Lactic acid bacteria that produce nicotinamide riboside, and lactic acid bacteria that produce nicotinamide mononucleotide and nicotinamide riboside

Abstract

To provide microorganisms having high production efficiency of nicotinamide riboside, and microorganisms capable of producing both nicotinamide mononucleotide and nicotinamide riboside.SOLUTION: Nicotinamide mononucleotide and nicotinamide riboside can be produced by culturing lactic acid bacteria belonging to the genus Fructobacillus.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lactic acid bacteria that produce nicotinamide riboside, and to lactic acid bacteria that produce nicotinamide mononucleotide and nicotinamide riboside. [Background technology]

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

[0003] NAD + While it has long been known as a coenzyme in redox reactions, in recent years, its role as a substrate for poly ADP-ribose polymerase, CD38 / CD157, and sirtuins has also become known (Non-Patent Document 1). In particular, NAD + The decomposition reaction of NAD to nicotinamide promotes the deacetylation of lysine residues by sirtuins, which are conjugated with NAD, and is involved in various life phenomena related to health and longevity (Non-Patent Documents 1 and 2). + The amount and activity of sirtuins declines, while nicotinamide is converted to NAD + Specifically, NAD, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), is produced by the enzyme reaction product of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), which resynthesizes NAD. +It is known that supplementation of intermediate metabolites effectively reactivates sirtuins (Non-Patent Document 1), and furthermore, it is known that a wide range of antioxidant effects are expressed through activation of sirtuins. Therefore, nicotinamide mononucleotide and nicotinamide riboside are known to contribute to many biological phenomena related to life extension and health promotion in calorie restriction.

[0004] Therefore, a method for efficiently producing nicotinamide mononucleotide or nicotinamide riboside is desired. It has been reported that nicotinamide mononucleotide can be obtained from yeasts that are consumed in the diet, 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, Bacillus subtilis, C. glutamicum, A. baylyi, and R. eutropha (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 200050 [Patent Document 2] International Publication No. 2017 / 083858 [Non-patent literature]

[0006] [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. Summary of the Invention [Problem to be solved by the invention]

[0007] Previous microbiological methods for producing nicotinamide mononucleotide or nicotinamide riboside still leave room for improvement in production efficiency.

[0008] Therefore, an object of the present invention is to provide a microorganism that has high efficiency in producing nicotinamide riboside, and a microorganism that can produce both nicotinamide mononucleotide and nicotinamide riboside. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have found that lactic acid bacteria belonging to a specific genus can efficiently produce nicotinamide riboside, and furthermore can produce both nicotinamide mononucleotide and nicotinamide riboside. The present invention was completed through further research based on this finding.

[0010] That is, the present invention provides the following aspects. Item 1. A method for producing nicotinamide riboside, comprising a step of culturing lactic acid bacteria belonging to the genus Fructobacillus and preparing a culture containing nicotinamide riboside. Item 2. The method according to Item 1, wherein the culture is carried out in a medium that does not contain fructose. Item 3. The method according to Item 1 or 2, wherein the lactic acid bacterium is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 4. The method according to Item 1 or 2, wherein the lactic acid bacteria is Fructobacillus tropaeoil RD012353 strain (deposit number NITE P-02765) and / or Fructobacillus fructosus NBRC3516 strain. Item 5. A nicotinamide riboside enrichment agent comprising a culture containing nicotinamide riboside obtained from lactic acid bacteria belonging to the genus Fructobacillus, a supernatant separated from the culture, and / or nicotinamide riboside isolated from the supernatant. Item 6. The agent according to Item 5, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 7. The fortifying agent according to Item 5, wherein the lactic acid bacteria is Fructobacillus tropaeoil RD012353 strain (deposit number NITE P-02765) and / or Fructobacillus fructosus NBRC3516 strain. Item 8. A food, beverage, cosmetic, or pharmaceutical comprising the nicotinamide riboside enhancer according to any one of Items 5 to 7. Item 9. A nicotinamide riboside-producing lactic acid bacterium selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 10. A method for producing nicotinamide mononucleotide and nicotinamide riboside, comprising a step of culturing lactic acid bacteria belonging to the genus Fructobacillus. Item 11. The method according to Item 10, wherein the culture is carried out in a medium containing fructose. Item 12. The method according to Item 10 or 11, wherein the lactic acid bacterium is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 13. The method according to Item 10 or 11, wherein the lactic acid bacteria is Fructobacillus tropaeoil RD012353 strain (deposit number NITE P-02765) and / or Fructobacillus fructosus NBRC3516 strain. Item 14. A nicotinamide mononucleotide and nicotinamide riboside enrichment agent comprising a culture containing nicotinamide mononucleotide and nicotinamide riboside obtained from lactic acid bacteria belonging to the genus Fructobacillus, bacterial cells separated from the culture, a disrupted product of the bacterial cells, and / or nicotinamide riboside and nicotinamide riboside isolated from the disrupted product. Item 15. The enhancer according to Item 14, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 16. The enhancer according to Item 14, wherein the lactic acid bacteria is Fructobacillus durionis strain RD011727 (deposit number NITE-P02764) and / or Fructobacillus fructosus strain NBRC3516. Item 17. A food, drink, cosmetic, or pharmaceutical product comprising the nicotinamide mononucleotide and nicotinamide riboside enhancer according to any one of Items 14 to 16. Item 18. A nicotinamide mononucleotide and nicotinamide riboside-producing lactic acid bacterium selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoil strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoil strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516. Item 19. Use of a culture containing nicotinamide riboside obtained from a lactic acid bacterium belonging to the genus Fructobacillus, a supernatant separated from the culture, and / or nicotinamide riboside isolated from the supernatant for the manufacture of a nicotinamide riboside enrichment agent. Item 20. Use of a culture containing nicotinamide mononucleotide and nicotinamide riboside obtained from a lactic acid bacterium belonging to the genus Fructobacillus, a bacterial cell separated from the culture, a disrupted product of the bacterial cell, and / or nicotinamide riboside and nicotinamide riboside isolated from the disrupted product, for the production of a nicotinamide mononucleotide and nicotinamide riboside enhancer. Effect of the Invention

[0011] According to the present invention, lactic acid bacteria belonging to the genus Fructobacillus are provided as microorganisms with high nicotinamide riboside production efficiency and capable of producing both nicotinamide mononucleotide and nicotinamide riboside. Therefore, a culture containing nicotinamide riboside or a culture containing nicotinamide mononucleotide and nicotinamide riboside obtained from lactic acid bacteria belonging to the genus Fructobacillus can be used as a food additive for enriching nicotinamide riboside or a food additive for enriching nicotinamide mononucleotide and nicotinamide riboside, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Method for producing nicotinamide riboside The method for producing nicotinamide riboside of the present invention is characterized by comprising a step of culturing lactic acid bacteria belonging to the genus Fructobacillus and preparing a culture containing nicotinamide riboside. The method for producing nicotinamide riboside of the present invention is described in detail below.

[0013] lactic acid bacteria The lactic acid bacteria used in the production method of the present invention are not particularly limited as long as they belong to the genus Fructobacillus. Lactic acid bacteria belonging to the genus Fructobacillus produce nicotinamide riboside.

[0014] Preferred examples of lactic acid bacteria include Fructobacillus durionis, Fructobacillus tropaeoil, and Fructobacillus fructosus, and more preferably Fructobacillus tropaeoil and Fructobacillus fructosus.

[0015] More preferred examples of lactic acid bacteria include Fructobacillus durionis RD011727 strain, Fructobacillus tropaeoil RD012353 strain, Fructobacillus tropaeoil RD012354 strain, Fructobacillus fructosus NBRC3516 strain, and Fructobacillus durionis NBRC113239 strain, and even more preferred examples include Fructobacillus durionis RD011727 strain, Fructobacillus tropaeoil RD012353 strain, Fructobacillus tropaeoil RD012354 strain, and Fructobacillus fructosus NBRC3516 strain.

[0016] In the method for producing nicotinamide riboside of the present invention, from the viewpoint of producing even larger amounts of nicotinamide riboside, preferred lactic acid bacteria include Fructobacillus fructosus and the Fructobacillus tropaeoil RD012353 strain, more preferred are the Fructobacillus tropaeoil RD012353 strain and the Fructobacillus fructosus NBRC3516 strain, and particularly preferred is the Fructobacillus tropaeoil RD012353 strain.

[0017] The Fructobacillus durionis RD011727 strain has been deposited domestically at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE-P02764 on August 21, 2018.

[0018] The Fructobacillus tropaeoil RD012353 strain was deposited domestically at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-02765 on August 21, 2018.

[0019] The Fructobacillus tropaeoil RD012354 strain was deposited domestically at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-02766 on August 21, 2018.

[0020] These Fructobacillus lactic acid bacteria strains may be used alone or in combination of two or more kinds.

[0021] Culture medium The medium used for culturing Fructobacillus lactic acid bacteria may be appropriately selected. Examples of the medium include those used for expansion culture (pre-culture medium) and those used for production culture (main culture medium). The main culture medium can be prepared based on the medium used as the pre-culture medium, and further containing additives. The medium is preferably a liquid medium, but may be an agar medium. In addition to a carbon source, the medium generally contains a nitrogen source, minerals, etc.

[0022] Examples of carbon sources include carbohydrates and carbohydrate materials. Examples of carbohydrates include sugars (monosaccharides, disaccharides, oligosaccharides), polysaccharides, and sugar alcohols. Examples of carbohydrates include lactose, sucrose, glucose, starch, xylitol, dextrose, and the like. The carbohydrate material may be an organic composition containing carbohydrates, and examples of such carbohydrate materials include foods 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. Examples of milk include milk derived from any mammal such as cows, goats, sheep, buffalo, camels, llamas, donkeys, yaks, horses, and reindeers. The carbohydrates may be isolated or may be contained in the carbohydrate material. For example, fructose (carbohydrate) may be used in the form contained in fruits (carbohydrate material). These carbon sources may be used alone or in combination of multiple types. Among these carbon sources, glucose is preferable. In the method for producing nicotinamide riboside of the present invention, it is preferable that the medium does not contain fructose.

[0023] The concentration of the carbon source in the medium is not particularly limited and may be appropriately set depending on the type of medium, the culture method, etc., and may be, for example, 0.5 to 4 w / w%, preferably 1 to 3 w / w%, and more preferably 1.5 to 2.5 w / w%.

[0024] As the nitrogen source, any inorganic or organic nitrogen source can be used. For example, proteins such as yeast extract (e.g., brewer's yeast), meat extract, and casein; protein hydrolysates such as peptone (e.g., protease peptone), peptides such as peptides; nitrogen-containing salts such as ammonium salts (e.g., ammonium citrate), and nitrates can be used. These nitrogen sources can be used alone or in combination.

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

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

[0027] The concentration of minerals in the medium is not particularly limited and may be set appropriately depending on the type of medium, culture method, etc. In the case of manganese, for example, the concentration is 0.001 to 0.01 w / w%, preferably 0.003 to 0.008 w / w%; in the case of sodium, the concentration is, for example, 0.05 to 1.5 w / w%, preferably 0.1 to 1 w / w%; in the case of magnesium, the concentration is, for example, 0.001 to 0.02 w / w%, preferably 0.005 to 0.015 w / w%; and in the case of potassium, the concentration is, for example, 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%.

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

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

[0030] Furthermore, when the culture obtained by the production method of the present invention is used as a nicotinamide riboside enrichment agent for food, the medium components are selected from the above-mentioned components that are approved as food additives.

[0031] Culture method The method for culturing the lactic acid bacteria of the genus Fructobacillus in the production method of the present invention is not particularly limited as long as the method is under growth conditions for the lactic acid bacteria of the genus Fructobacillus and allows the preparation of a culture containing nicotinamide riboside.

[0032] The culture temperature may be an optimum temperature for the Fructobacillus lactic acid bacteria to be cultured, and may be, for example, 26 to 40° C., preferably 27 to 38° C., more preferably 28 to 36° C., and even more preferably 29 to 34° C. The culture time may be appropriately set according to the type of Fructobacillus lactic acid bacteria to be actually cultured, and may be, for example, 4 to 48 hours, preferably 8 to 36 hours, and more preferably 12 to 24 hours.

[0033] In the production method of the present invention, the culture solution does not need to be stirred during the cultivation of the Fructobacillus lactic acid bacteria, and even under such conditions, nicotinamide riboside can be produced at a high level. Note that in the production method of the present invention, the culture solution may be stirred during the cultivation of the lactic acid bacteria in order to produce nicotinamide riboside at a higher level.

[0034] In the production method of the present invention, the above-mentioned culture is preferably carried out as a production culture (main culture) for a certain period of time, and before that, an expansion culture (preculture) can be carried out in a small amount of medium (for example, 1 / 6 to 1 / 4 of the main culture medium in terms of volume ratio). The culture conditions for the preculture can be appropriately set according to the type of Fructobacillus lactic acid bacteria, and the above-mentioned conditions can be adopted. In addition, in the main culture, the culture obtained in the preculture can be inoculated into the main culture medium so that the OD660 becomes, for example, 0.01 to 0.04, preferably 0.01 to 0.03.

[0035] A culture containing nicotinamide riboside is prepared as described above. This culture may contain other metabolites of Fructobacillus lactic acid bacteria co-produced in the culture.

[0036] The use form of the prepared culture containing nicotinamide riboside includes the use of the prepared culture as it is, the use of the culture by separating the supernatant, and the use of nicotinamide riboside by isolating it from the supernatant.The separation method includes, for example, centrifugation, membrane filtration, etc.The isolation method includes various chromatographic techniques such as ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, and high performance liquid chromatography, salting out, solvent precipitation, etc.

[0037] 2. Nicotinamide riboside enhancer; food, beverage, cosmetic or pharmaceutical The culture containing nicotinamide riboside obtained as described above, the supernatant separated from the culture, and / or the nicotinamide riboside isolated from the supernatant can be suitably used as a nicotinamide riboside enhancer to be incorporated as an additive in foods or as an active ingredient in cosmetics or pharmaceuticals for the purpose of enriching nicotinamide riboside.

[0038] When nicotinamide riboside enhancers are incorporated into foods, beverages, cosmetics, or pharmaceuticals and ingested, taken or applied, the nicotinamide riboside taken into the body is converted into nicotinamide adenine dinucleotide, a substance necessary for cells to use energy, resulting in a variety of useful effects.

[0039] The nicotinamide riboside enhancer may contain other Fructobacillus lactic acid bacteria metabolites in addition to nicotinamide riboside. In this case, the nicotinamide riboside enhancer can be used as an enhancer for at least one of the other Fructobacillus lactic acid bacteria metabolites in addition to nicotinamide riboside.

[0040] By incorporating a nicotinamide riboside enhancer into food and beverages, nicotinamide riboside can be efficiently ingested through eating and drinking. This allows for the health benefits of ingesting nicotinamide riboside (calorie restriction, anti-aging, health promotion, etc.). Note that food and beverages include not only food and beverages for humans, but also animal feed.

[0041] Examples of foods and beverages include, but are not limited to, fermented milk (drinkable yogurt, etc.), lactic acid bacteria drinks, milk drinks (coffee milk, fruit milk, etc.), tea drinks (green tea, black tea, oolong tea, etc.), fruit and vegetable drinks (drinks containing fruit juices such as orange, apple, grape, etc., and vegetable juices such as tomato, carrot, etc.), alcoholic drinks (beer, sparkling wine, wine, etc.), carbonated drinks, soft drinks, water-based drinks, and other drinks; and foods such as fermented milk (set yogurt, soft yogurt, etc.), sweets, instant foods, seasonings, and other processed foods.

[0042] In addition, functional foods are also included as food and drink. Functional foods refer to foods that have a certain functionality for the living body, and examples of such functional foods include health functional foods such as foods for specified health uses (including conditionally designated foods for specified health uses) and foods with nutritional functions, functional food, foods with functional claims, foods for special uses, dietary supplements, health supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquids, and other dosage forms), and beauty foods (for example, diet foods). Furthermore, functional foods may be foods for special uses such as foods for the sick, powdered milk for pregnant and lactating women, infant formula, foods for the elderly, and foods for care.

[0043] In addition, by incorporating the nicotinamide riboside enhancer into cosmetics, it is possible to expect the pharmacological effects of nicotinamide riboside (such as anti-aging) upon application.

[0044] Examples of cosmetics include basic cosmetics such as lotions, milky lotions, creams, essences, gels, packs, sheet masks, and lip balms; skin cleansers such as facial cleansers, makeup removers (including cleansing agents), exfoliants, and body shampoos; body care cosmetics such as sunscreens, body gels, body massage agents, antiperspirants, deodorants, hair removers, and bath additives; make-up cosmetics such as foundations, powders, lipsticks, blushers, eye shadows, eyeliners, mascaras, and eyebrow inks; nail cosmetics such as manicures and nail removers; hair cosmetics such as hair styling agents, shampoos, conditioners, rinses, and hair growth agents; and oral cosmetics such as liquid toothpaste, toothpaste, mouthwash, and mouth sprays.

[0045] Furthermore, by incorporating the nicotinamide riboside enhancer into a pharmaceutical product, nicotinamide riboside can be taken orally or transdermally as an active ingredient by taking or applying it, which is expected to have pharmacological effects (such as treatment or prevention of Cockayne syndrome and aging-related diseases, and treatment or prevention of oxidative damage to the skin) due to the intake of nicotinamide riboside.

[0046] Pharmaceuticals include oral preparations and topical preparations (including quasi-drugs). Oral preparations include tablets, coated tablets, sugar-coated tablets, capsules, and liquids, while topical preparations include liquids (including lotions, sprays, aerosols, and emulsions), foams, ointments, creams, gels, and patches.

[0047] The target subjects of the food, beverage, cosmetic or pharmaceutical containing a nicotinamide riboside enhancer are preferably mammals, for example, primates such as humans, chimpanzees and gorillas; pets or livestock such as dogs, cats, rabbits, cows, horses, pigs, goats, sheep and donkeys; and laboratory animals such as mice, rats, hamsters and monkeys.

[0048] 3. Method for producing nicotinamide mononucleotide and nicotinamide riboside Lactic acid bacteria belonging to the genus Fructobacillus produce nicotinamide mononucleotide as well as nicotinamide riboside. Therefore, the present invention also provides a method for producing nicotinamide mononucleotide and nicotinamide riboside.

[0049] In the method for producing nicotinamide mononucleotide and nicotinamide riboside, nicotinamide mononucleotide and nicotinamide riboside are co-produced by culturing Fructobacillus lactic acid bacteria under growth conditions under which a culture containing nicotinamide mononucleotide and nicotinamide riboside can be prepared. As a result, a culture containing nicotinamide mononucleotide and nicotinamide riboside is prepared.

[0050] The lactic acid bacteria used in the method for producing nicotinamide mononucleotide and nicotinamide riboside are as described above in "1. Method for producing nicotinamide riboside". Specifically, preferred examples of the lactic acid bacteria include Fructobacillus durionis, Fructobacillus tropaeoil, and Fructobacillus fructosus.

[0051] More preferred examples of lactic acid bacteria include Fructobacillus durionis strain RD011727, Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus durionis strain NBRC113239.

[0052] However, in the method for producing nicotinamide mononucleotide and nicotinamide riboside of the present invention, from the viewpoint of producing a larger amount of nicotinamide mononucleotide (i.e., a higher ratio of nicotinamide mononucleotide to nicotinamide riboside), preferred examples of the lactic acid bacteria include Fructobacillus durionis, Fructobacillus fructosus, and the Fructobacillus tropaeoil RD012353 strain, more preferred examples include Fructobacillus durionis and Fructobacillus fructosus, and even more preferred examples include the Fructobacillus durionis RD011727 strain and the Fructobacillus fructosus NBRC3516 strain.

[0053] Furthermore, in the method for producing nicotinamide mononucleotide and nicotinamide riboside of the present invention, from the viewpoint of producing both nicotinamide mononucleotide and nicotinamide riboside in a well-balanced manner, the lactic acid bacterium is preferably Fructobacillus tropaeoil, and more preferably the Fructobacillus tropaeoil RD012353 strain.

[0054] The medium used in the method for producing nicotinamide mononucleotide and nicotinamide riboside is as described above in "1. Method for producing nicotinamide riboside", except that it preferably further contains fructose as a carbon source. The concentration of fructose in the medium is not particularly limited and may be appropriately set depending on the type of medium, the culture method, etc., and may be, for example, 0.5 to 4 w / w%, preferably 1 to 3.5 w / w%, and more preferably 1.5 to 2.5 w / w%. By including fructose in the medium, lactic acid bacteria belonging to the genus Fructobacillus accumulate nicotinamide riboside and nicotinamide riboside in the bacterial cells.

[0055] The culture method and the like used in the production method of nicotinamide mononucleotide and nicotinamide riboside are also as described above in "1. Production method of nicotinamide riboside". As a result, both nicotinamide mononucleotide and nicotinamide riboside are produced. Examples of the use form of the prepared culture containing both nicotinamide mononucleotide and nicotinamide riboside include a form in which the prepared culture is used as is, a form in which the culture is separated and used, a form in which the culture is disrupted and used, and a form in which nicotinamide riboside is isolated and used. Examples of the separation method include centrifugation and membrane filtration. Examples of the cell disruption method include bead disruption. Examples of the isolation method include various chromatographic techniques such as ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, and high performance liquid chromatography, salting out, solvent precipitation, and the like.

[0056] 4. Nicotinamide mononucleotide and nicotinamide riboside enhancers; food, beverages, cosmetics or pharmaceuticals A culture containing nicotinamide mononucleotide and nicotinamide riboside obtained by the method for producing nicotinamide mononucleotide and nicotinamide riboside, bacterial cells separated from the culture, a disrupted product of the bacterial cells, and / or nicotinamide mononucleotide and nicotinamide riboside isolated from the disrupted product can be suitably used as a nicotinamide mononucleotide and nicotinamide riboside enhancer to be incorporated as an additive in food or as an active ingredient in cosmetics or pharmaceuticals for the purpose of enhancing nicotinamide mononucleotide and nicotinamide riboside.

[0057] When nicotinamide mononucleotide and nicotinamide riboside enhancers are incorporated into foods, beverages, cosmetics, or pharmaceuticals and ingested, taken or applied, the nicotinamide mononucleotide and nicotinamide riboside taken into the body are converted into nicotinamide adenine dinucleotide, a substance necessary for cells to use energy, thereby achieving a variety of useful effects.

[0058] The nicotinamide mononucleotide and nicotinamide riboside enrichment agent may contain other Fructobacillus lactic acid bacteria metabolites in addition to nicotinamide mononucleotide and nicotinamide riboside. In this case, the nicotinamide mononucleotide and nicotinamide riboside enrichment agent can be used as an enrichment agent for the other Fructobacillus lactic acid bacteria metabolites in addition to nicotinamide mononucleotide and nicotinamide riboside.

[0059] By incorporating a nicotinamide mononucleotide and nicotinamide riboside enhancer into food and beverages, nicotinamide mononucleotide and nicotinamide riboside can be efficiently ingested through eating and drinking.

[0060] The foods and beverages in which nicotinamide mononucleotide and nicotinamide riboside enhancers are formulated are as described above in “2. Nicotinamide riboside enhancers; foods, beverages, cosmetics, or pharmaceuticals.”

[0061] Furthermore, by incorporating the nicotinamide mononucleotide and nicotinamide riboside enhancer into cosmetics, it is possible to expect pharmacological effects due to nicotinamide riboside (such as anti-aging) and pharmacological effects due to nicotinamide mononucleotide (such as an increase in intracellular nicotinamide mononucleotide and anti-aging).

[0062] Cosmetics containing nicotinamide mononucleotide and nicotinamide riboside enhancers are as described above in “2. Nicotinamide riboside enhancers; foods, beverages, cosmetics, or pharmaceuticals.”

[0063] Furthermore, by incorporating the nicotinamide mononucleotide and nicotinamide riboside enhancer of the food into a medicine, nicotinamide mononucleotide and nicotinamide riboside can be taken orally or transdermally as active ingredients by taking or applying it, which is expected to produce pharmacological effects by taking nicotinamide mononucleotide (treatment or prevention of age-related diseases, etc.) and nicotinamide riboside (treatment or prevention of Cockayne syndrome and age-related diseases, and treatment or prevention of oxidative damage to the skin, etc.).

[0064] The dosage forms and applications of pharmaceuticals containing nicotinamide mononucleotide and nicotinamide riboside enhancers are as described above in “2. Nicotinamide riboside enhancers; foods, beverages, cosmetics, or pharmaceuticals.” EXAMPLES

[0065] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0066] Example 1: Production of nicotinamide riboside using Fructobacillus lactic acid bacteria Fructobacillus durionis RD011727 strain (deposit number NITE-P02764), Fructobacillus tropaeoil RD012353 strain (deposit number NITE P-02765), Fructobacillus tropaeoil RD012354 strain (deposit number NITE P-02766), and Fructobacillus fructosus NBRC3516 strain were each independently inoculated into 3 ml of MRS medium (preculture medium) manufactured by Difco, and expanded at 30°C for 24 hours at 30°C. The resulting culture was inoculated into 15 ml of MRS medium (main culture medium) so that the OD660 was 0.02, and production culture was performed at 30°C for 12 hours at 30°C. The resulting culture was centrifuged to recover the supernatant and the bacterial cells. Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) were quantified in the collected culture supernatant and the collected bacterial cells.

[0067] (MRS medium composition) 2w / w% glucose 1w / w% protease peptone 1w / w% beef extract 0.5w / w% yeast extract 0.2w / w% ammonium citrate 0.1w / w% Tween80 0.5w / w% sodium acetate 0.01w / w% magnesium sulfate 0.005w / w% manganese sulfate 0.2w / w% dipotassium hydrogen phosphate

[0068] (Quantification of Nicotinamide Mononucleotide and Nicotinamide Riboside in Supernatant) 15 ml of the collected supernatant was mixed with the same volume of 4 w / w% sodium bicarbonate solution, and the resulting mixture was passed through a BondElut PBA column (1 ml, manufactured by Agilent). Furthermore, 3 ml of 2 w / w% sodium bicarbonate solution was passed through the column to wash it, and then 3 ml of 2 v / v% formic acid solution was passed through to collect the eluate. 150 μl of 1.3 M potassium hydroxide aqueous solution and 100 μl of 20 w / w% acetophenone were added to 250 μl of the eluate, and reacted at 4 ° C for 30 minutes, and then 400 μl of 98 v / v% formic acid aqueous solution was added and reacted at 110 ° C for 7 minutes to perform a fluorescent derivatization treatment of nicotinamide mononucleotide and nicotinamide riboside, and analyzed under the HPLC conditions described below, and the amount of nicotinamide mononucleotide and nicotinamide riboside produced in the supernatant (amount produced per culture solution; mg / L) was measured. The results are shown in Table 1. In Table 1, nd indicates that no detection was found.

[0069] (Quantitative determination of intracellular nicotinamide mononucleotide and nicotinamide riboside) The collected cells were washed with 10 ml of 0.85 w / w% KCl aqueous solution. The washed cells were centrifuged again to collect the cells. The collected cells were suspended in 0.5 ml of 0.1 M potassium phosphate buffer (pH 7.0), and an equal amount of 0.1 mm zirconia beads was added, followed by disruption of the cells using a bead crusher. The disrupted cells were separated by centrifugation, and the supernatant (cell disruption extract) was collected. 150μl of 1.3M potassium hydroxide aqueous solution and 100μl of 20w / w% acetophenone were added to 250μl of the cell disruption extract, and the mixture was reacted at 4℃ for 30 minutes, followed by adding 400μl of 98v / v% formic acid aqueous solution and reacting at 110℃ for 7 minutes to perform fluorescent derivatization of cotinamide mononucleotide and nicotinamide riboside. The mixture was analyzed under the HPLC conditions described below, and the amount of nicotinamide mononucleotide and nicotinamide riboside produced in the collected cells (production amount per cell; mg / g of dry cells) was measured. The results are shown in Table 1.

[0070] (HPLC analysis conditions) Column: YMC Triart C18 (4.6 x 150 mm) Column temperature: 30℃ Eluent A: 0.1v / v% formic acid in water Eluent B: 0.1 v / v% formic acid in acetonitrile Gradient conditions: Gradient from 0 min (eluent A: eluent B = 9:1 (volume ratio)) to 15 min (eluent A: eluent B = 3:7 (volume ratio)) Flow rate: 1ml / min Detector: Fluorescence detector (Ex: 320 nm, Em: 458 nm) Detection time: 4.3 min (for nicotinamide mononucleotide) 5.1 min (for nicotinamide riboside)

[0071] [Table 1]

[0072] As is clear from Table 1, in the culture obtained using a medium containing no fructose, neither nicotinamide mononucleotide (NMN) nor nicotinamide riboside (NR) was detected in the cells, and nicotinamide mononucleotide (NMN) was not detected in the supernatant, while specific production of nicotinamide riboside (NR) was confirmed in the supernatant. In particular, production of a large amount of nicotinamide riboside (NR) was confirmed from Fructobacillus tropaeoil RD012353 strain and Fructobacillus fructosus NBRC3516 strain, especially Fructobacillus tropaeoil RD012353 strain.

[0073] Example 2 Production of nicotinamide mononucleotide and nicotinamide riboside using Fructobacillus lactic acid bacteria Except for adding 2 w / w% fructose to the main culture medium, the culture, the culture supernatant and the bacterial cells were collected, and nicotinamide mononucleotide and nicotinamide riboside were quantified in the same manner as in Example 1. The results are shown in Table 2.

[0074] [Table 2]

[0075] As is clear from Table 2, in the culture obtained using a medium containing fructose, neither nicotinamide mononucleotide (NMN) nor nicotinamide riboside (NR) was detected in the supernatant, while specific production of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) was confirmed in the cells. In addition, both nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) were produced in a well-balanced manner from Fructobacillus tropaeoil RD012353 and Fructobacillus tropaeoil RD012354, especially from Fructobacillus tropaeoil RD012353. On the other hand, it was confirmed that Fructobacillus durionis RD011727 strain and Fructobacillus fructosus NBRC3516 strain, especially Fructobacillus durionis RD011727 strain, produced both nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), with the ratio of nicotinamide mononucleotide (NMN) being particularly high.

Claims

1. A method for producing nicotinamide riboside, comprising a step of culturing lactic acid bacteria belonging to the genus Fructobacillus and preparing a culture containing nicotinamide riboside.

2. The method according to claim 1 , wherein the culture is carried out in a medium containing no fructose.

3. The method according to claim 1 or 2, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis RD011727 strain (deposit number NITE-P02764), Fructobacillus tropaeoily RD012353 strain (deposit number NITE P-02765), Fructobacillus tropaeoily RD012354 strain (deposit number NITE P-02766), and Fructobacillus fructosus NBRC3516 strain.

4. The method according to claim 1 or 2, wherein the lactic acid bacteria is Fructobacillus tropaeoi RD012353 strain (deposit number NITE P-02765) and / or Fructobacillus fructosus NBRC3516 strain.

5. A nicotinamide riboside enrichment agent comprising a culture containing nicotinamide riboside obtained from a lactic acid bacterium belonging to the genus Fructobacillus, a supernatant separated from the culture, and / or nicotinamide riboside isolated from the supernatant.

6. The fortifying agent according to claim 5, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoilu strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoilu strain RD012354 (deposit number NITE P-02766) and Fructobacillus fructosus strain NBRC3516.

7. The fortifying agent according to claim 5, wherein the lactic acid bacteria is Fructobacillus tropaeoi strain RD012353 (deposit number NITE P-02765) and / or Fructobacillus fructosus strain NBRC3516.

8. A food, drink, cosmetic or pharmaceutical comprising the nicotinamide riboside enhancer according to any one of claims 5 to 7.

9. A nicotinamide riboside-producing lactic acid bacterium selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoilu strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoilu strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516.

10. A method for producing nicotinamide mononucleotide and nicotinamide riboside, comprising a step of culturing lactic acid bacteria belonging to the genus Fructobacillus.

11. The method according to claim 10 , wherein the culture is carried out in a medium containing fructose.

12. The method according to claim 10 or 11, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis RD011727 strain (deposit number NITE-P02764), Fructobacillus tropaeoily RD012353 strain (deposit number NITE P-02765), Fructobacillus tropaeoily RD012354 strain (deposit number NITE P-02766), and Fructobacillus fructosus NBRC3516 strain.

13. The method according to claim 10 or 11, wherein the lactic acid bacteria is Fructobacillus tropaeoi RD012353 strain (deposit number NITE P-02765) and / or Fructobacillus fructosus NBRC3516 strain.

14. A nicotinamide mononucleotide and nicotinamide riboside enrichment agent comprising a culture containing nicotinamide mononucleotide and nicotinamide riboside obtained from lactic acid bacteria belonging to the genus Fructobacillus, bacterial cells separated from the culture, a disrupted product of the bacterial cells, and / or nicotinamide riboside and nicotinamide riboside isolated from the disrupted product.

15. The fortifying agent according to claim 14, wherein the lactic acid bacteria is selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoilu strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoilu strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516.

16. The fortifying agent according to claim 14, wherein the lactic acid bacteria is Fructobacillus durionis strain RD011727 (deposit number NITE-P02764) and / or Fructobacillus fructosus strain NBRC3516.

17. A food, drink, cosmetic, or pharmaceutical comprising the nicotinamide mononucleotide and nicotinamide riboside enhancer according to any one of claims 14 to 16.

18. A lactic acid bacterium capable of producing nicotinamide mononucleotide and nicotinamide riboside, selected from the group consisting of Fructobacillus durionis strain RD011727 (deposit number NITE-P02764), Fructobacillus tropaeoilu strain RD012353 (deposit number NITE P-02765), Fructobacillus tropaeoilu strain RD012354 (deposit number NITE P-02766), and Fructobacillus fructosus strain NBRC3516.

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