TW201742924A - [beta]-NMN-rich yeast extract - Google Patents

Abstract

To produce a [beta]-NMN-containing yeast extract from yeast having eating experience, and to produce a yeast-originated [beta]-NMN composition. It is found that a yeast extract containing [beta]-NMN in an amount of 2.0% (w/w) or more relative to a dried solid content can be produced by subjecting an extract from yeast to a reaction with a crude enzyme prepared from a microorganism belonging to the genus Rhizopus, e.g., Rhizopus oryzae. A yeast-originated [beta]-NMN-containing composition can be produced by purifying [beta]-NMN from a yeast extract.

Description

Yeast extract with high content of β-NMN

The present invention provides a method for producing a yeast extract having a high content of "β-nicotinamide mononucleotide (β-NM N)" as a food specification, by making Rhizopus oryzae The crude enzyme prepared by Rhiz opus is produced by the action of an extract obtained by culturing a high protein Candida utilis. Among them, "β-nicotinamide mononucleotide (β-NMN)" is a molecule (Sirtuin activator) that activates a longevity gene (Sirtuin).

Beta-nicotine indoleamine mononucleotide (β-NMN), a beta-nicotine indoleamine adenine that is a metabolite of the de novo pathw ay or the salvage pathway in the living organism. Intermediate metabolite of dinucleotide (NA D) (Patent Documents 1 to 4, Non-Patent Document 1). By administering β-NMN to a living body, biosynthesis of NAD can be directly induced, and the NAD concentration in the tissue is increased (Non-Patent Document 2). With respect to the protein encoded by the Sirt uin gene, the presence of a protein family of SIRT1, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7 has been confirmed in humans (Non-Patent Document 1). These Sirtuin families are NAD-dependent deacetylated enzymes which are activated by NAD as a matrix to exhibit a broad anti-aging effect (Non-Patent Document 1). As described above, "the improvement of abnormal glucose metabolism (Non-Patent Document 2)" and "participation in circadian rhythm (Non-Patent Documents 3 and 4)" have been described for the function of β-NMN which is a Sirtuin activator. "Improvement of the function of aging mitochondria (Non-Patent Document 5)", "protecting the heart from ischemia-reperfusion (Non-Patent Document 6)", and "reducing the reduction of neural stem cells caused by aging (Non-Patent Document 7)" "Inhibition of the presentation of Claudin-1 by epigenetic control mechanisms, reduction of proteinuria in diabetic nephropathy (Non-Patent Document 8)", "Control of planned cell death (Non-Patent Document 9)", "Parkinson" Report on the improvement of the disease (Non-Patent Document 10), "Oxidative stress caused by aging, or recovery of vascular dysfunction (Non-Patent Document 11)". As mentioned above, there are numerous negative life phenomena related to the aging of cells or tissues and organs. By administering β-NMN to improve the biosynthesis of NAD, the Sirtuin family centered on SIRT1 is activated, and it is expected to have a reply or The effect of prevention. With such a situation, it is expected that the aging of the individual will be delayed as a whole, and finally the life will be prolonged (longevity).

In addition, yeast is used in various foods, and Candida utilis is a food yeast that is recognized by the US Food and Drug Administration (FDA) for its high nutritional function and dietary experience. Therefore, it has been effectively used for pharmaceuticals or nutritional supplements, seasonings, and the like for many years. [Prior Technical Literature] [Patent Literature]

[Patent Document 1] International Publication No. WO2014/146044 [Patent Document 2] Chinese Patent Publication No. 101601679 B [Patent Document 3] US Patent Publication No. 2011-0123510 A1 [Patent Document 4] US Patent Registration No. 7737158 [Non-Patent Literature ]

[Non-Patent Document 1] Liana, R Stein. et al. The dynamic regulation of NAD metabolism in mitochondria. Trends in Endocrinology and Metabolism. 2012, Vol. 23, No. 9 [Non-Patent Document 2] J, Yoshino. et al Nicotinamide Mononucleotide, a Key NAD+ Intermediate, Treats the Pathophysiology of Diet-and Age-Induced Diabetes in Mice. Cell Metab. 2011, 14(4), P. 528-536. [Non-Patent Document 3] Clara Bien Peek1. et Al. Circadian Clock NAD+ Cycle Drives Mitochondrial Oxidative Metabolism in Mice. Science. 2013, 342(6158), 1243417. [Non-Patent Document 4] Ramsey, KM. et al. Circadian clock feedback cycle through NAM PT-mediated NAD+ biosynthesis. Science 2009, 324 (5927), P. 651-654. [Non-Patent Document 5] Ana, P. Gomes. et al. Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging. Cell. 2013, 155(7 ), P. 1624-1638. [Non-Patent Document 6] T, Yamamoto. et al. Nicotinamide mononucleotide, an intermed iate of NAD+ synthesis, protects the heart from ischemia and reperfusion. PLoS One. 2014, 9(6), e98972. [Non-Patent Document 7] Liana, R Stein. et al. Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging. EMBO J. 2014, 33(12 ), P. 1321-1340 [Non-Patent Document 8] K, Hasegawa. et al. Renal tubular Sirt1 attenuates diabetic albumi nuria by epigenetically suppressing Claudin-1 overexpression in podocytes. Nat Med. 2013, 19(11), P. 1496 -1504 [Non-Patent Document 9] Nicolas Preyat. et al. Complex role of nicotinamide adenine dinuc leotide in the regulation of programmed cell death pathways. Biochem Pharmacology. 2015, S0006-2952 (15) [Non-Patent Document 10] Lei lu. Et al. Nicotinamide mononucleotide improves energy acti vity and survival rate in an in vitro model of Parkinson's disease. Exp Ther Med. 2014, 8(3), P. 943-950. [Non-Patent Document 11] Natalie E.de Picciotto. Et al. Nicotinamide mononucleotide supp lementation reverses vascular dysfunction and oxidative stress with aging in mice. Aging cell. 2016, 15, P. 522-530.

[Problems to be Solved by the Invention] Currently, β-NMN is only sold for research purposes, and no food specifications are sold. Therefore, the subject of the present invention is to obtain a yeast extract containing β-NMN from a yeast having dietary experience, and to obtain a composition having a high β-NMN content derived from yeast. [Means for solving the problem]

The inventors of the present invention found that the yeast extract was extracted from yeast and the optimum conditions (temperature) were obtained by using an enzyme or a crude enzyme obtained from a microorganism belonging to the genus Rhizopus such as Rhizop us oryzae. The yeast extract was obtained by performing an enzyme reaction at 45 to 60 ° C and a pH of 4.5 to 6.0 to obtain a yeast extract having a high content of β-NMN, and completed the present invention.

Specifically, it is an invention as described below. (1) A yeast extract having a high content of β-nicotine guanamine mononucleotide, which contains 2.0% (w/w) or more of β-nicotine guanamine mononucleotide per unit of dry solid content. (2) A method for producing a yeast extract containing β-nicotine indoleamine mononucleotide as in (1), comprising the step of carrying out a reaction using an enzyme having the following physicochemical properties; (a) action: smoking Alkaline indoleamine adenine dinucleotide hydrolysis to nicotinamide mononucleotide; (b) optimum pH: pH 4.5~6.0; (c) optimum temperature: 45 ° C ~ 60 ° C; (d) source: belongs to A microorganism of the genus Rhizopus. (3) A method for producing a yeast extract containing β-nicotine decylamine mononucleotide according to (2), wherein the enzyme is a protein extracted from a microorganism belonging to the genus Rhizopus. [Effects of the Invention]

According to the present invention, the β-nicotinamide mononucleotide can be obtained simply from a yeast extract having dietary experience. In particular, the round yeast is a yeast having a dietary experience from the past, and the yeast extract obtained therefrom has high safety. Such a yeast extract having a high content of β-nicotine guanamine mononucleotide can be ingested as a medicine, a nutritional supplement, a functional food or the like.

In the present invention, edible yeast can be used in the case of yeast. For example, a yeast belonging to the genus Saccharo myces, Kluyveromyces, Candida, Pichia, etc., wherein Candida is preferred (Candida) high protein Candida utilis. More specifically, there are high protein Candida utilis IAM 4264, Candida utilis ATCC 9950, Candida utilis ATCC 9550, Candida utilis IAM 4233, Candida utilis AHU 3259, and the like. If a yeast having a high glutathione content is used, the content of the β-nicotine guanamine mononucleotide is increased, which is more desirable.

In the medium for cultivating yeast, glucose, acetic acid, ethanol, glycerin, molasses, sulfite pulp waste liquid, etc. are used as the carbon source, and urea, ammonia, ammonium sulfate, ammonium chloride, and the like are used for the nitrogen source. Nitrate, etc. Phosphate, potassium, and magnesium sources may also be used as usual industrial materials such as superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium chloride, and other inorganic salts such as zinc, copper, manganese, and iron ions may be added. . Other than this, it can be cultured without using vitamins, amino acids, nucleic acid-related substances, etc., but it may be added. Organic substances such as corn steep liquor, casein, yeast extract, meat extract, peptone may also be added.

The culture conditions such as the culture temperature or the pH are not particularly limited, and the culture may be carried out in accordance with the yeast strain used. In general, the culture temperature is 21 to 37 ° C, preferably 25 to 34 ° C, and the pH is 3.0 to 8.0, particularly preferably 3.5 to 7.0.

In the case of the culture form of the present invention, it may be either batch culture or continuous culture, and the latter is industrially preferable. Conditions such as stirring and aeration during the culture are not particularly limited, and may be a general method.

The cultured cells are subjected to pretreatment to prepare an extract. The wet yeast cells after the culture of the cells are washed by repeating suspension in distilled water and centrifuging, followed by extraction. The extraction method can be appropriately adjusted depending on the type of the yeast cell to be used, and in order to increase the content of β-NMN, it is desirable to make NAD (nicotinamide adenine dinucleotide) in yeast, The β-NMN is not subject to the conditions of decomposition. This is carried out by autolysis, alkali extraction, warm water extraction or a combination of these. The method using Candida utilis is resuspended in distilled water in a manner of 7 to 10%, preferably 8 to 9%, based on the dry weight of the bacterial concentration. When the bacterial suspension is extracted, the pH is adjusted according to the demand. It is most desirable to adjust the pH at the time of extraction to around 6.0. The adjustment of the pH can be a well-known method.

The extraction temperature is 50 to 90 ° C, preferably 50 to 65 ° C. The method of adjusting the temperature is not particularly limited as long as the extract is at the above temperature, and a known method can be used.

The extraction time is 5 minutes or more. In the extraction, stirring is desired. The stirring speed or the like can be appropriately adjusted without particular limitation. Further, if the extraction time is set to 40 to 50 minutes, it is more preferable because the content of β-NMN is increased.

After the extraction, the bacterial suspension was removed by centrifugation to obtain a supernatant. The supernatant is used as an extract to provide a substrate solution for the enzyme reaction of the present invention.

The enzyme used is an enzyme which produces β-NMN using NAD contained in the solution obtained in the preceding paragraph as a substrate. Specifically, an enzyme derived from a filamentous fungus belonging to the genus Rhizopus is used. Examples of the Rhizopus genus include Rhizopus oryzae, Risopus microspores, Rhizopus oligosporus, and the like, and an enzyme derived from Rhizopus having dietary experience can be used.

The enzyme used in the present invention can be obtained by using a crude enzyme prepared from a microorganism of the genus Rhizopus as described above. The microorganism of Rhizopus may be a strain used in the food industry or the like. Such strains are particularly excellent for use in the production of enzymes such as proteases (JP-A-2010-004760, etc.) for Rhizopus oryzae. Further, the filamentous fungus of the genus Rhizo pus may be a strain obtained from a strain distribution company such as ATCC or NBRC, or a commercially available strain strain company. The preparation of the crude enzyme used in the invention may be a general enzyme preparation method, for example, by bacterial culture, a fraction of a protein group containing an enzyme or the like is obtained by a crude purification step of chromatography. In the present invention, since a crude enzyme can be used, a fraction containing a protein group derived from a culture solution can be used, or the culture solution can be disrupted with Rhizopus oryzae, and a fraction containing a protein group in the cell can be used. . It can also be a dried product obtained in the drying step. Further, various enzymes derived from Rhizopus are commercially available, and since such commercially available enzymes mostly contain a mixed enzyme, an enzyme which can be used in the method of the present invention can be obtained.

The above enzymes use NAD as a matrix to produce NMN enzymes, and NND enzymes can be produced using not only NAD in yeast but also pure NAD as a matrix. NAD can use generally available.

Regarding the amount of the enzyme to be used in the reaction, although it is not the same according to the preparation method of the enzyme, 0.05% (w/v) to 0.25% (w/v) is usually added, and 0.1% (w/v) is preferably added. Among them, the β-NMN measurement method used in the study of the optimum reaction conditions for the enzyme in the present case is based on the measurement conditions of the LC-MS described in the examples.

The optimum temperature for the reaction of the crude enzyme is 45 to 60 ° C, preferably 50 ° C to 55 ° C, preferably 55 ° C. Among them, the method for detecting β-NMN used in the study of the optimum reaction conditions for the enzyme in the present case is based on the measurement conditions of the LC-MS described in the examples.

The optimum pH of the reaction of the crude enzyme is 4.5 to 6.0, preferably 5.0 to 5.5, and preferably pH 5.0. Among them, the detection method of β-NMN used in the study of the optimum reaction conditions for the enzyme in the present case is based on the measurement conditions of the LC-MS described in the examples.

By adding a crude enzyme from Rhizopus oryzae to an extract obtained by preparing the yeast cultured as described above, an enzyme reaction is carried out under optimum reaction conditions to obtain a solid component relative to the yeast extract. A yeast extract containing 2.0% (w/w) or more of β-NMN. The β-NMN of the present invention causes the content of β-NMN to be different due to the NAD content in the yeast. The content of β-NMN can be further increased by increasing the NAD content contained in the substrate solution of the enzyme reaction extracted from the enzyme cells. Among them, the method for detecting β-NMN used in the study of the optimum reaction conditions for the enzyme in the present case is based on the measurement conditions of the LC-MS described in the examples.

The extract of the enzyme reaction is concentrated, and after freeze-drying or hot air drying, a dried product of the yeast extract containing β-NMN can be obtained. Further, by purifying β-NMN from a yeast extract containing β-NMN, a composition having a higher β-NMN content from yeast can be obtained. Further, by purifying β-NMN from the yeast extract before drying in the preceding stage, a composition derived from yeast having a high β-NMN content can also be obtained. As the purification method, a general purification method such as an ion exchange resin can be used.

The method for ingesting the yeast extract of the present invention or the composition containing β-NMN derived from yeast is not particularly limited, and can be administered by parenteral administration such as oral administration, intravenous, intraperitoneal or subcutaneous injection. Specifically, it may be an oral preparation such as a tablet, a powder, a granule, a pill, a suspension, an emulsion, an infusion, a decoction, a capsule, a syrup, a liquid, an elixir, an extract, an elixir, or a fluid extract. Or any of the parenteral agents such as injections, drips, creams, suppositories, and the like.

The yeast extract is not only used as a medicine, but also as a food, and can be ingested as a functional food, a nutritional supplement, a nutritional supplement, or the like.

Further, the present invention may also be used in combination with other compositions which do not degrade the Sirtuin activity of β-NMN or increase the Sirtuin activity of β-NMN. Examples thereof include maltitol, sorbitol, starch, and the like which are excipients and diluents.

The uptake amount of the present invention may be administered in an amount exhibiting the Sirtuin activity of β-NMN. Generally, the amount of administration required for the activity of β-NMN is determined according to the selection of the composition to be administered, the age, weight and reaction of the ingestor, the state of the ingestor, and the like. [Examples]

The present invention is specifically shown below, but the present invention is not limited to these examples.

(Measurement conditions of β-NMN used in the study of optimum enzyme reaction conditions) Mass spectrometry (MS) measurement condition analysis instrument by LC-MS: amaZon speed (Bruker daltonics) Ionization method: electrospray free Electrospray ionization (ESI) Separation section: Ion trap detection section: Positive mode (MRM mode) β-NMN→For precursor ion m/z335, trace fragment ion m/z123 NAD→ for precursor ion m/z664, trace debris Ion m/z 524, 542 Capillary voltage 4.5 kV Nebulizer: 30.0 psi Dry gas: 10.0 L/min Drying temperature: 250 ° C High performance liquid chromatography (HPLC) Determination of conditional pump: LC-20AD (Shimadzu Corporation) Degassing machine: DGU-20A3 (Shimadzu Corporation) Autosampler: SIL-20AC HT (Shimadzu Corporation) Diode Array Detector: SPD-M20A (Shimadzu Corporation) Column oven: CTO-20AC ( Shimadzu Corporation) Mobile phase A: Formic acid (Wako Pure Chemical Industries, Ltd.) for LC-MS was added to ultra-pure water (Wako Pure Chemical Co., Ltd.) for LC-MS to make it 0.1% (v/v) (pH 2.5) ). Mobile phase B: 0.1% LC-MS formic acid acetonitrile (Wako Pure Chemical Industries Co., Ltd.) Column: Inertsil ODS-3 (particle size 3um, length 150mm, inner diameter 2.1mm) (GL science) Column oven temperature: 45°C Flow rate: 0.2 mL/min Sample injection volume: 5 uL Sample cooler temperature: 4 ° C Dissolution method: Linear gradient gradient conditions: 0 min (0% mobile phase B) -20 min (100% mobile phase B) - 25 min (100% mobile phase B)- 25.1min (0% mobile phase B)-40min (0% mobile phase B) Material analysis 20min, column cleaning 5min, column equilibration 15min total 40min analysis and analysis of the sample preparation method: the reaction solution is The mobile phase was diluted 100-fold with 0.1% (v/v) formic acid. Thereafter, the diluted solution was filtered through a DISMIC 13CP020AS 0.22 um filter (ADVAN TEC) connected to a syringe to supply an insoluble matter, and supplied to the LC-MS.

(Quantitative analysis conditions of β-NMN and NAD in the dried product obtained by optimum conditions) Determination of pump and degasser by HPLC: Chromaster 5110 (Hitachi High-Tech Science Corporation.) Autosampler: Chromaster 5210 (Hitachi High-Tech Science Corporation.) UV-VIS detector: Chromaster 5420 (Hitachi High-Tech Science Corporation.) Column oven: Chromaster 5310 (Hitachi High-Tech Science Corporation.) Mobile phase: 75 mM ammonium dihydrogen phosphate (pH 2.3) (Wako Pure Chemical Company). Degassing was carried out for 60 minutes with an air blower. Column: According to Wakosil-II 5C18 RS (particle size 5um, length 30mm, inner diameter 4.6mm) (Wako Pure Chemical Company) → Wakosil-II 5C18 RS (particle size 5um, length 150mm, inner diameter 4.6mm) (and pure light Pharmaceutical company) → Wakosil-II 5C18 RS (particle size 5um, length 250mm, inner diameter 4.6mm) (Wako Pure Chemicals Co., Ltd.) The three columns are connected in series. Column oven temperature: 26 ° C Flow rate: 1.0 mL / min (0.0 min) → 1.0 mL / min (7.0 min) → 0.2 mL / min (8.0 min) → 0.2 mL / min (20.0 min) → 1.5 mL / min ( 21.0min)→1.5mL/min(55.0min)→1.0mL/min(56.0min)→1.0mL/min(60.0min) Dissolution method: isocratic Detection wavelength: abs 260nm Analysis time: 60min Sample injection amount : 5 uL sample cooler temperature: 2 ° C Analytical sample preparation method: The yeast extract dried product obtained by the present invention was dissolved in a mobile phase of 75 mM ammonium dihydrogen phosphate (pH 2.3), and adjusted to a final concentration of 1 %(w/w). Thereafter, the insoluble matter was filtered by a DISMIC 13CP020AS 0.22 um filter (ADVANTEC) connected to a syringe, and supplied to HPLC. Standard materials used for quantitative analysis: β-NMN (Sigma-Aldrich), NAD (Sigma-Aldr ich), from the calibration curve shown in Fig. 8, the dried yeast extract obtained by the present invention was calculated. The content of β-NMN.

(Cultivation of yeast) Candida utilis IAM 4264 was preliminarily cultured in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%). It was inoculated with 1 to 2% in 18 L medium of a 30 L volume fermentation tank. The medium composition used was 4% of glucose, 0.3% of monoammonium phosphate, 0.161% of ammonium sulfate, 0.137% of potassium chloride, 0.08% of magnesium sulfate, 1.6 ppm of copper sulfate, 14 ppm of iron sulfate, 16 ppm of manganese sulfate, and 14 ppm of zinc sulfate. The culture conditions were carried out at pH 4.0, a culture temperature of 30 ° C, aeration amount of 1 vvm, and stirring at 600 rpm, and ammonia was added to control the pH. After 16 hours of bacterial culture, the culture solution was collected, and the cells were collected by centrifugation to obtain 180 g of the moist yeast cells. The washing was carried out by repeatedly suspending the obtained yeast cells in distilled water and centrifuging. The solid content was resuspended in distilled water so that the solid content concentration became 82.88 g/L. At this time, it was pH 5.8.

<Example 1> (Extraction of yeast extract) The suspension of the above-mentioned cell suspension was slowly stirred at 90 ° C in a water bath at 95 ° C to raise the temperature to 90 ° C, and subjected to extraction treatment for 10 minutes while stirring. . After the extraction treatment, 25 mL of the bacterial suspension obtained by sampling was cooled in ice, and centrifuged at 10,000 rpm for 10 minutes at 4 ° C to obtain a supernatant. The precipitate was added and suspended in an amount of ultrapure water equivalent to the supernatant, and centrifuged again to obtain a supernatant. The supernatant obtained by the initial centrifugation and the supernatant obtained by the second centrifugation were collected and supplemented to 50 mL with ultrapure water, which was an extract.

<Example 2> (Mass Spectrometry of β-NMN and NAD Reference Material) The mass spectrum of β-NMN and NAD was obtained by the above measurement conditions. As shown in Fig. 2, β-NMN is a fragment ion of m/z 123 of nicotinic acid amide (Nam) when MS/MS is carried out using m/z 334 of β-NMN as a precursor ion. When the precursor ion was m/z 335 and m/z 123 was present among the fragment ions, when LC-MS/MS was carried out under the above conditions, as shown in Fig. 2, β-NMN was detected at 3.0 min. As shown in Figure 3, if MS/MS is used as the precursor ion of m/z664 of NAD, m/z524 and m/z542, adenosine diphosphate from adenosine diphosphate ribose (ADP-ribose) will be detected. (aDP) m/z 428, fragment ion of m/z 232 of ribose-5-phosphate (R5P). The precursor ion m/z 664 was a substance having m/z 524 and 542 among the fragment ions, and LC-MS/MS was carried out under the above conditions. As shown in Fig. 4, NAD was detected at 8.5 min.

<Example 3> (Exploration of optimum enzyme reaction temperature) The yeast was cultured in the same manner as in Example 1 and subjected to extraction treatment, and the temperature of the yeast extract was adjusted to 30 ° C, 35 ° C, 40 ° C, and 45 ° C, respectively. 50 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 58 ° C, 60 ° C, 62 ° C, 65 ° C, 70 ° C, the reaction pH was adjusted to 6.5 with 9 N HCl or 9 N NaOH, added from After 0.1% (w/v) of the crude enzyme prepared by Rhizopus oryzae, the enzyme reaction was carried out for 1 hour. According to the measurement using LC-MS, the production rate of β-NMN at each temperature is shown in Fig. 5. The reaction temperature is 45 to 60 ° C, especially showing the highest β-NMN production rate around 55 ° C. The β-NMN production rate is a relative value when the ionic strength of β-NMN in the extract in which the enzyme is not reacted is set to 1. The ionic strength was measured by LC-MS.

<Example 4> (Exploration of optimum enzyme reaction pH) The yeast was cultured in the same manner as in Example 1 and subjected to extraction treatment to adjust the temperature of the yeast extract to 54 ° C, and the reaction pH was 9 N HCl or 9 N NaOH was adjusted to pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, and crude was prepared by adding Rhizopus oryzae. After the enzyme was 0.1% (w/v), the enzyme reaction was carried out for 1 hour. The production rate of β-NMN at each pH was as shown in Fig. 6 according to the measurement using LC-MS. The reaction pH is in the vicinity of 4.5 to 6.0, and the highest β-NMN production rate is exhibited particularly near pH 5.0. The β-NMN production rate is a relative value when the ionic strength of β-NMN in the extract in which the enzyme is not reacted is set to 1. The ionic strength was measured by LC-MS.

<Example 5> (Exploration of metal ion demand of enzyme) The yeast was cultured in the same manner as in Example 1 and subjected to extraction treatment, and the temperature of the yeast extract was adjusted to 55 ° C, and the reaction pH was 9 N HCl or 9 N NaOH. Adjusted to pH 5.0, and added manganese chloride (MnCl ₂ · 4H ₂ O), zinc chloride (ZnCl ₂ ), copper chloride (CuSO ₄ · 5H ₂ O), respectively, at a final concentration of 100 mM. Magnesium chloride (MgCl ₂ · 6H ₂ O), calcium chloride (CaCl ₂ · 2H ₂ O), ferric chloride (FeCl ₃ · 6H ₂ O), ethylenediaminetetraacetic acid (EDTA, 2Na). Further, after adding 0.1% (w/v) of the crude enzyme prepared from Rhizopus oryzae, the enzyme reaction was carried out for 1 hour. According to the measurement using LC-MS, the production rate of β-NMN in the presence of each metal ion is as shown in FIG. The presence of Zn ions, Cu ions, and Fe ions in the reaction solution is shown to hinder the production of β-NMN. Further, since the activity of β-NMN is also exhibited in the presence of EDT A as a chelating agent in the reaction liquid, it is not necessary to add a metal compound in the present enzyme reaction. The production rate of β-NMN is a relative value when the ionic strength of β-NMN in the liquid obtained by reacting the enzyme to which no metal compound is added is set to 100. The ionic strength was measured by LC-MS.

<Example 6> (Study of optimum enzyme addition amount and enzyme reaction time) The yeast was cultured in the same manner as in Example 1 and subjected to extraction treatment, and the temperature of the yeast extract was adjusted to 55 ° C, and the reaction pH was 9 N HCl or 9 N NaOH was adjusted to pH 5.0, and the amount of crude enzyme prepared from Rhizopus oryzae was 0.010% (w/v), 0.025% (w/v), and 0.050% (w/v). ), 0.10% (w/v), 0.25% (w/v), 0.50% (w/v), 0.75% (w/v), 1.0% (w/v), and the enzyme reaction was carried out for 5 hours after the addition. . During the reaction, samples were taken every 1 hour at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours to examine the change in the production of each β-NMN over time. According to the measurement using LC-MS, the production rate of β-NMN in the yeast extract of each addition amount and reaction time is shown in FIG. When the amount of the crude enzyme added is 0.25% (w/v) or more, the β-NMN which is produced is decomposed in about 30 minutes to 1 hour. The addition of 0.010% (w/v) and 0.025% (w/v) exhibited slow and linear production of β-NMN. The highest rate of β-NMN production was the addition of 0.10% (w/v) of the enzyme reaction for 3 hours. The β-NMN production rate is a relative value when the ionic strength of β-NMN in the unreacted extract of the enzyme is set to 1. The ionic strength was measured by LC-MS.

<Example 7> (Measurement of β-NMN content under optimum enzyme reaction conditions) Using yeast Candida utilis IAM 4264, yeast was cultured in the same manner as in Example 1 to carry out extraction treatment, and yeast extraction was carried out. The temperature of the liquid was adjusted to 55 ° C, the pH of the reaction was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and 0.1% (w/v) of the crude enzyme prepared from Rhizopus oryzae was added, and 3 was carried out. The best response for the hour. Thereafter, a dried product of the yeast extract containing β-NMN is obtained by a drying step. The dried product was quantitatively analyzed under the above measurement conditions. The chromatogram is shown in Figure 10. In the optimum enzyme reaction conditions, the content of β-NMN in the yeast extract was 2.16% (w/w) per unit dry solid content as shown in Fig. 11 when the β-NMN was quantified using the calibration line of Fig. 9. Before the reaction, the solid content per unit contained 2.29% (w/w) of NAD, and the NAD per unit dry solid content after the reaction was reduced by 0.18% (w/w), and β-NMN was produced. Considering this situation, it is predicted that the production of β-NMN by the present enzyme reaction is a mechanism as shown in FIG.

<Example 8> The production of β-NMN was confirmed using a commercially available enzyme of Rhizopus. A yeast extract was prepared in the same manner as in Example 1, and a commercially available enzyme "Lilipase A-10D" (manufactured by Nagase ChemteX Corp.) (0.1% (w/v) was added, and the optimum reaction was carried out for 3 hours. The reaction conditions were carried out at pH 5.0 and at a temperature of 55 °C. As a result, a yeast extract containing β-NMN 2.01% (w/w) was obtained. [Industrial use]

β-NMN can be obtained from yeast which is safe for consumption, and can be ingested not only as a pharmaceutical but also as a functional food or a nutritional supplement. By ingesting the present invention, the functionality of β-NMN can be obtained.

no.

Fig. 1 shows the molecular structure, composition formula and molecular weight of β-NMN and NAD. Fig. 2 is a chromatogram showing the MS/MS mass spectrum of the β-NMN reference material and the retention time measured by LC-MS/MS. [Fig. 3] MS/MS mass spectrum of NAD reference material. Fig. 4 is a chromatogram showing the retention time measured by LC-MS/MS. Fig. 5 shows the results of Example 3. A diagram showing the optimum reaction temperature for enzyme reactions. Fig. 6 shows the results of Example 4. A diagram showing the optimum pH for the reaction of the enzyme. Fig. 7 shows the results of Example 5. A diagram showing the metal demand for crude enzymes in the reaction of enzymes. Fig. 8 shows the results of Example 6. To show the optimal reaction time and the optimum amount of enzyme added in the enzyme reaction. [Fig. 9] A calibration curve showing β-NMN and NAD for quantifying an extract obtained by preparing from Candida utilis IAM 4264 and a crude extract from Rhizopus oryzae. The reaction of the enzyme, the β-NMN and NAD in the yeast extract obtained. Fig. 10 shows the results of Example 7. To demonstrate the reaction of the extract obtained from the preparation of Candida utilis IAM 4264 with the crude enzyme from Rhizopus oryzae, the β-NMN and NAD in the obtained yeast extract. Chromatogram. Fig. 11 shows the results of Example 7. To demonstrate the reaction of the extract obtained from the preparation of Candida utilis IAM 4264 with the crude enzyme from Rhizopus oryzae, the β-NMN and NAD in the obtained yeast extract. The map of the content. Fig. 12 is a view showing the present reaction mechanism for producing β-NMN by adding a crude enzyme derived from Rhizopus oryzae to an extract prepared from Candida utilis IAM 4264.

no

Claims (6)

A method for producing a yeast extract containing β-nicotine indoleamine mononucleotide, comprising the step of carrying out a reaction using an enzyme having the following physicochemical properties; (1) Action: Nicotine indoleamine adenine dinucleoside Acid hydrolysis to nicotinamide mononucleotide; (2) Optimum pH: pH 4.5~6.0; (3) Optimum temperature: 45 ° C ~ 60 ° C; (4) Source: microorganism belonging to Rhizopus . A method for producing a yeast extract containing β-nicotine indoleamine mononucleotide according to the first aspect of the invention, wherein the yeast extract containing β-nicotine indoleamine mononucleotide per unit dry solid component Contains 2.0% (w/w) or more of β-nicotine indoleamine mononucleotide. A method for producing a yeast extract containing β-nicotine decylamine mononucleotide according to the first aspect of the invention, wherein the enzyme is a protein extracted from a microorganism belonging to the genus Rhizopus. A composition comprising: a β-nicotine amide monoamine obtained by a method for producing a yeast extract containing β-nicotine guanamine mononucleotide according to any one of claims 1 to 3; Yeast extract of nucleotides. An enzyme having the following physicochemical properties: (1) Action: hydrolyzing nicotine indoleamine adenine dinucleotide to nicotinamide mononucleotide; (2) optimum pH: pH 4.5 to 6.0; 3) Optimum temperature: 45 ° C ~ 60 ° C; (4) Source: microorganisms belonging to the genus Rhizopus. A yeast extract having a high content of β-nicotine guanamine mononucleotide containing 2.0% (w/w) or more of β-nicotine guanamine mononucleotide per unit of dry solid content.