diseases have been shown to be closely related to a decrease in the amount of nicotinamide adenine dinucleotide (NAD + ) and a decrease in the activity of NAD + -dependent deacetylase sirtuin (Non-Patent Documents 1 and 2). In addition, activation of sirtuin is considered to explain many of the effects related to lifespan extension or health promotion under calorie restriction (Non-Patent Document 2). + ) and + + It has been shown that the activity of NAD + -dependent deacetylase sirtuin is closely related to the decrease in the amount of nicotinamide adenine dinucleotide (NAD + ) and the decrease in the activity of NAD + -dependent deacetylase sirtuin (Non-Patent Documents 1 and 2). In addition, activation of sirtuin is considered to explain many of the effects related to lifespan extension or health promotion under calorie restriction (Non-Patent Document 2).

[0003] NAD + + has long been known as a coenzyme for redox reactions, but in recent years, it has also been known to play roles as substrates for poly(ADP-ribose) polymerase, CD38 / CD157, sirtuin, etc. (Non-Patent Document 1). In particular, the decomposition reaction of NAD + by sirtuin to nicotinamide promotes the lysine residue deacetylation reaction by the conjugated sirtuin, and is involved in various life phenomena related to health and longevity (Non-Patent Documents 1 and 2). While the amount of NAD + and sirtuin activity decrease with aging, the enzyme reaction products of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) that resynthesizes NAD + from nicotinamide, specifically, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), which are NAD + + from NAD + to nicotinamide promotes the lysine residue deacetylation reaction by the conjugated sirtuin, and is involved in various life phenomena related to health and longevity (Non-Patent Documents 1 and 2). While the amount of NAD + and sirtuin activity decrease with aging, the enzyme reaction products of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) that resynthesizes NAD + from nicotinamide, specifically, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), which are NAD + + amount and sirtuin activity decrease with aging, while the enzyme reaction products of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) that resynthesizes NAD + from nicotinamide, specifically, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), which are NAD + + to NAD + +It is known that supplementing with intermediate metabolites effectively reactivates sirtuins (Non-Patent Literature 1), and furthermore, that sirtuin activation exerts a wide range of antioxidant effects. For this reason, nicotinamide mononucleotide is known to contribute to many biological phenomena related to life extension and health promotion in calorie restriction.

[0004] Therefore, there is a need for an efficient method for producing nicotinamide mononucleotide. To date, it has been reported that nicotinamide mononucleotide can be obtained from yeasts with a history of being consumed as food, such as Torula yeast (Patent Document 1), and that nicotinamide riboside can be obtained from genetically modified bacteria selected from the group consisting of Escherichia coli (E. coli), B. subtilis, C. glutamicum, A. baylyi, and R. eutropha (Patent Document 2).

[0005] Furthermore, a method for producing nicotinamide mononucleotide is known (Patent Document 3), which includes a step of reacting β-nicotinamide adenine dinucleotide as a substrate with a metabolic composition of a microorganism belonging to the genus Aspergillus. More specifically, nicotinamide mononucleotide is produced by an enzymatic reaction of β-nicotinamide adenine dinucleotide contained in cultured yeast. The patented method requires the use of enzymes in the reaction, raising concerns about safety when the produced nicotinamide mononucleotide is consumed as food. In addition, the production volume of nicotinamide mononucleotide is not considered sufficient. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2017 / 200050 [Patent Document 2] International Publication No. 2017 / 083858 [Patent Document 3] International Publication No. 2019 / 181961 [Non-patent literature]

[0007] [Non-Patent Document 1] Imai, S. & Guarente, L. (2014) Trends Cell Biol., 24, 464-471. [Non-Patent Document 2] Guarente, L. (2013) Genes Dev., 27, 2072-2085. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Existing microbiological methods for producing nicotinamide mononucleotide still have room for improvement in production efficiency.

[0009] Therefore, the present invention aims to provide a highly efficient method for producing nicotinamide mononucleotide. [Means for solving the problem]

[0010] As a result of diligent research, the inventors discovered that the production of nicotinamide mononucleotide increases when a microorganism capable of producing nicotinamide mononucleotide is reacted in a reaction solution with a pH of 4.0 to 10.0 (for example, a reaction solution with pH adjusted using a buffer). The present invention was completed by further research based on this finding.

[0011] Item 1. A method for producing nicotinamide mononucleotide, comprising a nicotinamide mononucleotide enrichment step in which a microorganism capable of producing nicotinamide mononucleotide is reacted in a reaction solution with a pH of 4 to 10. Item 2. The method for producing nicotinamide mononucleotide as described in Item 1, wherein the microorganism capable of producing nicotinamide mononucleotide is a lactic acid bacterium. Item 3. The manufacturing method according to item 1 or 2, wherein the reaction solution with a pH of 4 to 10 contains a buffer selected from the group consisting of acetate buffer, phosphate buffer, borate buffer, carbonate buffer, Tris buffer, HEPES buffer, and MES buffer. Item 4. A manufacturing method according to any one of items 1 to 3, wherein the lactic acid bacteria belong to the genus Fructobacillus. Item 5. A method for producing lactic acid bacteria as described in any of Items 1 to 4, wherein the lactic acid bacteria are selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE BP02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE BP-02766), and Fructobacillus fructosus strain NBRC3516. [Effects of the Invention]

[0012] According to the present invention, by reacting a microorganism capable of producing nicotinamide mononucleotide in a reaction solution with a pH of 4 to 10, the desired nicotinamide mononucleotide can be easily and efficiently obtained at high concentrations even under very mild conditions, at room temperature and pressure. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below. The present invention is a method for producing nicotinamide mononucleotide, comprising a nicotinamide mononucleotide enrichment step in which a microorganism capable of producing nicotinamide mononucleotide is reacted in a reaction solution with a pH of 4 to 10 (for example, a reaction solution whose pH has been adjusted with a buffer).

[0014] Microorganisms capable of producing nicotinamide mononucleotide The microorganisms capable of producing nicotinamide mononucleotide used in the present invention are not particularly limited, but examples include yeast and lactic acid bacteria.

[0015] Edible yeast can be used as yeast. For example, yeasts belonging to the genus Saccharomyces, Kluyveromyces, Candida, Pichia, etc. can be mentioned. Among them, Candida utilis belonging to the genus Candida is preferable. More specifically, Candida utilis IAM4264, Candida utilis ATCC9950, Candida utilis ATCC9550, Candida utilis IAM4233, Candida utilis AHU3259, etc. can be exemplified.

[0016] Examples of lactic acid bacteria include Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Melissococcus, Enterococcus, Trichococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Oenococcus, Abiotrophia, and Desemedia. Examples include the genera Zia, Paralactobacillus, Granulicatella, Alkalibacterium, Olsenella, Isobaculum, Marinelactibacillus, Atopostipes, Lactovum, Pilibacter, Fructobacillus, Lacticigemium, Bavariicoccus, and Bifidobacterium, with the genus Fructobacillus being preferred.

[0017] Among them, examples of the genus Fructobacillus include Fructobacillus durionis, Fructobacillus tropaeoil, and Fructobacillus fructosus. More preferably, Fructobacillus tropaeoil and Fructobacillus fructosus are included.

[0018] Examples of more preferred lactic acid bacteria include Fructobacillus durionis strain RD011727 (deposit number NITE BP-02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE BP-02766), Fructobacillus fructosus strain NBRC3516, and Fructobacillus durionis strain NBRC113239. More preferably, Fructobacillus durionis strain RD011727 (deposit number NITE BP-02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE BP-02766), and Fructobacillus fructosus strain NBRC3516 are included.

[0019] In the present invention, one or a combination of multiple species of the above microorganisms having the ability to produce nicotinamide mononucleotide can be used.

[0020] For the microorganism having the ability to produce nicotinamide mononucleotide used in the present invention, the cells of the microorganism cultured in a separate culturing step can be used.

[0021] The culture medium used for culturing microorganisms can be any medium that contains a carbon source, a nitrogen source, and minerals, without any particular restrictions.

[0022] Carbon sources include carbohydrates and carbohydrate materials. Carbohydrates include sugars (monosaccharides, disaccharides, oligosaccharides), polysaccharides, and sugar alcohols. Examples of carbohydrates include lactose, sucrose, glucose, starch, xylitol, and dextrose. Carbohydrate materials can be any organic composition containing carbohydrates, such as milk and its processed products (skim milk powder, whey, milk powder, condensed milk, etc.), soy milk and its processed products (soy milk hydrolysate, etc.), grains, fruits, and vegetables. Milk can be derived from any mammal such as cows, goats, sheep, buffalo, camels, llamas, donkeys, yaks, horses, and reindeer. Carbohydrates may be isolated or contained in carbohydrate materials. For example, fructose (carbohydrate) may be used in the form contained in fruit (carbohydrate material). These carbon sources may be used individually or in combination of multiple types.

[0023] The concentration of the carbon source in the culture medium is not particularly limited and can be set appropriately depending on the type of medium and culture method, but for example, 0.5 to 15 w / w%, preferably 1 to 10 w / w%, and more preferably 1.5 to 8.5 w / w% are given.

[0024] Any inorganic or organic nitrogen source can be used as the nitrogen source. Examples include yeast extract (such as brewer's yeast), meat extract, proteins such as casein; protein hydrolysates such as peptone (such as protease peptone), peptides, and nitrogen-containing salts such as ammonium salts (such as ammonium citrate) and nitrates. These nitrogen sources may be used individually or in combination.

[0025] The concentration of the nitrogen source in the culture medium is not particularly limited and can be set appropriately according to the type of medium and culture method, but for proteins, for example, 0.3-4 w / w%, preferably 0.5-3 w / w%, and more preferably 1-2 w / w%; for peptides, for example, 0.1-2 w / w%, preferably 0.3-1.8 w / w%, and more preferably 0.5-1.5 w / w%; and for nitrogen-containing salts, for example, 0.03-1.5 w / w%, preferably 0.05-1 w / w%, and more preferably 0.1-0.5 w / w%.

[0026] Examples of minerals include manganese (manganese sulfate, etc.), zinc, iron, sodium (sodium acetate, etc.), potassium (dipotassium bisulfate, potassium phosphate, etc.), magnesium (magnesium sulfate, etc.), calcium, phosphorus (potassium phosphate, etc.), sulfur (manganese sulfate, potassium bisulfate, magnesium sulfate, etc.), and trace elements. These minerals may be used individually or in combination. Among these minerals, manganese, sodium, magnesium, and potassium are preferred.

[0027] The concentration of minerals in the culture medium is not particularly limited and can be set appropriately according to the type of medium and culture method, but examples include: for manganese, 0.001 to 0.01 w / w%, preferably 0.003 to 0.008 w / w%; for sodium, 0.05 to 1.5 w / w%, preferably 0.1 to 1 w / w%; for magnesium, 0.001 to 0.02 w / w%, preferably 0.005 to 0.015 w / w%; and for potassium, 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%.

[0028] In addition to the components mentioned above, the culture medium may also contain other components such as vitamins (e.g., B vitamins), surfactants (e.g., nonionic surfactants (e.g., Tween), anionic surfactants (e.g., SDS), antibacterial agents (e.g., triclosan), and antibiotics (e.g., monesin). These other components may be used individually or in combination. Among these other components, surfactants are preferred, and nonionic surfactants are more preferred.

[0029] The concentrations of other components in the culture medium are not particularly limited and can be set appropriately depending on the type of other components, the type of culture medium, the culture method, etc. However, if a surfactant is included, the concentration of the surfactant can be, for example, 0.01 to 0.5 w / w%, preferably 0.05 to 0.3 w / w%.

[0030] Culture conditions such as culture temperature and pH can be applied without particular restrictions and should be set according to the microorganism being used. Generally, a culture temperature of 21 to 37°C, preferably 25 to 34°C, and a pH of 3.0 to 8.0, particularly 3.5 to 7.0, is preferred.

[0031] The culture method can be batch culture, fed-batch culture, or continuous culture, but the latter is preferable for industrial purposes. There are no particular restrictions on conditions such as stirring and aeration during culture; general methods are acceptable.

[0032] Microorganisms obtained by culture can be used as is, or after solid-liquid separation by filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressure dehydration, belt press, centrifugal concentration dehydration, multi-disc dehydration, etc., to recover bacterial cells from the culture medium, or after washing the recovered bacterial cells, they can be used in the nicotinamide mononucleotide enrichment reaction. Preferably, bacterial cells recovered from the culture medium, and more preferably bacterial cells recovered from the culture medium and washed, can be used in the nicotinamide mononucleotide enrichment reaction.

[0033] By using microorganisms capable of producing nicotinamide mononucleotides obtained in the culture process in the nicotinamide mononucleotide enrichment reaction process, the nicotinamide mononucleotide content in the microorganisms can be increased.

[0034] In the nicotinamide mononucleotide enrichment reaction step, a reaction solution is prepared containing a microorganism capable of producing nicotinamide mononucleotide and a suitable reaction liquid, and adjusted to a pH of 4 to 10. The reaction to enrich nicotinamide mononucleotide is then carried out by maintaining the reaction solution at a suitable temperature. In this invention, the pH of the reaction solution refers to the pH at the temperature applied during the nicotinamide mononucleotide enrichment reaction.

[0035] The pH of the reaction solution in the nicotinamide mononucleotide enrichment reaction step is preferably 4.8 to 9.2, more preferably 5.8 to 9.2, and even more preferably 6.8 to 9.2, 6.8 to 8.2, or 7.8 to 9.2.

[0036] Furthermore, when using Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765) as a microorganism capable of producing nicotinamide mononucleotide, the pH of the reaction solution in the nicotinamide mononucleotide enrichment reaction step is preferably 4.8 to 9.2, more preferably 5.8 to 8.2, even more preferably 6.8 to 8.2, and even more preferably 7.8 to 8.2.

[0037] When using Fructobacillus durionis strain RD011727 (deposit number NITE BP-02764) as a microorganism capable of producing nicotinamide mononucleotide, the pH of the reaction solution in the nicotinamide mononucleotide enrichment reaction step is preferably 5.8 to 8.2, more preferably 5.8 to 8.2, and even more preferably 6.8 to 8.2.

[0038] When using Fructobacillus fructosus strain NBRC3516 as a microorganism capable of producing nicotinamide mononucleotide, the pH of the reaction solution in the nicotinamide mononucleotide enrichment reaction step is preferably 4.8 to 9.2, more preferably 5.8 to 9.2, even more preferably 6.8 to 9.2, even more preferably 7.8 to 9.2, and even more preferably 7.8 to 8.2.

[0039] The reaction liquid described above can be used without particular limitations, as long as the pH of the reaction solution can be adjusted within the above range. For example, when microorganisms obtained by culture are used directly in the nicotinamide mononucleotide enrichment reaction, the reaction liquid can be the culture medium used for culturing microorganisms capable of producing nicotinamide mononucleotide. When microorganisms obtained by culture are recovered from the culture medium and more preferably washed before being used in the nicotinamide mononucleotide enrichment reaction, the reaction liquid can be water (including adjusting the pH to an optimal range using a pH adjusting agent during the reaction), buffer solution, organic solvent, or a mixture of two or more of these.

[0040] Examples of buffer solutions include acetate buffer, phosphate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer. These buffer solutions can be used individually or in combination.

[0041] More specifically, examples include KHC8H4O4-NaOH (pH4.0), CH3COOH-CH3COONa (pH4.0), MES-NaOH (pH5.0), CH3COOH-CH3COONa (pH5.0), KH2PO4-K2HPO4 (pH6.0), MES-NaOH (pH6.0), KH2PO4-K2HPO4 (pH7.0), PIPES-NaOH (pH7.0), HEPES-NaOH (pH8.0), H3BO4-NaOH (pH8.0), CHES-NaOH (pH9.0), H3BO4-NaOH (pH9.0), H2CO3-NaHCO3 (pH10.0), and CHES-NaOH (pH10.0). Among these, CH3COOH-CH3COONa, KH2PO4-K2HPO4, and H3BO4-NaOH are preferred, with KH2PO4-K2HPO4 being more preferred.

[0042] Organic solvents include aromatic compounds such as benzene and benzonitrile, ketones such as acetone, acetylacetone, and methyl ethyl ketone, fatty acid esters such as ethyl acetate, butyl acetate, ethyl butyrate, and ethyl formate, ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and 1,4-dioxane, halogenated hydrocarbons such as dichloromethane, chloroform, and dichloroethane, and 1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, and 2,3 Examples of organic solvents include diols such as -butanediol, 1,2-hexanediol, 1,6-hexanediol, 1,2-pentanediol, 1,5-pentanediol, 2-methyl-2,4-pentanediol, and 3-methyl-1,5-pentanediol; alcohols having linear or branched alkyl groups with 1 to 7 carbon atoms; and alcohols such as cyclohexanol, 3-methoxy-3-methyl-1-butanol, and 3-methoxy-1-butanol. These organic solvents can be used individually or in combination.

[0043] As a pH adjusting agent, one that can adjust the pH to the optimal level during the reaction should be appropriately selected. Examples include inorganic acids such as hydrochloric acid, organic acids such as citric acid, inorganic bases such as hydroxides such as sodium hydroxide and potassium hydroxide, and organic bases such as organic amines. One of these compounds can be used alone, or a combination of several compounds can be used.

[0044] The reaction temperature in the nicotinamide mononucleotide enrichment reaction step of the present invention is, for example, 10 to 55°C, preferably 15 to 45°C, more preferably 20 to 37°C, and even more preferably 20 to 35°C. The reaction time is, for example, 0.1 to 48 hours, preferably 1 to 24 hours, and more preferably 3 to 20 hours.

[0045] The nicotinamide mononucleotide enrichment reaction can be carried out by suspending a microorganism capable of producing nicotinamide mononucleotide in the above-mentioned reaction liquid and allowing it to stand, stir, or shake.

[0046] Microorganisms containing nicotinamide mononucleotide obtained by the nicotinamide mononucleotide enrichment reaction can be dried as is by freeze-drying, shelf-drying, spray-drying, etc., to obtain a cell powder which can then be incorporated as a food additive or as an active ingredient in cosmetics or pharmaceuticals.

[0047] Furthermore, microorganisms containing nicotinamide mononucleotide obtained by the nicotinamide mononucleotide enrichment reaction can be recovered by separating the buffer from the solid-liquid by methods such as centrifugation and membrane filtration. The recovered microbial cells can then be dried by methods such as shelf drying or freeze-drying to obtain a microbial powder, which can then be incorporated as an additive in food products or as an active ingredient in cosmetics or pharmaceuticals. [Examples]

[0048] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0049] Test Example 1 [1-1] Culture of Fructobacillus tropaeoil strain RD012353 Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765), a lactic acid bacterium of the Fructobacillus genus capable of producing nicotinamide mononucleotide, was inoculated into 3 ml of MRS medium (pre-culture medium) manufactured by Difco and cultured statically at 30°C for 24 hours. The resulting culture solution was inoculated into 100 ml of MRS medium (main culture medium) to achieve an OD660 of 0.02 and cultured with shaking at 30°C for 12 hours. The obtained culture medium was subjected to centrifugation to recover the bacterial cells. The recovered bacterial cells were washed with 100 ml of 0.85 w / w% KCl aqueous solution. The washed bacterial cells were subjected to centrifugation again to recover the bacterial cells.

[0050] [1-2] Nicotinamide mononucleotide enrichment reaction step (NMN enrichment reaction) The bacterial cells recovered in [1-1] above were each suspended in 20 ml of the reaction liquid described below to prepare the reaction solution, which was then subjected to an NMN enrichment reaction by standing at 25°C for 8 hours. The pH of the reaction solution during the NMN enrichment reaction is as shown in Table 1.

[0051] The reaction liquids used in the NMN enrichment reaction are shown below. The reaction liquids with pH 3.0 and pH 11.0 were used for comparison. Comparative Example 1: 0.1M citrate buffer (pH 3.0) Example 1: 0.1M acetate buffer (pH 4.0) Example 2: 0.1M acetate buffer (pH 5.0) Example 3: 0.1M phosphate buffer (pH 6.0) Example 4: 0.1M phosphate buffer (pH 7.0) Example 5: 0.1M phosphate buffer (pH 8.0) Example 6: 0.1M borate buffer (pH 9.0) • Example 7: 0.1M carbonate buffer (pH 10.0) • Comparative Example 2: 0.1M phosphate buffer (pH 11.0)

[0052] After the reaction was complete, the bacterial cells were recovered by centrifugation. The recovered bacterial cells were washed with 100 ml of 0.85 w / w% KCl aqueous solution. The washed bacterial cells were subjected to centrifugation again, and the bacterial cells were recovered.

[0053] [1-3] Nicotinamide mononucleotide (NMN) level measurement The bacterial cells collected in [1-1] above and the bacterial cells collected in [1-2] above were suspended in 20 ml of deionized water, an equal amount of glass beads were added, and the bacterial cells were crushed using a bead crusher. The crushed bacterial cells were separated by centrifugation, and the supernatant (bacterial cell crushing extract) was collected. The recovered supernatant was analyzed by HPLC under the following analytical conditions, and the amount of nicotinamide mononucleotide (NMN) produced in the recovered bacterial cells was measured. The relative value of the NMN production in the bacterial cells recovered in [1-2] above was derived, with the NMN production in the bacterial cells recovered in [1-1] above (without NMN enrichment) set to 100. The results are shown in Table 1.

[0054] (MRS medium composition) 2w / w% glucose 1 w / w% protease peptone 1 w / w% beef extract 0.5 w / w% yeast extract 0.2 w / w% ammonium citrate 0.1w / w% Tween80 0.5 w / w% sodium acetate 0.01 w / w% magnesium sulfate 0.005 w / w% manganese sulfate 0.2 w / w% dipotassium hydrogen phosphate

[0055] (HPLC analysis conditions) Columns: DaisoPak SP-100-5-ODS-P (4.6 x 150 mm) x 2 Column temperature: 25℃ Eluent: 75 mM ammonium phosphate aqueous solution (pH 6.0) Flow rate: 0.6ml / min Detector: UV detector (260nm) Detection time: 15.1 minutes

[0056] [Table 1]

[0057] As shown in Table 1, the productivity of NMN can be significantly improved by reacting the bacterial cells after cultivation in a solution with a pH of 4 to 10.

[0058] Test Example 2 [2-1] Culture of Fructobacillus durionis strain RD011727 Fructobacillus durionis strain RD011727 (deposit number NITEBP-02764), a lactic acid bacterium of the Fructobacillus genus capable of producing nicotinamide mononucleotide, was inoculated into 3 ml of MRS medium (pre-culture medium) manufactured by Difco and cultured statically at 30°C for 24 hours. The resulting culture solution was then inoculated into 100 ml of MRS medium (main culture medium) to achieve an OD660 of 0.02 and cultured with shaking at 30°C for 12 hours. The obtained culture medium was subjected to centrifugation to recover the bacterial cells. The recovered bacterial cells were washed with 100 ml of 0.85 w / w% KCl aqueous solution. The washed bacterial cells were subjected to centrifugation again to recover the bacterial cells.

[0059] [2-2] Nicotinamide mononucleotide enrichment reaction step (NMN enrichment reaction) The NMN enrichment reaction was carried out in the same manner as in [1-2] of Test Example 1, except that the bacterial cells recovered in [2-1] above and the reaction liquids used in Examples 1-7 of Test Example 1 were used. The pH of the reaction solution during the NMN enrichment reaction was about the same as that of the reaction liquid, as shown in Table 2.

[0060] [2-3] Nicotinamide mononucleotide (NMN) level measurement The amount of NMN was measured in the same manner as in [1-3] of Test Example 1, except that the bacterial cells recovered in [2-1] and the bacterial cells recovered in [2-2] were used. The relative value of the NMN production of the bacterial cells recovered in [2-2] was derived, with the NMN production of the bacterial cells recovered in [2-1] (without NMN enrichment reaction) set to 100. The results are shown in Table 2.

[0061] [Table 2]

[0062] As shown in Table 2, the productivity of NMN can be significantly improved by reacting the bacterial cells after cultivation in a solution with a pH of 4 to 10.

[0063] Test Example 3 [3-1] Culture of Fructobacillus fructosus strain NBRC3516 Fructobacillus fructosus NBRC3516 strain, a lactic acid bacterium of the Fructobacillus genus capable of producing nicotinamide mononucleotide, was inoculated into 3 ml of Difco's MRS medium (pre-culture medium) and cultured statically at 30°C for 24 hours. The resulting culture solution was then inoculated into 100 ml of MRS medium (main culture medium) to achieve an OD660 of 0.02 and cultured with shaking at 30°C for 12 hours. The resulting culture medium was subjected to centrifugation to recover the bacterial cells.

[0064] [3-2] Nicotinamide mononucleotide enrichment reaction step (NMN enrichment reaction) The NMN enrichment reaction was carried out in the same manner as in [1-2] of Test Example 1, except that the bacterial cells recovered in [3-1] above and the reaction liquids used in Examples 1 to 6 of Test Example 1 were used. The pH of the reaction solution during the NMN enrichment reaction was about the same as that of the reaction liquid, as shown in Table 3.

[0065] [3-3] Measurement of nicotinamide mononucleotide (NMN) levels The amount of NMN was measured in the same manner as in [1-3] of Test Example 1, except that the bacterial cells recovered in [3-1] and [3-2] above were used. The relative value of the NMN production of the bacterial cells recovered in [3-2] above was derived, with the NMN production of the bacterial cells recovered in [3-1] above (without NMN enrichment reaction) set to 100. The results are shown in Table 3.

[0066] [Table 3]

[0067] As shown in Table 3, the productivity of NMN can be significantly improved by reacting the bacterial cells after cultivation in a solution with a pH of 4 to 10.

[0068] Test Example 4 Except for changing the reaction liquid used in the NMN enrichment reaction to the buffer solution described below (the pH of the reaction solution during the NMN enrichment reaction was approximately the same as that of the reaction liquid, as shown in Table 4), the same procedure as in Test Examples 1-3 was followed, and the relative value of the NMN production was derived. The results are shown in Table 4.

[0069] The reaction solution (buffer) used in the NMN enrichment reaction is shown below. Examples 21, 24, 27: 0.1M MES buffer (pH 5.0) ·Example 22, 25, 28: 0.1M HEPES buffer (pH 7.0) Examples 23, 26, 29: 0.1M Tris buffer (pH 9.0)

[0070] [Table 4]

[0071] As shown in Table 4, by reacting the bacterial cells after cultivation in a solution with a pH of 4 to 10, the productivity of NMN can be significantly improved regardless of the type of buffer used.

[0072] Test Example 5 The reaction liquid used in the NMN enrichment reaction was changed to deionized water, and the pH during the reaction was adjusted to 7 using a 24 wt% NaOH aqueous solution (the pH of the reaction solution during the NMN enrichment reaction is shown in Table 5). The procedure was the same as in Test Example 3, and the relative value of the NMN production was derived. The results are shown in Table 5.

[0073] [Table 5]

Claims

1. A method for producing nicotinamide mononucleotide, comprising a nicotinamide mononucleotide enrichment step in which a lactic acid bacterium of the genus Fructobacillus, capable of producing nicotinamide mononucleotide, is reacted in a buffer solution with a pH of 4 to 10 at a temperature of 10 to 55°C for 1 to 48 hours.

2. The manufacturing method according to claim 1, wherein the buffer solution with a pH of 4 to 10 includes a buffer solution selected from the group consisting of acetate buffer solution, phosphate buffer solution, borate buffer solution, carbonate buffer solution, Tris buffer solution, HEPES buffer solution, and MES buffer solution.

3. The manufacturing method according to claim 1 or 2, wherein the lactic acid bacteria are selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE BP-02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE BP-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE BP-02766), and Fructobacillus fructosus NBRC3516.

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