WO2017200050A1 - Β-nmn-rich yeast extract - Google Patents
Β-nmn-rich yeast extract Download PDFInfo
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- WO2017200050A1 WO2017200050A1 PCT/JP2017/018709 JP2017018709W WO2017200050A1 WO 2017200050 A1 WO2017200050 A1 WO 2017200050A1 JP 2017018709 W JP2017018709 W JP 2017018709W WO 2017200050 A1 WO2017200050 A1 WO 2017200050A1
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- yeast extract
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- HPMMZWHQVQTFCS-UHFFFAOYSA-N C[O](CC1(CCOP(O)(O)=O)O)C([N]2(C3)C3=CC(C(N)=O)=CC=C2)=C1O Chemical compound C[O](CC1(CCOP(O)(O)=O)O)C([N]2(C3)C3=CC(C(N)=O)=CC=C2)=C1O HPMMZWHQVQTFCS-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
- A23L31/10—Yeasts or derivatives thereof
- A23L31/15—Extracts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
Definitions
- the present invention relates to a molecule that activates a food standard longevity gene (sirtuin), which comprises a step of allowing a crude enzyme prepared from Rhizopus genus such as Rhizopus oryzae to act on an extract obtained by culturing Candida utilis (provided is a method for producing a yeast extract containing a high content of “ ⁇ -Nicotinamide mononucleotide ( ⁇ -NMN)”, a sirtuin activator).
- ⁇ -N ⁇ -Nicotinamide mononucleotide
- ⁇ -Nicotinamide mononucleotide is an intermediate metabolite of ⁇ -Nicotinamide adenine dinucleotide (NAD), which is a metabolite of the de novo pathway and the Salvage pathway in vivo (Patent Documents 1 to 4, Non-Patent Document 1) ).
- NAD ⁇ -Nicotinamide adenine dinucleotide
- ⁇ -NMN can directly induce biosynthesis of NAD and improve the NAD concentration in tissues when administered to a living body (Non-patent Document 2).
- Non-patent Document 2 the presence of SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7 protein families has been confirmed in humans as proteins encoded by sirtuin genes (Non-patent Document 1).
- Non-patent Document 1 functions related to ⁇ -NMN, which is a sirtuin activator, include “improvement of abnormal sugar metabolism (Non-patent Document 2)”, “participation in Circadian rhythm (Non-patent Document 3) 4” ) '', ⁇ Functional improvement of aging mitochondria (Non-patent document 5) '', ⁇ Protection of heart from ischemia-reperfusion (Non-patent document 6) '', ⁇ Inhibition of neural stem cell decrease due to aging (Non-patent document 7) '' ”,“ Epigenetic control mechanism suppresses claudin-1 expression and diminishes albuminuria in diabetic nephropathy (Non-patent document 8) ”,“ Control of programmed cell death (Non-patent document 9)
- Torula yeast (Candida utilis) is an edible yeast that has been evaluated for higher nutritional functionality and safety from eating experience than the US Food and Drug Administration (FAD). For this reason, it has been used effectively for many years in medicines, supplements and seasonings.
- ⁇ -NMN is sold only for research purposes, and no food-standard products are sold. Therefore, it is an object to obtain a yeast extract containing ⁇ -NMN from yeast with experience of eating, and to obtain a composition with a high content of ⁇ -NMN derived from yeast.
- the present inventor extracted yeast extract from yeast and optimized (temperature 45 to 60 ° C., pH 4.5 to 6.0) with an enzyme or a crude enzyme obtained from a microorganism belonging to the genus Rhizopus such as Rhizopus oryzae. It has been found that a yeast extract enriched with ⁇ -NMN can be obtained by carrying out an enzymatic reaction, and the present invention has been completed.
- the invention is as follows.
- B Optimum pH: pH 4.5 to 6.0.
- Origin A microorganism belonging to the genus Rhizopus.
- ⁇ -Nicotinamide mononucleotide can be easily obtained from yeast with experience in food.
- Torula yeast is a yeast that has been eating for a long time, and the yeast extract obtained from this is highly safe.
- Such yeast extract containing a high content of ⁇ -Nicotinamide mononucleotide can be ingested as pharmaceuticals, supplements, functional foods and the like.
- the calibration curve used for the determination of ⁇ -NMN and NAD in the yeast extract obtained by the reaction of the extract prepared from Candida utilis IAM 4264 and the crude enzyme derived from Rhizopus oryzae is shown.
- Results of Example 7. It is a chromatogram showing ⁇ -NMN and NAD in a yeast extract obtained by the reaction of an extract prepared from Candida utilis IAM 4264 and a crude enzyme derived from Rhizopus oryzae.
- Results of Example 7. 3 is a graph showing ⁇ -NMN and NAD contents in a yeast extract obtained by the reaction of an extract prepared from Candida utilis IAM 4264 ⁇ and a crude enzyme derived from Rhizopus oryzae. This reaction mechanism of ⁇ -NMN production by adding an extract prepared from Candida utilis IAM 4264 and a crude enzyme derived from Rhizopus oryzae is shown.
- edible yeast can be used as yeast.
- yeast belonging to the genus Saccharomyces, Kluyveromyces genus, Candida genus, Pichia genus and the like can be mentioned, among which Candida genus Candida utilis is preferable.
- Candidaandutilis IAM 4264, Candida utilis ATCC 9950, Candida utilis ATCC 9550, Candida utilis IAM 4233, Candida utilis AHU 3259 and the like More preferably, the use of yeast with a high glutathione content increases the ⁇ -NMN content.
- yeast having a high glutathione content yeasts obtained by known methods can be used (Japanese Patent Laid-Open Nos. 59-151894, 60-156379, etc.).
- glucose, acetic acid, ethanol, glycerol, molasses, sulfite pulp waste liquid, etc. are used as carbon sources, and nitrogen, urea, ammonia, ammonium sulfate, ammonium chloride, Nitrate is used.
- Phosphoric acid, potassium, and magnesium sources may be ordinary industrial raw materials such as lime perphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, and other zinc, copper, manganese, iron ions, etc.
- Add inorganic salt Others can be cultured without using vitamins, amino acids, nucleic acid-related substances, etc., but these may be added.
- Organic substances such as cone-briker, casein, yeast extract, meat extract and peptone may be added.
- Culture conditions such as culture temperature and pH can be applied without particular limitation, and may be set according to the yeast strain used and cultured.
- the culture temperature is 21 to 37 ° C., preferably 25 to 34 ° C.
- the pH is 3.0 to 8.0, particularly 3.5 to 7.0.
- the culture format of the present invention may be either batch culture or continuous culture, but the latter is desirable industrially. Conditions such as agitation and aeration during culture are not particularly limited and may be a general method.
- an extract is prepared by pretreatment. Extraction is performed after washing the wet yeast cells after culturing the cells by suspending them in distilled water and repeating centrifugation.
- the extraction method may be appropriately adjusted according to the type of yeast used, but in order to increase the content of ⁇ -NMN, NAD (nicotinamide adenine dinucleotide nucleotide), ⁇ - It is desirable to carry out under the condition that NMN is not decomposed.
- the self-digestion method, the alkali extraction method, the hot water extraction method, or a combination thereof is used.
- the suspension is resuspended in distilled water so that the bacterial cell concentration is 7 to 10%, preferably 8 to 9%, in terms of dry weight.
- pH adjustment is performed as necessary. Most preferably, the pH during extraction is adjusted to around 6.0. The pH may be adjusted by a known method.
- Extraction temperature is 50 to 90 ° C, preferably 50 to 65 ° C.
- a known method can be used without particular limitation as long as the extract reaches the above temperature.
- Extraction time may be 5 minutes or more. It is desirable to stir during extraction. The stirring speed and the like may be adjusted as appropriate and are not particularly limited. Further, when the extraction time is 40 to 50 minutes, the content of ⁇ -NMN increases, which is more preferable.
- the cell suspension After extraction, the cell suspension is removed by centrifugation to obtain a supernatant. This supernatant was used as an extract and a substrate solution for the enzyme reaction according to the present invention.
- the enzyme used is an enzyme that produces ⁇ -NMN using NAD contained in the solution obtained up to the previous stage as a substrate.
- an enzyme derived from a filamentous fungus belonging to the genus Rhizopus is used.
- the Rhizopus genus include Rhizopus oryzae, Risopus microsporus, Rhizopus oligosporus, etc., and an enzyme derived from the genus zoRhizopus genus with experience in eating can be used.
- a crude enzyme prepared from a microorganism belonging to the genus Rhizopus as described above can be used.
- the microorganism belonging to the genus Rhizopus may be a strain used in the food industry.
- Rhizopus genus fungi such as Rhizopus oryzae are particularly preferred because they are used in the production of enzyme production such as protease (JP 2010-004760 A, etc.).
- the filamentous fungus belonging to the genus Rhizopus may be a strain obtained from strain distribution agencies such as ATCC, NBRC, or a commercially available seed strain sales company.
- a crude enzyme used in the present invention can be prepared by a general enzyme preparation method.
- a fraction containing a protein group such as an enzyme is obtained through a cell culture and a crude purification step by chromatography. Since a crude enzyme can be used in the present application, a fraction containing a protein group from a culture solution, or a fraction containing an intracellular protein group by crushing the culture solution and Rhizopus oryzae may be used. It is good also as a dried material by obtaining a drying process. Furthermore, various enzymes derived from the genus Rhizopus are commercially available, and since many of these commercially available enzymes contain contaminating enzymes, enzymes that can be used in the method of the present application are also available.
- the above enzymes are enzymes that produce NMN using NAD as a substrate, and can be used not only for NAD in yeast, but also for enzymes that produce NMN using NAD pure products as substrates. NAD can use what is generally available.
- the amount of the enzyme used for the reaction varies depending on the method for preparing the enzyme, but is usually 0.05% (w / v) to 0.25% (w / v), preferably 0.1% (w / V) Add.
- the ⁇ -NMN measurement method used for studying the optimal reaction conditions for the enzyme depends on the LC-MS measurement conditions described in the Examples.
- the optimum temperature for the reaction of the crude enzyme is 45 to 60 ° C, preferably 50 to 55 ° C, and most preferably 55 ° C.
- the detection method of ⁇ -NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
- the optimum pH for the reaction of the crude enzyme is 4.5 to 6.0, preferably 5.0 to 5.5, most preferably pH 5.0.
- the detection method of ⁇ -NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
- a yeast extract containing the above ⁇ -NMN can be obtained.
- the ⁇ -NMN of the present invention differs in the content of ⁇ -NMN produced by the NAD content in yeast. Increasing the NAD content contained in the substrate solution of the enzyme reaction extracted from the yeast cells can further increase ⁇ -NMN.
- the detection method of ⁇ -NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
- the extract subjected to the enzyme reaction can be concentrated and then freeze-dried or hot-air dried to obtain a dried product of ⁇ -NMN-containing yeast extract. Further, by purifying ⁇ -NMN from the ⁇ -NMN-containing yeast extract, it is possible to obtain a composition further enriched in yeast-derived ⁇ -NMN. In addition, a composition enriched in yeast-derived ⁇ -NMN can also be obtained by purifying ⁇ -NMN from the yeast extract before drying in the previous stage. As a purification method, a general purification method using an ion exchange resin or the like can be used.
- the method of ingesting the yeast extract or yeast-derived ⁇ -NMN-containing composition of the present invention is not particularly limited, and examples include oral administration, parenteral administration such as intravenous, intraperitoneal or subcutaneous administration.
- parenteral administration such as intravenous, intraperitoneal or subcutaneous administration.
- oral preparations such as tablets, powders, granules, pills, suspensions, emulsions, soaking and decoction, capsules, syrups, solutions, elixirs, extracts, tinctures, fluid extracts, Or any of parenterals such as injections, drops, creams, suppositories, etc. may be used.
- the yeast yeast extract can be ingested not only as a medicine but also as a food, and can also be ingested as a functional food, a nutritional supplement, a supplement and the like.
- the present invention can also be used in combination with other compositions that do not decrease the sirtuin activity of ⁇ -NMN or enhance the sirtuin activity of ⁇ -NMN.
- excipients for example, excipients, diluents, dextrins, malt tols, sorbitol, starch and the like.
- the amount of intake according to the present invention may be administered in such an amount that the sirtuin activity of ⁇ -NMN is expressed.
- the dose required for ⁇ -NMN activity is determined by the condition of the consumer, the choice of composition administered, the age, weight and response of the consumer, the condition of the consumer, etc. Is done.
- Wakosil-II 5C18 RS particle size 5 um, length 30 mm, inner diameter 4.6 mm
- Wakosil-II 5C18 RS particle diameter 5 um, length 150 mm, inner diameter 4.6 mm
- Wakosil-II 5C18 RS particle diameter 5 um, length 250 mm, inner diameter 4.6 mm
- Candida utilis IAM 4264 is pre-cultured in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in advance, and this is 1-2% in 18 L medium in a 30 L fermentor. Inoculated. Medium composition is 4% glucose, 0.3% monoammonium phosphate, 0.161% ammonium sulfate, 0.137% potassium chloride, 0.08% magnesium sulfate, 1.6 ppm copper sulfate, 14 ppm iron sulfate, Manganese sulfate 16 ppm and zinc sulfate 14 ppm were used.
- the culture conditions were pH 4.0, culture temperature 30 ° C., aeration rate 1 vvm, stirring 600 rpm, and ammonia was added to control the pH. After culturing the cells for 16 hours, the culture solution was collected and collected by centrifugation to obtain 180 g of wet yeast cells. The obtained yeast cells were washed by suspending them in distilled water and repeating the centrifugation. Resuspended in distilled water to a dry solids concentration of 82.88 g / L. At this time, the pH was 5.8.
- Example 1 (Yeast extract extraction) The cell suspension is heated to 90 ° C. while gently stirring the suspension in a water bath at 95 ° C. and subjected to extraction for 10 minutes while stirring. After the extraction treatment, 25 mL of the sampled cell suspension was cooled under ice and centrifuged at 10,000 rpm for 10 minutes at 4 ° C. to obtain a supernatant. The same amount of ultrapure water as the supernatant was added to the precipitate, suspended, and centrifuged again to obtain the supernatant. The supernatant obtained by the first centrifugation and the supernatant obtained by the second centrifugation were pooled, and the extract was filled up to 50 mL with ultrapure water.
- Example 2 Mass spectrum of ⁇ -NMN and NAD standard
- mass spectra of ⁇ -NMN and NAD were obtained.
- FIG. 2 when ⁇ / NMN was subjected to MS / MS using ⁇ / NMN m / z 334 as a precursor ion, a fragment ion of Nicotinamide (Nam) m / z 123 was detected.
- this precursor ion m / z 335 and fragment ion m / z 123 were subjected to LC-MS / MS under the above-mentioned conditions, ⁇ -NMN was detected at 3.0 min as shown in FIG. As shown in FIG.
- NAD is obtained by performing MS / MS using NAD m / z 664 as a precursor ion, m / z 524 and m / z 542 derived from Adenosine diphosphate ribose (ADP-ridose), Adenosine Diphosphate (ADP) m / z 428 and Ribose 5-phosphate (R5P) m / z 232 fragment ions were detected.
- ADP-ridose Adenosine diphosphate ribose
- ADP Adenosine Diphosphate
- R5P Ribose 5-phosphate
- Example 3 (Examination of optimal enzyme reaction temperature) Yeast was cultured and extracted in the same manner as in Example 1, and the temperature of the yeast extract was 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, respectively. , 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, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae ) After the addition amount, the enzyme reaction was carried out for 1 hour.
- the production rate of ⁇ -NMN at each temperature as measured by LC-MS was as shown in FIG.
- the highest ⁇ -NMN production rate was exhibited when the reaction temperature was 45-60 ° C, particularly around 55 ° C.
- the ⁇ -NMN production rate indicates a relative value when the ionic strength of ⁇ -NMN in the extract not reacted with the enzyme is 1.
- the ionic strength was measured by LC-MS.
- Example 4 (Examination of optimum enzyme reaction pH) Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was 54 ° C., the reaction pH was 9 N HCl or 9 N NaOH, pH 4.0, pH 4.5, pH 5.0, pH 5.5, The pH was adjusted to 6.0, pH 6.5, pH 7.0, and pH 7.5, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae was added, and the enzyme reaction was performed for 1 hour. The production rate of ⁇ -NMN at each pH as measured by LC-MS was as shown in FIG.
- the highest ⁇ -NMN production rate was exhibited when the reaction pH was in the range of 4.5 to 6.0, particularly around pH 5.0.
- the ⁇ -NMN production rate indicates a relative value when the ionic strength of ⁇ -NMN in the extract not reacted with the enzyme is 1.
- the ionic strength was measured by LC-MS.
- Example 5 (Examination of metal ion requirement of enzyme) Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the final concentration was 100 mM.
- the presence of Zn ions, Cu ions, and Fe ions in the reaction solution was shown to inhibit the formation of ⁇ -NMN. Furthermore, since EDTA, which is a chelating agent, is present in the reaction solution, it exhibits ⁇ -NMN-forming activity, so that the addition of a metal compound is not necessary for this enzyme reaction.
- the production rate of ⁇ -NMN is a relative value when the ionic strength of ⁇ -NMN in the solution after the enzyme reaction without addition of a metal compound is taken as 100. The ionic strength was measured by LC-MS.
- Example 6 (Examination of optimal enzyme addition amount and enzyme reaction time) Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the crude enzyme prepared from Rhizopus oryzae was adjusted to 0. .010% (w / v), 0.025% (w / v), 0.050% (w / v), 0.10% (w / v), 0.25% (w / v), 0 Enzymatic reaction was carried out for 5 hours after addition of 50% (w / v), 0.75% (w / v) and 1.0% (w / v) respectively.
- the highest ⁇ -NMN production rate was the enzyme reaction for 3 hours with the addition of 0.10% (w / v).
- the ⁇ -NMN production rate indicates a relative value when the ionic strength of ⁇ -NMN in the extract not reacted with the enzyme is 1.
- the ionic strength was measured by LC-MS.
- Example 7 (Measurement of ⁇ -NMN content under optimal enzyme reaction conditions) Using Candida utilis IAM 4264, yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and Rhizopus After adding 0.1% (w / v) of crude enzyme prepared from oryzae, an optimal reaction was performed for 3 hours. Thereafter, a dried yeast extract containing ⁇ -NMN was obtained by a drying step. The dried product was quantitatively analyzed under the above measurement conditions. The chromatogram is as shown in FIG.
- the ⁇ -NMN content in the yeast extract under the optimal enzyme reaction conditions was determined by quantifying ⁇ -NMN using the calibration curve crude of FIG. 8, and as shown in FIG. w). Before the reaction, NAD was contained at 2.29% (w / w) per dry solid content, and after the reaction, NAD decreased to 0.18% (w / w) per dry solid content, and ⁇ -NMN Has been generated. From this, the mechanism as shown in FIG. 11 is predicted for the production of ⁇ -NMN by this enzyme reaction.
- Example 8 Production of ⁇ -NMN was confirmed using a commercially available enzyme belonging to the genus Rhizopus.
- a yeast extract was prepared, and 0.1% (w / v) of commercially available enzyme “Lipase A-10D” (manufactured by Nagase ChemteX) was added for 3 hours for optimal reaction. went.
- the reaction conditions were pH 5.0 and temperature 55 ° C.
- a yeast extract containing 2.01% (w / w) of ⁇ -NMN could be obtained.
- ⁇ -NMN can be obtained from edible safe yeast and can be ingested not only as a pharmaceutical product, but also as a functional food and a dietary supplement. By ingesting the product of the present invention, the functionality possessed by ⁇ -NMN is obtained. I can do it.
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Abstract
[Problem] To produce a β-NMN-containing yeast extract from yeast having eating experience, and to produce a yeast-originated β-NMN composition.
[Solution] It is found that a yeast extract containing β-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 β-NMN-containing composition can be produced by purifying β-NMN from a yeast extract.
Description
本発明は、Candida utilisを培養して得られた抽出液にRhizopus oryzae等のRhizopus属から調製した粗酵素を作用させる工程を含む、食品規格の長寿遺伝子 (サ－チュイン)を活性化させる分子 (サ－チュインアクティベ－タ－)である「β－Nicotinamide mononucleotide （β－ＮＭＮ）」を高含有した酵母エキスの製造法を提供する。
The present invention relates to a molecule that activates a food standard longevity gene (sirtuin), which comprises a step of allowing a crude enzyme prepared from Rhizopus genus such as Rhizopus oryzae to act on an extract obtained by culturing Candida utilis ( Provided is a method for producing a yeast extract containing a high content of “β-Nicotinamide mononucleotide (β-NMN)”, a sirtuin activator).
The present invention relates to a molecule that activates a food standard longevity gene (sirtuin), which comprises a step of allowing a crude enzyme prepared from Rhizopus genus such as Rhizopus oryzae to act on an extract obtained by culturing Candida utilis ( Provided is a method for producing a yeast extract containing a high content of “β-Nicotinamide mononucleotide (β-NMN)”, a sirtuin activator).
β－Nicotinamide mononucleotide（β－ＮＭＮ）は、生体内のde novo経路やSalvage経路の代謝物質であるβ－Nicotinamide adenine dinucleotide（ＮＡＤ）の中間代謝物質である (特許文献１～４、非特許文献1)。β－ＮＭＮは、生体に投与することによりＮＡＤの生合成を直接に誘導、組織中のNAD濃度を向上させることが出来る (非特許文献２)。サ－チュイン遺伝子がコ－ドしているタンパク質にはヒトでは、SIRT１を中心にSIRT２、SIRT３、SIRT４、SIRT５、SIRT６、SIRT７のタンパク質ファミリ－の存在が確認されている (非特許文献1)。これらサ－チュインファミリ－は、NAD依存性脱アセチル化酵素であり、NADを基質に活性化され、幅広い抗老化作用を発現させる (非特許文献1)。このように、サ－チュインアクティベ－タ－であるβ－ＮＭＮが関わる機能として、「糖代謝異常の改善 (非特許文献2)」、「サ－カディアンリズムへの関与 (非特許文献3)4)」、「老化ミトコンドリアの機能改善 (非特許文献5)」、「虚血再灌流からの心臓の保護 (非特許文献6)」、「老化による神経幹細胞の減少の抑制 (非特許文献7)」、「エピジェネティク制御機構によりClaudin－1の発現を抑制し、糖尿病性腎症のアルブミン尿の低下 (非特許文献8)」、「プログラム細胞死の制御 (非特許文献9)」、「パ－キンソン病の改善 (非特許文献10)」、「老化による酸化ストレスや血管機能障害の回復 (非特許文献11)」などが報告されている。このように、細胞または組織、器官レベルの老化が関与する数多くのネガティブな生体現象は、β－ＮＭＮの投与によってＮＡＤの生合成を高め、SIRT1を中心としたサ－チュインファミリ－の活性化によって、回復、予防が期待できる。このことにより、個体の老化を総合的に遅らせ、最終的には延命 (長寿)に繋がることが期待できる。
β-Nicotinamide mononucleotide (β-NMN) is an intermediate metabolite of β-Nicotinamide adenine dinucleotide (NAD), which is a metabolite of the de novo pathway and the Salvage pathway in vivo (Patent Documents 1 to 4, Non-Patent Document 1) ). β-NMN can directly induce biosynthesis of NAD and improve the NAD concentration in tissues when administered to a living body (Non-patent Document 2). In humans, the presence of SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7 protein families has been confirmed in humans as proteins encoded by sirtuin genes (Non-patent Document 1). These sirtuin families are NAD-dependent deacetylases, which are activated by using NAD as a substrate and exhibit a wide range of anti-aging effects (Non-patent Document 1). As described above, functions related to β-NMN, which is a sirtuin activator, include “improvement of abnormal sugar metabolism (Non-patent Document 2)”, “participation in Circadian rhythm (Non-patent Document 3) 4” ) '', `` Functional improvement of aging mitochondria (Non-patent document 5) '', `` Protection of heart from ischemia-reperfusion (Non-patent document 6) '', `` Inhibition of neural stem cell decrease due to aging (Non-patent document 7) '' ”,“ Epigenetic control mechanism suppresses claudin-1 expression and diminishes albuminuria in diabetic nephropathy (Non-patent document 8) ”,“ Control of programmed cell death (Non-patent document 9) ”,“ There have been reports such as “Improvement of Parkinson's disease (Non-patent Document 10)” and “Recovery of oxidative stress and vascular dysfunction due to aging (Non-patent Document 11)”. As described above, many negative biological phenomena involving aging at the cell, tissue, or organ level increase the biosynthesis of NAD by the administration of β-NMN, and the activation of the sirtuin family centering on SIRT1. Recovery, prevention can be expected. By this, it can be expected that the aging of the individual will be delayed overall and eventually lead to life prolongation (longevity).
また、酵母は各種食品等に使用されており、トルラ酵母（Candida utilis）はアメリカ食品医薬品局 （ＦＡＤ）より高い栄養機能性と食経験からの安全性が評価されている食用酵母である。このことより、長年にわたって医薬品やサプリメント、調味料などに有効活用されている。
Yeast is used in various foods, and Torula yeast (Candida utilis) is an edible yeast that has been evaluated for higher nutritional functionality and safety from eating experience than the US Food and Drug Administration (FAD). For this reason, it has been used effectively for many years in medicines, supplements and seasonings.
現在、β－ＮＭＮは研究用途のみの販売で、食品規格のものは販売されていない。よって、食経験のある酵母からβ－ＮＭＮを含有した酵母エキスを得ること、酵母由来のβ－ＮＭＮを高含有化させた組成物を得ることを課題とする。
Currently, β-NMN is sold only for research purposes, and no food-standard products are sold. Therefore, it is an object to obtain a yeast extract containing β-NMN from yeast with experience of eating, and to obtain a composition with a high content of β-NMN derived from yeast.
本発明者は、酵母から酵母エキスを抽出し、Rhizopus oryzaeなどのRhizopus属に属する微生物から得られた酵素又は粗酵素で、最適化（温度４５～６０℃、ｐＨ４．５～６．０）した酵素反応を行うことで、β－ＮＭＮを高含有化させた酵母エキスを得られることを見出し、本発明を完成させた。
The present inventor extracted yeast extract from yeast and optimized (temperature 45 to 60 ° C., pH 4.5 to 6.0) with an enzyme or a crude enzyme obtained from a microorganism belonging to the genus Rhizopus such as Rhizopus oryzae. It has been found that a yeast extract enriched with β-NMN can be obtained by carrying out an enzymatic reaction, and the present invention has been completed.
具体的には、以下のような発明である。
（１）β‐ニコチンアミドモノヌクレオチドを乾燥固形分あたり２．０％（ｗ／ｖ）以上含有するβ‐ニコチンアミドモノヌクレオチド高含有酵母エキス。
（２）次の理化学的性質を有する酵素を用いて反応させる工程を含む前記（１）のβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。
（ａ）作用：ニコチンアミドアデニンジヌクレオチドをニコチンアミドモノヌクレオチドに加水分解する。
（ｂ）至適ｐＨ：ｐＨ４．５～６．０。
（ｃ）至適温度：４５℃～６０℃。
（ｄ）由来：Rhizopus 属に属する微生物。
（３）前記（１）の製造方法において、使用する酵素が、Rhizopus 属に属する微生物から抽出したタンパク質であるβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。 Specifically, the invention is as follows.
(1) A yeast extract containing a high content of β-nicotinamide mononucleotide containing 2.0% (w / v) or more of β-nicotinamide mononucleotide per dry solid content.
(2) The method for producing a yeast extract containing β-nicotinamide mononucleotide according to (1), which comprises a step of reacting with an enzyme having the following physicochemical properties.
(A) Action: Hydrolyzes nicotinamide adenine dinucleotide to nicotinamide mononucleotide.
(B) Optimum pH: pH 4.5 to 6.0.
(C) Optimal temperature: 45 ° C to 60 ° C.
(D) Origin: A microorganism belonging to the genus Rhizopus.
(3) A method for producing a yeast extract containing β-nicotinamide mononucleotide, wherein the enzyme used in the production method of (1) is a protein extracted from a microorganism belonging to the genus Rhizopus.
（１）β‐ニコチンアミドモノヌクレオチドを乾燥固形分あたり２．０％（ｗ／ｖ）以上含有するβ‐ニコチンアミドモノヌクレオチド高含有酵母エキス。
（２）次の理化学的性質を有する酵素を用いて反応させる工程を含む前記（１）のβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。
（ａ）作用：ニコチンアミドアデニンジヌクレオチドをニコチンアミドモノヌクレオチドに加水分解する。
（ｂ）至適ｐＨ：ｐＨ４．５～６．０。
（ｃ）至適温度：４５℃～６０℃。
（ｄ）由来：Rhizopus 属に属する微生物。
（３）前記（１）の製造方法において、使用する酵素が、Rhizopus 属に属する微生物から抽出したタンパク質であるβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。 Specifically, the invention is as follows.
(1) A yeast extract containing a high content of β-nicotinamide mononucleotide containing 2.0% (w / v) or more of β-nicotinamide mononucleotide per dry solid content.
(2) The method for producing a yeast extract containing β-nicotinamide mononucleotide according to (1), which comprises a step of reacting with an enzyme having the following physicochemical properties.
(A) Action: Hydrolyzes nicotinamide adenine dinucleotide to nicotinamide mononucleotide.
(B) Optimum pH: pH 4.5 to 6.0.
(C) Optimal temperature: 45 ° C to 60 ° C.
(D) Origin: A microorganism belonging to the genus Rhizopus.
(3) A method for producing a yeast extract containing β-nicotinamide mononucleotide, wherein the enzyme used in the production method of (1) is a protein extracted from a microorganism belonging to the genus Rhizopus.
本発明によると、β－Nicotinamide mononucleotide（β－ＮＭＮ）を食経験のある酵母から簡便に取得できる。特にトルラ酵母は古くから食経験のある酵母であり、これから取得した酵母エキスは安全性が高い。このような、β－Nicotinamide mononucleotideを高含有する酵母エキスは、医薬品、サプリメント、機能性食品等として摂取できる。
According to the present invention, β-Nicotinamide mononucleotide (β-NMN) can be easily obtained from yeast with experience in food. In particular, Torula yeast is a yeast that has been eating for a long time, and the yeast extract obtained from this is highly safe. Such yeast extract containing a high content of β-Nicotinamide mononucleotide can be ingested as pharmaceuticals, supplements, functional foods and the like.
本発明では、酵母として食用酵母が使用できる。例えばSaccharomyces属に属する酵母、Kluyveromyces属、Candida属、Pichia属などが挙げられ、中でも、Candida属のCandida utilisが好ましい。より具体的には、Candida utilis IAM 4264、Candida utilis ATCC 9950、Candida utilis ATCC 9550、Candida utilis IAM 4233、Candida utilis AHU 3259などである。さらに好ましくは、グルタチオンを高含有する酵母を使用すると、β－ＮＭＮの含量が高まる。グルタチオンを高含有する酵母は、公知の方法で得られる酵母を使用可能である（特開昭５９－１５１８９４、特開昭６０－１５６３７９など）。
In the present invention, edible yeast can be used as yeast. For example, yeast belonging to the genus Saccharomyces, Kluyveromyces genus, Candida genus, Pichia genus and the like can be mentioned, among which Candida genus Candida utilis is preferable. More specifically, Candidaandutilis IAM 4264, Candida utilis ATCC 9950, Candida utilis ATCC 9550, Candida utilis IAM 4233, Candida utilis AHU 3259 and the like. More preferably, the use of yeast with a high glutathione content increases the β-NMN content. As the yeast having a high glutathione content, yeasts obtained by known methods can be used (Japanese Patent Laid-Open Nos. 59-151894, 60-156379, etc.).
酵母を培養する際の培地には、炭素源として、ブドウ糖、酢酸、エタノ－ル、グリセロ－ル、糖蜜、亜硫酸パルプ廃液等が用いられ、窒素源としては、尿素、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸塩などが使用される。リン酸、カリウム、マグネシウム源も過リン酸石灰、リン酸アンモニウム、塩化カリウム、水酸化カリウム、硫酸マグネシウム、塩化マグネシウム等の通常の工業用原料でよく、その他亜鉛、銅、マンガン、鉄イオン等の無機塩を添加する。その他は、ビタミン、アミノ酸、核酸関連物質等を使用しないでも培養可能であるが、これらを添加しても良い。コ－ンスチ－ブリカ－、カゼイン、酵母エキス、肉エキス、ペプトン等の有機物を添加しても良い。
In the medium for culturing yeast, glucose, acetic acid, ethanol, glycerol, molasses, sulfite pulp waste liquid, etc. are used as carbon sources, and nitrogen, urea, ammonia, ammonium sulfate, ammonium chloride, Nitrate is used. Phosphoric acid, potassium, and magnesium sources may be ordinary industrial raw materials such as lime perphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, and other zinc, copper, manganese, iron ions, etc. Add inorganic salt. Others can be cultured without using vitamins, amino acids, nucleic acid-related substances, etc., but these may be added. Organic substances such as cone-briker, casein, yeast extract, meat extract and peptone may be added.
培養温度やｐH等の培養条件は、特に制限なく適用でき、使用する酵母菌株に合わせて設定し、培養すれば良い。一般的には、培養温度は２１～３７℃、好ましくは２５～３４℃が良く、ｐＨは３．０～８．０、特に３．５～７．０が好ましい。
Culture conditions such as culture temperature and pH can be applied without particular limitation, and may be set according to the yeast strain used and cultured. 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 3.5 to 7.0.
本発明の培養形式としては、バッチ培養、あるいは連続培養のいずれでも良いが、工業的には後者が望ましい。培養時の撹拌、通気等の条件は特に制限なく、一般的な方法でよい。
The culture format of the present invention may be either batch culture or continuous culture, but the latter is desirable industrially. Conditions such as agitation and aeration during culture are not particularly limited and may be a general method.
培養後の菌体は、前処理により抽出液の調製を行う。菌体培養後の湿潤酵母菌体を蒸留水に懸濁して遠心分離を繰り返すことで洗浄した後に、抽出を行う。抽出法は、使用する酵母菌体の種類に応じて適宜調整すればよいが、β－ＮＭＮの含量を高めるには、酵母中のＮＡＤ（ニコチンアミドアデニンジヌクレオチド (nicotinamide adenine dinucleotide)）、β－ＮＭＮが分解されないような条件で行うことが望ましい。自己消化法、アルカリ抽出法、温水抽出法、又はこれらの組み合わせにより行う。Candida utilisを用いた場合の方法は菌体濃度が乾燥重量換算７～１０％、好ましくは８～９％になるように蒸留水に再懸濁する。この菌体懸濁液の抽出の際に、必要に応じてｐＨ調整を行う。最も好ましくは抽出時のｐＨを６．０付近に調整する。ｐＨ調整は、公知の方法でよい。
For the cultured cells, an extract is prepared by pretreatment. Extraction is performed after washing the wet yeast cells after culturing the cells by suspending them in distilled water and repeating centrifugation. The extraction method may be appropriately adjusted according to the type of yeast used, but in order to increase the content of β-NMN, NAD (nicotinamide adenine dinucleotide nucleotide), β- It is desirable to carry out under the condition that NMN is not decomposed. The self-digestion method, the alkali extraction method, the hot water extraction method, or a combination thereof is used. When Candida utilis is used, the suspension is resuspended in distilled water so that the bacterial cell concentration is 7 to 10%, preferably 8 to 9%, in terms of dry weight. During the extraction of the cell suspension, pH adjustment is performed as necessary. Most preferably, the pH during extraction is adjusted to around 6.0. The pH may be adjusted by a known method.
抽出温度は５０～９０℃、好ましくは、５０～６５℃とする。温度の調整法は、抽出液が前記の温度になれば特に制限なく公知の方法が利用できる。
Extraction temperature is 50 to 90 ° C, preferably 50 to 65 ° C. As a method for adjusting the temperature, a known method can be used without particular limitation as long as the extract reaches the above temperature.
抽出時間は、５分以上行えばよい。抽出中は、撹拌することが望ましい。撹拌速度等は、適宜調整すればよく、特に制限はない。また、抽出時間を４０～５０分とすると、β‐ＮＭＮの含量が高まるので、さらに好ましい。
* Extraction time may be 5 minutes or more. It is desirable to stir during extraction. The stirring speed and the like may be adjusted as appropriate and are not particularly limited. Further, when the extraction time is 40 to 50 minutes, the content of β-NMN increases, which is more preferable.
抽出後は、菌体懸濁液を遠心分離で除去し、上清を得る。この上清を抽出液とし、本発明である酵素反応の基質溶液とした。
After extraction, the cell suspension is removed by centrifugation to obtain a supernatant. This supernatant was used as an extract and a substrate solution for the enzyme reaction according to the present invention.
使用する酵素は、前段までで得られた溶液中に含まれるＮＡＤを基質とし、β－ＮＭＮを生成する酵素を用いる。具体的には、Rhizopus 属に属する糸状菌類由来の酵素を用いる。Rhizopus 属は、Rhizopus oryzae、Risopus microsporus、Rhizopus oligosporusなどがあげられ、食経験のある Rhizopus属由来の酵素を用いることができる。
The enzyme used is an enzyme that produces β-NMN using NAD contained in the solution obtained up to the previous stage 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 microsporus, Rhizopus oligosporus, etc., and an enzyme derived from the genus zoRhizopus genus with experience in eating can be used.
本発明で使用する酵素は、前述のようにRhizopus属の微生物から調製した粗酵素を用いることができる。Rhizopus属の微生物は、食品工業等で使用される株で良い。Rhizopus oryzae等のRhizopus属菌類は、プロテア－ゼ等の酵素生産の製造に用いられているため（特開２０１０－００４７６０など）、そのような株が特に良い。さらに、Rhizopus属の糸状菌は、ATCC、NBRC等の菌株分譲機関、又は市販の種菌株販売会社等から入手した株でも良い。本発明で用いる粗酵素の調製は、一般的な酵素調製法で可能であり、例えば、菌体培養、クロマトグラフィ－による粗精製工程を経て酵素などのタンパク質群を含む画分を取得する。本願は、粗酵素を用いることができるため、培養液からタンパク質群を含む画分、又は、培養液とRhizopus oryzaeを破砕し、細胞内のタンパク質群を含む画分を用いても良い。乾燥工程を得て乾燥物としても良い。さらに、Rhizopus属由来の酵素は、各種市販されており、このような市販酵素の多くは、夾雑酵素を含んでいるため、本願の方法に用いることができる酵素も入手可能である。
As the enzyme used in the present invention, a crude enzyme prepared from a microorganism belonging to the genus Rhizopus as described above can be used. The microorganism belonging to the genus Rhizopus may be a strain used in the food industry. Rhizopus genus fungi such as Rhizopus oryzae are particularly preferred because they are used in the production of enzyme production such as protease (JP 2010-004760 A, etc.). Furthermore, the filamentous fungus belonging to the genus Rhizopus may be a strain obtained from strain distribution agencies such as ATCC, NBRC, or a commercially available seed strain sales company. A crude enzyme used in the present invention can be prepared by a general enzyme preparation method. For example, a fraction containing a protein group such as an enzyme is obtained through a cell culture and a crude purification step by chromatography. Since a crude enzyme can be used in the present application, a fraction containing a protein group from a culture solution, or a fraction containing an intracellular protein group by crushing the culture solution and Rhizopus oryzae may be used. It is good also as a dried material by obtaining a drying process. Furthermore, various enzymes derived from the genus Rhizopus are commercially available, and since many of these commercially available enzymes contain contaminating enzymes, enzymes that can be used in the method of the present application are also available.
以上のような酵素は、ＮＡＤを基質としてＮＭＮを生成する酵素であり、酵母中のＮＡＤだけでなく、ＮＡＤ純品を基質として、ＮＭＮを生成する酵素にも用いることができる。ＮＡＤは、一般的に入手可能なものを利用できる。
The above enzymes are enzymes that produce NMN using NAD as a substrate, and can be used not only for NAD in yeast, but also for enzymes that produce NMN using NAD pure products as substrates. NAD can use what is generally available.
反応に用いる酵素の添加量に関しては、酵素の調製方法によって異なるが、通常は、０．０５％（ｗ／ｖ）～０．２５％（ｗ／ｖ）添加、好ましくは０．１％（ｗ／ｖ）添加する。なお、本願で酵素の至適反応条件の検討に用いたβ－ＮＭＮの測定方法は、実施例中に記載したＬＣ－ＭＳの測定条件による。
The amount of the enzyme used for the reaction varies depending on the method for preparing the enzyme, but is usually 0.05% (w / v) to 0.25% (w / v), preferably 0.1% (w / V) Add. In this application, the β-NMN measurement method used for studying the optimal reaction conditions for the enzyme depends on the LC-MS measurement conditions described in the Examples.
The amount of the enzyme used for the reaction varies depending on the method for preparing the enzyme, but is usually 0.05% (w / v) to 0.25% (w / v), preferably 0.1% (w / V) Add. In this application, the β-NMN measurement method used for studying the optimal reaction conditions for the enzyme depends on the LC-MS measurement conditions described in the Examples.
粗酵素の反応の至適温度は、４５～６０℃、好ましくは５０℃～５５℃、もっとも好ましくは５５℃である。なお、本願で酵素の至適反応条件の検討に用いたβ－ＮＭＮの検出方法は、実施例中に記載したＬＣ－ＭＳの測定条件による。
The optimum temperature for the reaction of the crude enzyme is 45 to 60 ° C, preferably 50 to 55 ° C, and most preferably 55 ° C. In addition, the detection method of β-NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
粗酵素の反応の至適ｐＨは、４．５～６．０、好ましくは５．０～５．５、もっとも好ましくはｐＨ５．０である。なお、本願で酵素の至適反応条件の検討に用いたβ－ＮＭＮの検出方法は、実施例中に記載したＬＣ－ＭＳの測定条件による。
The optimum pH for the reaction of the crude enzyme is 4.5 to 6.0, preferably 5.0 to 5.5, most preferably pH 5.0. In addition, the detection method of β-NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
前述のように培養した酵母から調製した抽出液にRhizopus oryzae由来の粗酵素を添加し、至適反応条件で酵素反応を行うことで、酵母エキスの固形分に対して２．０％（ｗ／ｗ）以上のβ－ＮＭＮを含有する酵母エキスを得ることが出来る。本発明β－NMNは、酵母中のNAD含量により生成されるβ－ＮＭＮの含量は異なる。酵母菌体から抽出する酵素反応の基質溶液中に含まれるＮＡＤ含量を高めると、さらにβ－ＮＭＮを高含有化することができる。なお、本願で酵素の至適反応条件の検討に用いたβ－ＮＭＮの検出方法は、実施例中に記載したＬＣ－ＭＳの測定条件による。
By adding a crude enzyme derived from Rhizopus zaoryzae to the extract prepared from the yeast cultured as described above, and performing an enzyme reaction under the optimal reaction conditions, 2.0% (w / w w) A yeast extract containing the above β-NMN can be obtained. The β-NMN of the present invention differs in the content of β-NMN produced by the NAD content in yeast. Increasing the NAD content contained in the substrate solution of the enzyme reaction extracted from the yeast cells can further increase β-NMN. In addition, the detection method of β-NMN used for the examination of the optimum reaction conditions of the enzyme in the present application is based on the LC-MS measurement conditions described in the Examples.
酵素反応を施した抽出液は、濃縮後、凍結乾燥又は熱風乾燥することで、β－ＮＭＮ含有酵母エキスの乾燥物を得ることが出来る。
さらに、β－ＮＭＮ含有酵母エキスから、β－ＮＭＮを精製することで、酵母由来のβ－ＮＭＮをさらに高含有化した組成物を得ることができる。また、前段の乾燥前の酵母抽出液からβ－ＮＭＮを精製することでも、酵母由来のβ－ＮＭＮを高含有化した組成物を得ることが出来る。精製法は、イオン交換樹脂等を用いた一般的な精製法が利用できる。 The extract subjected to the enzyme reaction can be concentrated and then freeze-dried or hot-air dried to obtain a dried product of β-NMN-containing yeast extract.
Further, by purifying β-NMN from the β-NMN-containing yeast extract, it is possible to obtain a composition further enriched in yeast-derived β-NMN. In addition, a composition enriched in yeast-derived β-NMN can also be obtained by purifying β-NMN from the yeast extract before drying in the previous stage. As a purification method, a general purification method using an ion exchange resin or the like can be used.
さらに、β－ＮＭＮ含有酵母エキスから、β－ＮＭＮを精製することで、酵母由来のβ－ＮＭＮをさらに高含有化した組成物を得ることができる。また、前段の乾燥前の酵母抽出液からβ－ＮＭＮを精製することでも、酵母由来のβ－ＮＭＮを高含有化した組成物を得ることが出来る。精製法は、イオン交換樹脂等を用いた一般的な精製法が利用できる。 The extract subjected to the enzyme reaction can be concentrated and then freeze-dried or hot-air dried to obtain a dried product of β-NMN-containing yeast extract.
Further, by purifying β-NMN from the β-NMN-containing yeast extract, it is possible to obtain a composition further enriched in yeast-derived β-NMN. In addition, a composition enriched in yeast-derived β-NMN can also be obtained by purifying β-NMN from the yeast extract before drying in the previous stage. As a purification method, a general purification method using an ion exchange resin or the like can be used.
本発明の酵母エキス又は酵母由来のβ－ＮＭＮ含有組成物の摂取方法は、特に限定されず、経口投与、静脈内、腹膜内もしくは皮下投与等の非経口投与をあげることが出来る。具体的には、錠剤、散剤、顆粒剤、丸剤、懸濁剤、乳剤、浸剤・煎剤、カプセル剤、シロップ剤、液剤、エリキシル剤、エキス剤、チンキ剤、流エキス剤等の経口剤、又は注射剤、点滴剤、クリ－ム剤、坐剤等の非経口剤のいずれでもよい。
摂 取 The method of ingesting the yeast extract or yeast-derived β-NMN-containing composition of the present invention is not particularly limited, and examples include oral administration, parenteral administration such as intravenous, intraperitoneal or subcutaneous administration. Specifically, oral preparations such as tablets, powders, granules, pills, suspensions, emulsions, soaking and decoction, capsules, syrups, solutions, elixirs, extracts, tinctures, fluid extracts, Or any of parenterals such as injections, drops, creams, suppositories, etc. may be used.
酵母エキスは、医薬品だけでなく、食品として摂取可能であり、機能性食品、栄養補助食品、サプリメント等としても摂取出来る。
The yeast yeast extract can be ingested not only as a medicine but also as a food, and can also be ingested as a functional food, a nutritional supplement, a supplement and the like.
また、本発明は、β－ＮＭＮのサ－チュイン活性を低下させない又はβ－ＮＭＮのサ－チュイン活性を増強させる他の組成物と併用することも可能である。例えば、賦形剤、希釈剤となるデキストリン、マルチト－ル、ソルビト－ル、デンプンなどである。
The present invention can also be used in combination with other compositions that do not decrease the sirtuin activity of β-NMN or enhance the sirtuin activity of β-NMN. For example, excipients, diluents, dextrins, malt tols, sorbitol, starch and the like.
本発明の摂取量は、β－ＮＭＮのサ－チュイン活性が発現される量を投与すればよい。一般的に、β－ＮＭＮの活性に必要な投与量を決定するには、摂取者の状態、投与される組成物の選択、摂取者の年齢、体重、および応答、摂取者の状態などによって決定される。
The amount of intake according to the present invention may be administered in such an amount that the sirtuin activity of β-NMN is expressed. Generally, the dose required for β-NMN activity is determined by the condition of the consumer, the choice of composition administered, the age, weight and response of the consumer, the condition of the consumer, etc. Is done.
以下に、本願発明を具体的に示すが、本願発明は、これに限定されるものではない。
Hereinafter, the present invention will be specifically described, but the present invention is not limited thereto.
（至適酵素反応条件の検討に使用したβ－ＮＭＮの測定条件）
ＬＣ－ＭＳにより測定
質量分析計 (MS)測定条件
分析機器: amaZon speed (Bruker daltonics 社)
イオン化法: Electro spray ionization (ESI)
分離部: イオントラップ
検出部: Positive mode (MRM mode)
β－NMN → プレカ－サ－イオンm/z 335にフラグメントイオン m/z 123をトレ－ス NAD → プレカ－サ－イオンm/z 664にフラグメントイオン m/z 524、542をトレ－ス
キャピラリ－電圧: 4.5 kV
ネブライザ－: 30.0 psi
ドライガス: 10.0 L/min
ドライ温度: 250℃
高速液体クロマトグラフィ－ (HPLC)測定条件
ポンプ: LC－20AD (島津製作所 社)
デガッサ－: DGU－20A3 (島津製作所 社)
オ－トサンプラ－: SIL－20AC HT (島津製作所 社)
ダイオ－ドアレイ検出器: SPD－M20A (島津製作所 社)
カラムオ－ブン: CTO－20AC (島津製作所 社)
移動相A: LC－MS用ギ酸 (和光純薬 社)をLC－MS用超純水 (和光純薬 社) で0.1%(v/v) になるように添加 (pH 2.5)。
移動相 B : 0.1% LC－MS用ギ酸アセトニトリル (和光純薬 社)
カラム: Inertsil ODS－3 (粒子径3 um、長さ150 mm、内径2.1 mm) (GL science 社)
カラムオ－ブン温度: 45℃
流速: 0.2 mL/min
サンプル注入量: 5 uL
サンプルク－ラ－温度: 4℃
溶出法: リニアグラジエント
グラジエント条件: 0 min (0% 移動相 B)－20 min (100% 移動相 B)－25 min (100% 移動相B)－25.1 min (0% 移動相 B)－40 min (0% 移動相 B)
物質分析 20 min、カラム洗浄 5 min、カラム平衡化 15 minの計40 min分析
分析検体の調製法： 反応溶液を移動相0.1%(v/v) ギ酸で100倍希釈をした。その後、希釈溶液は、シリンジに装着したDISMIC 13CP020AS 0.22 umフィルタ－ (ADVANTEC 社)によって不溶性物質をろ過し、LC－MSに供した。 (Measurement conditions of β-NMN used for examination of optimum enzyme reaction conditions)
Mass spectrometer (MS) by LC-MS Measurement condition analyzer: amaZon speed (Bruker daltonics)
Ionization method: Electro spray ionization (ESI)
Separator: Ion trap detector: Positive mode (MRM mode)
β-NMN → Tracer ion m /z 123 to precursor ion m / z 335 Trace NAD → Fragment ion m / z 524 and 542 to precursor ion m / z 664 Trace capillary Voltage: 4.5 kV
Nebulizer: 30.0 psi
Dry gas: 10.0 L / min
Drying temperature: 250 ℃
High performance liquid chromatography (HPLC) measurement conditions
Pump: LC-20AD (Shimadzu Corporation)
Degasser: 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: LC-MS formic acid (Wako Pure Chemical Industries, Ltd.) was added to LC-MS ultrapure water (Wako Pure Chemical Industries, Ltd.) to 0.1% (v / v) (pH 2.5).
Mobile phase B: 0.1% LC-MS formic acid acetonitrile (Wako Pure Chemical Industries, Ltd.)
Column: Inertsil ODS-3 (particle size 3 um,length 150 mm, inner diameter 2.1 mm) (GL science)
Column oven temperature: 45 ° C
Flow rate: 0.2 mL / min
Sample injection volume: 5 uL
Sample cooler temperature: 4 ℃
Elution method: Linear gradient Gradient condition: 0 min (0% mobile phase B) −20 min (100% mobile phase B) −25 min (100% mobile phase B) −25.1 min (0% mobile phase B) −40 min (0% mobile phase B)
Material analysis 20 min, column washing 5 min, column equilibration 15 min total Preparation method for analysis sample: Reaction solution was diluted 100 times with mobile phase 0.1% (v / v) formic acid. Thereafter, the diluted solution was subjected to LC-MS by filtering insoluble substances with a DISMIC 13CP020AS 0.22 um filter (ADVANTEC) attached to a syringe.
ＬＣ－ＭＳにより測定
質量分析計 (MS)測定条件
分析機器: amaZon speed (Bruker daltonics 社)
イオン化法: Electro spray ionization (ESI)
分離部: イオントラップ
検出部: Positive mode (MRM mode)
β－NMN → プレカ－サ－イオンm/z 335にフラグメントイオン m/z 123をトレ－ス NAD → プレカ－サ－イオンm/z 664にフラグメントイオン m/z 524、542をトレ－ス
キャピラリ－電圧: 4.5 kV
ネブライザ－: 30.0 psi
ドライガス: 10.0 L/min
ドライ温度: 250℃
高速液体クロマトグラフィ－ (HPLC)測定条件
ポンプ: LC－20AD (島津製作所 社)
デガッサ－: DGU－20A3 (島津製作所 社)
オ－トサンプラ－: SIL－20AC HT (島津製作所 社)
ダイオ－ドアレイ検出器: SPD－M20A (島津製作所 社)
カラムオ－ブン: CTO－20AC (島津製作所 社)
移動相A: LC－MS用ギ酸 (和光純薬 社)をLC－MS用超純水 (和光純薬 社) で0.1%(v/v) になるように添加 (pH 2.5)。
移動相 B : 0.1% LC－MS用ギ酸アセトニトリル (和光純薬 社)
カラム: Inertsil ODS－3 (粒子径3 um、長さ150 mm、内径2.1 mm) (GL science 社)
カラムオ－ブン温度: 45℃
流速: 0.2 mL/min
サンプル注入量: 5 uL
サンプルク－ラ－温度: 4℃
溶出法: リニアグラジエント
グラジエント条件: 0 min (0% 移動相 B)－20 min (100% 移動相 B)－25 min (100% 移動相B)－25.1 min (0% 移動相 B)－40 min (0% 移動相 B)
物質分析 20 min、カラム洗浄 5 min、カラム平衡化 15 minの計40 min分析
分析検体の調製法： 反応溶液を移動相0.1%(v/v) ギ酸で100倍希釈をした。その後、希釈溶液は、シリンジに装着したDISMIC 13CP020AS 0.22 umフィルタ－ (ADVANTEC 社)によって不溶性物質をろ過し、LC－MSに供した。 (Measurement conditions of β-NMN used for examination of optimum enzyme reaction conditions)
Mass spectrometer (MS) by LC-MS Measurement condition analyzer: amaZon speed (Bruker daltonics)
Ionization method: Electro spray ionization (ESI)
Separator: Ion trap detector: Positive mode (MRM mode)
β-NMN → Tracer ion m /
Nebulizer: 30.0 psi
Dry gas: 10.0 L / min
Drying temperature: 250 ℃
High performance liquid chromatography (HPLC) measurement conditions
Pump: LC-20AD (Shimadzu Corporation)
Degasser: 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: LC-MS formic acid (Wako Pure Chemical Industries, Ltd.) was added to LC-MS ultrapure water (Wako Pure Chemical Industries, Ltd.) to 0.1% (v / v) (pH 2.5).
Mobile phase B: 0.1% LC-MS formic acid acetonitrile (Wako Pure Chemical Industries, Ltd.)
Column: Inertsil ODS-3 (particle size 3 um,
Column oven temperature: 45 ° C
Flow rate: 0.2 mL / min
Sample injection volume: 5 uL
Sample cooler temperature: 4 ℃
Elution method: Linear gradient Gradient condition: 0 min (0% mobile phase B) −20 min (100% mobile phase B) −25 min (100% mobile phase B) −25.1 min (0% mobile phase B) −40 min (0% mobile phase B)
（至適条件反応により取得した乾燥物中のβ－ＮＭＮならびにＮＡＤの定量分析条件）
ＨＰＬＣにより測定
ポンプ、デガッサ－： Chromaster 5110 (日立ハイテクサイエンス 社)
オ－トサンプラ－： Chromaster 5210 (日立ハイテクサイエンス 社)
UV－VIS 検出器： Chromaster 5420 (日立ハイテクサイエンス 社)
カラムオ－ブン： Chromaster 5310 (日立ハイテクサイエンス 社)
移動相： 75 mM リン酸二水素アンモニウム (pH 2.3) (和光純薬 社)。アスピレ－タ－で60 分間の脱気処理を行った。
カラム： Wakosil－II 5C18 RS (粒子径5 um、長さ30 mm、内径4.6 mm) (和光純薬 社)
→ Wakosil－II 5C18 RS (粒子径5 um、長さ150 mm、内径4.6 mm) (和光純薬 社) → Wakosil－II 5C18 RS (粒子径5 um、長さ250 mm、内径4.6 mm) (和光純薬 社)の順でカラムをタンデムに３連結した。
カラムオ－ブン温度： 26℃
流速： 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 (21.0 min) → 1.5 mL/min (55.0 min) → 1.0 mL/min (56.0 min) → 1.0 mL/min (60.0 min)
溶出法： アイソクラティック
検出波長： abs 260 nm
分析時間： 60 min
サンプル注入量： 5 uL
サンプルク－ラ－温度： 2℃
分析検体の調製法： 本発明によって得られた酵母エキス乾燥物を移動相７５ ｍＭ リン酸二水素アンモニウム （ｐＨ ２．３）で終濃度１％ （ｗ／ｗ）になるように溶解、調整した。その後、シリンジに装着したDISMIC 13CP020AS ０．２２ μｍフィルタ－ (ADVANTEC 社)によって不溶性物質をろ過し、HPLCに供した。
定量分析に使用した標準物質： β－ＮＭＮ （Sigma－Aldrich 社）、ＮＡＤ （Sigma－Aldrich 社）図８に示す検量線から、本発明によって得られた酵母エキス乾燥物中のβ－ＮＭＮの含量を算出した。 (Conditions for quantitative analysis of β-NMN and NAD in dry matter obtained by optimal reaction)
Measuring pump by HPLC, degasser: Chromaster 5110 (Hitachi High-Tech Science)
Autosampler: Chromaster 5210 (Hitachi High-Tech Science)
UV-VIS detector: Chromaster 5420 (Hitachi High-Tech Science)
Column oven: Chromaster 5310 (Hitachi High-Tech Science)
Mobile phase: 75 mM ammonium dihydrogen phosphate (pH 2.3) (Wako Pure Chemical Industries, Ltd.). Deaeration treatment was performed for 60 minutes with an aspirator.
Column: Wakosil-II 5C18 RS (particle size 5 um, length 30 mm, inner diameter 4.6 mm) (Wako Pure Chemical Industries, Ltd.)
→ Wakosil-II 5C18 RS (particle diameter 5 um, length 150 mm, inner diameter 4.6 mm) (Wako Pure Chemical Industries) → Wakosil-II 5C18 RS (particle diameter 5 um, length 250 mm, inner diameter 4.6 mm) (sum Three columns were connected in tandem in the order of Kojun Pharmaceutical).
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 (21.0 min) → 1.5 mL / min (55.0 min) → 1.0 mL / min (56.0 min) → 1.0 mL / min (60.0 min)
Elution method: Isocratic
Detection wavelength: abs 260 nm
Analysis time: 60 min
Sample injection volume: 5 uL
Sample cooler temperature: 2 ° C
Preparation method of analytical sample: The dried yeast extract obtained by the present invention was dissolved and adjusted with a mobile phase of 75 mM ammonium dihydrogen phosphate (pH 2.3) to a final concentration of 1% (w / w). . Thereafter, the insoluble material was filtered through a DISMIC 13CP020AS 0.22 μm filter (ADVANTEC) attached to a syringe and subjected to HPLC.
Standard substances used for quantitative analysis: β-NMN (Sigma-Aldrich), NAD (Sigma-Aldrich) From the calibration curve shown in FIG. 8, the content of β-NMN in the dried yeast extract obtained by the present invention Was calculated.
ＨＰＬＣにより測定
ポンプ、デガッサ－： Chromaster 5110 (日立ハイテクサイエンス 社)
オ－トサンプラ－： Chromaster 5210 (日立ハイテクサイエンス 社)
UV－VIS 検出器： Chromaster 5420 (日立ハイテクサイエンス 社)
カラムオ－ブン： Chromaster 5310 (日立ハイテクサイエンス 社)
移動相： 75 mM リン酸二水素アンモニウム (pH 2.3) (和光純薬 社)。アスピレ－タ－で60 分間の脱気処理を行った。
カラム： Wakosil－II 5C18 RS (粒子径5 um、長さ30 mm、内径4.6 mm) (和光純薬 社)
→ Wakosil－II 5C18 RS (粒子径5 um、長さ150 mm、内径4.6 mm) (和光純薬 社) → Wakosil－II 5C18 RS (粒子径5 um、長さ250 mm、内径4.6 mm) (和光純薬 社)の順でカラムをタンデムに３連結した。
カラムオ－ブン温度： 26℃
流速： 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 (21.0 min) → 1.5 mL/min (55.0 min) → 1.0 mL/min (56.0 min) → 1.0 mL/min (60.0 min)
溶出法： アイソクラティック
検出波長： abs 260 nm
分析時間： 60 min
サンプル注入量： 5 uL
サンプルク－ラ－温度： 2℃
分析検体の調製法： 本発明によって得られた酵母エキス乾燥物を移動相７５ ｍＭ リン酸二水素アンモニウム （ｐＨ ２．３）で終濃度１％ （ｗ／ｗ）になるように溶解、調整した。その後、シリンジに装着したDISMIC 13CP020AS ０．２２ μｍフィルタ－ (ADVANTEC 社)によって不溶性物質をろ過し、HPLCに供した。
定量分析に使用した標準物質： β－ＮＭＮ （Sigma－Aldrich 社）、ＮＡＤ （Sigma－Aldrich 社）図８に示す検量線から、本発明によって得られた酵母エキス乾燥物中のβ－ＮＭＮの含量を算出した。 (Conditions for quantitative analysis of β-NMN and NAD in dry matter obtained by optimal reaction)
Measuring pump by HPLC, degasser: Chromaster 5110 (Hitachi High-Tech Science)
Autosampler: Chromaster 5210 (Hitachi High-Tech Science)
UV-VIS detector: Chromaster 5420 (Hitachi High-Tech Science)
Column oven: Chromaster 5310 (Hitachi High-Tech Science)
Mobile phase: 75 mM ammonium dihydrogen phosphate (pH 2.3) (Wako Pure Chemical Industries, Ltd.). Deaeration treatment was performed for 60 minutes with an aspirator.
Column: Wakosil-II 5C18 RS (
→ Wakosil-II 5C18 RS (
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 (21.0 min) → 1.5 mL / min (55.0 min) → 1.0 mL / min (56.0 min) → 1.0 mL / min (60.0 min)
Elution method: Isocratic
Detection wavelength: abs 260 nm
Analysis time: 60 min
Sample injection volume: 5 uL
Sample cooler temperature: 2 ° C
Preparation method of analytical sample: The dried yeast extract obtained by the present invention was dissolved and adjusted with a mobile phase of 75 mM ammonium dihydrogen phosphate (pH 2.3) to a final concentration of 1% (w / w). . Thereafter, the insoluble material was filtered through a DISMIC 13CP020AS 0.22 μm filter (ADVANTEC) attached to a syringe and subjected to HPLC.
Standard substances used for quantitative analysis: β-NMN (Sigma-Aldrich), NAD (Sigma-Aldrich) From the calibration curve shown in FIG. 8, the content of β-NMN in the dried yeast extract obtained by the present invention Was calculated.
（酵母の培養）
Candida utilis IAM 4264を予めＹＰＤ培地（酵母エキス１％、ポリペプトン２％、グルコ－ス２％）を含む三角フラスコで種母培養し、これを３０ Ｌ容発酵槽に１８ Ｌ培地に１～２％植菌した。培地組成は、グルコ－ス４％、燐酸一アンモニウム０．３％、硫酸アンモニウム０．１６１％、塩化カリウム０．１３７％、硫酸マグネシウム０．０８％、硫酸銅１．６ ｐｐｍ、硫酸鉄１４ ｐｐｍ、硫酸マンガン１６ ｐｐｍ、硫酸亜鉛１４ ｐｐｍを用いた。培養条件は、ｐＨ４．０、培養温度３０℃、通気量１ ｖｖｍ、撹拌６００ ｒｐｍで行い、アンモニアを添加しｐＨのコントロ－ルを行った。１６時間の菌体培養した後、培養液を回収し、遠心分離により集菌し、１８０ｇの湿潤酵母菌体を得た。
得られた酵母菌体を蒸留水に懸濁して遠心分離を繰り返すことで洗浄した。乾燥固形分濃度８２．８８ ｇ／Ｌとなるよう蒸留水に再懸濁した。この時ｐＨ ５．８であった。 (Yeast culture)
Candida utilis IAM 4264 is pre-cultured in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in advance, and this is 1-2% in 18 L medium in a 30 L fermentor. Inoculated. Medium composition is 4% glucose, 0.3% monoammonium phosphate, 0.161% ammonium sulfate, 0.137% potassium chloride, 0.08% magnesium sulfate, 1.6 ppm copper sulfate, 14 ppm iron sulfate, Manganese sulfate 16 ppm and zinc sulfate 14 ppm were used. The culture conditions were pH 4.0, culture temperature 30 ° C., aeration rate 1 vvm, stirring 600 rpm, and ammonia was added to control the pH. After culturing the cells for 16 hours, the culture solution was collected and collected by centrifugation to obtain 180 g of wet yeast cells.
The obtained yeast cells were washed by suspending them in distilled water and repeating the centrifugation. Resuspended in distilled water to a dry solids concentration of 82.88 g / L. At this time, the pH was 5.8.
Candida utilis IAM 4264を予めＹＰＤ培地（酵母エキス１％、ポリペプトン２％、グルコ－ス２％）を含む三角フラスコで種母培養し、これを３０ Ｌ容発酵槽に１８ Ｌ培地に１～２％植菌した。培地組成は、グルコ－ス４％、燐酸一アンモニウム０．３％、硫酸アンモニウム０．１６１％、塩化カリウム０．１３７％、硫酸マグネシウム０．０８％、硫酸銅１．６ ｐｐｍ、硫酸鉄１４ ｐｐｍ、硫酸マンガン１６ ｐｐｍ、硫酸亜鉛１４ ｐｐｍを用いた。培養条件は、ｐＨ４．０、培養温度３０℃、通気量１ ｖｖｍ、撹拌６００ ｒｐｍで行い、アンモニアを添加しｐＨのコントロ－ルを行った。１６時間の菌体培養した後、培養液を回収し、遠心分離により集菌し、１８０ｇの湿潤酵母菌体を得た。
得られた酵母菌体を蒸留水に懸濁して遠心分離を繰り返すことで洗浄した。乾燥固形分濃度８２．８８ ｇ／Ｌとなるよう蒸留水に再懸濁した。この時ｐＨ ５．８であった。 (Yeast culture)
Candida utilis IAM 4264 is pre-cultured in an Erlenmeyer flask containing YPD medium (
The obtained yeast cells were washed by suspending them in distilled water and repeating the centrifugation. Resuspended in distilled water to a dry solids concentration of 82.88 g / L. At this time, the pH was 5.8.
＜実施例１＞
（酵母エキスの抽出）
上記菌体懸濁液に９５℃のウォ－タ－バス下で緩やかに懸濁液を撹拌しながら９０℃まで昇温し、撹拌しながら１０分間の抽出処理を行う。抽出処理後、サンプリングした菌体懸濁液２５ ｍＬを氷中下で冷却し、１００００ ｒｐｍで１０分、４℃下で遠心分離し、上清を取得した。沈殿物に上清と等量の超純水を添加、懸濁し、再び度遠心分離し、上清を取得した。最初の遠心分離で取得した上清と2回目に遠心分離して取得した上清をプ－ルし、超純水で５０ ｍＬにフィルアップしたものを抽出液とした。 <Example 1>
(Yeast extract extraction)
The cell suspension is heated to 90 ° C. while gently stirring the suspension in a water bath at 95 ° C. and subjected to extraction for 10 minutes while stirring. After the extraction treatment, 25 mL of the sampled cell suspension was cooled under ice and centrifuged at 10,000 rpm for 10 minutes at 4 ° C. to obtain a supernatant. The same amount of ultrapure water as the supernatant was added to the precipitate, suspended, and centrifuged again to obtain the supernatant. The supernatant obtained by the first centrifugation and the supernatant obtained by the second centrifugation were pooled, and the extract was filled up to 50 mL with ultrapure water.
（酵母エキスの抽出）
上記菌体懸濁液に９５℃のウォ－タ－バス下で緩やかに懸濁液を撹拌しながら９０℃まで昇温し、撹拌しながら１０分間の抽出処理を行う。抽出処理後、サンプリングした菌体懸濁液２５ ｍＬを氷中下で冷却し、１００００ ｒｐｍで１０分、４℃下で遠心分離し、上清を取得した。沈殿物に上清と等量の超純水を添加、懸濁し、再び度遠心分離し、上清を取得した。最初の遠心分離で取得した上清と2回目に遠心分離して取得した上清をプ－ルし、超純水で５０ ｍＬにフィルアップしたものを抽出液とした。 <Example 1>
(Yeast extract extraction)
The cell suspension is heated to 90 ° C. while gently stirring the suspension in a water bath at 95 ° C. and subjected to extraction for 10 minutes while stirring. After the extraction treatment, 25 mL of the sampled cell suspension was cooled under ice and centrifuged at 10,000 rpm for 10 minutes at 4 ° C. to obtain a supernatant. The same amount of ultrapure water as the supernatant was added to the precipitate, suspended, and centrifuged again to obtain the supernatant. The supernatant obtained by the first centrifugation and the supernatant obtained by the second centrifugation were pooled, and the extract was filled up to 50 mL with ultrapure water.
＜実施例２＞
（β－ＮＭＮならびにＮＡＤ標準物質のマススペクトル）
前記の測定条件でβ－ＮＭＮとＮＡＤのマススペクトルを取得した。β－ＮＭＮは図２で示すように、β－ＮＭＮのm/z 334をプレカ－サ－イオンにMS/MSを行うと、Nicotinamide (Nam)のm/z 123のフラグメントイオンを検出した。このプレカ－サ－イオン m/z 335、フラグメントイオンにm/z 123を前記の条件でLC－MS/MSを行ったところ、図２に示すように、3.0 minにβ－ＮＭＮを検出した。ＮＡＤは図３で示すように、ＮＡＤのm/z ６６４をプレカ－サ－イオンにMS/MSを行うと、Adenosine diphosphate ribose (ＡＤＰ－ｒｉｄｏｓｅ)由来のm/z ５２４とm/z ５４２、Adenosine diphosphate (ADP)のm/z 428、Ribose 5－phosphate (R5P)のm/z 232のフラグメントイオンを検出した。このプレカ－サ－イオン m/z 664、フラグメントイオンにm/z 524、542を前記の条件でLC－MS/MSを行ったところ、図３に示すように、８．５ｍｉｎにＮＡＤを検出した。 <Example 2>
(Mass spectrum of β-NMN and NAD standard)
Under the above measurement conditions, mass spectra of β-NMN and NAD were obtained. As shown in FIG. 2, when β / NMN was subjected to MS / MS using β / NMN m / z 334 as a precursor ion, a fragment ion of Nicotinamide (Nam) m /z 123 was detected. When this precursor ion m / z 335 and fragment ion m / z 123 were subjected to LC-MS / MS under the above-mentioned conditions, β-NMN was detected at 3.0 min as shown in FIG. As shown in FIG. 3, NAD is obtained by performing MS / MS using NAD m / z 664 as a precursor ion, m / z 524 and m / z 542 derived from Adenosine diphosphate ribose (ADP-ridose), Adenosine Diphosphate (ADP) m / z 428 and Ribose 5-phosphate (R5P) m / z 232 fragment ions were detected. When this precursor ion m / z 664 and fragment ions m / z 524 and 542 were subjected to LC-MS / MS under the above-mentioned conditions, NAD was detected at 8.5 min as shown in FIG. .
（β－ＮＭＮならびにＮＡＤ標準物質のマススペクトル）
前記の測定条件でβ－ＮＭＮとＮＡＤのマススペクトルを取得した。β－ＮＭＮは図２で示すように、β－ＮＭＮのm/z 334をプレカ－サ－イオンにMS/MSを行うと、Nicotinamide (Nam)のm/z 123のフラグメントイオンを検出した。このプレカ－サ－イオン m/z 335、フラグメントイオンにm/z 123を前記の条件でLC－MS/MSを行ったところ、図２に示すように、3.0 minにβ－ＮＭＮを検出した。ＮＡＤは図３で示すように、ＮＡＤのm/z ６６４をプレカ－サ－イオンにMS/MSを行うと、Adenosine diphosphate ribose (ＡＤＰ－ｒｉｄｏｓｅ)由来のm/z ５２４とm/z ５４２、Adenosine diphosphate (ADP)のm/z 428、Ribose 5－phosphate (R5P)のm/z 232のフラグメントイオンを検出した。このプレカ－サ－イオン m/z 664、フラグメントイオンにm/z 524、542を前記の条件でLC－MS/MSを行ったところ、図３に示すように、８．５ｍｉｎにＮＡＤを検出した。 <Example 2>
(Mass spectrum of β-NMN and NAD standard)
Under the above measurement conditions, mass spectra of β-NMN and NAD were obtained. As shown in FIG. 2, when β / NMN was subjected to MS / MS using β / NMN m / z 334 as a precursor ion, a fragment ion of Nicotinamide (Nam) m /
＜実施例３＞
（至適酵素反応温度の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度をそれぞれ３０℃、３５℃、４０℃、４５℃、５０℃、５２℃、５３℃、５４℃、５５℃、５６℃、５８℃、６０℃、６２℃、６５℃、７０℃ 、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨで６．５に調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各温度でのβ－ＮＭＮの生成率は、図４に示すようになった。反応温度が４５～６０℃、特に５５℃付近で最も高いβ－ＮＭＮ生成率を示した。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 3>
(Examination of optimal enzyme reaction temperature)
Yeast was cultured and extracted in the same manner as in Example 1, and the temperature of the yeast extract was 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, respectively. , 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, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae ) After the addition amount, the enzyme reaction was carried out for 1 hour. The production rate of β-NMN at each temperature as measured by LC-MS was as shown in FIG. The highest β-NMN production rate was exhibited when the reaction temperature was 45-60 ° C, particularly around 55 ° C. The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
（至適酵素反応温度の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度をそれぞれ３０℃、３５℃、４０℃、４５℃、５０℃、５２℃、５３℃、５４℃、５５℃、５６℃、５８℃、６０℃、６２℃、６５℃、７０℃ 、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨで６．５に調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各温度でのβ－ＮＭＮの生成率は、図４に示すようになった。反応温度が４５～６０℃、特に５５℃付近で最も高いβ－ＮＭＮ生成率を示した。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 3>
(Examination of optimal enzyme reaction temperature)
Yeast was cultured and extracted in the same manner as in Example 1, and the temperature of the yeast extract was 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, respectively. , 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, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae ) After the addition amount, the enzyme reaction was carried out for 1 hour. The production rate of β-NMN at each temperature as measured by LC-MS was as shown in FIG. The highest β-NMN production rate was exhibited when the reaction temperature was 45-60 ° C, particularly around 55 ° C. The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
＜実施例４＞
（至適酵素反応pHの検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５４℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ４．０、ｐＨ４．５、ｐＨ５．０、ｐＨ５．５、ｐＨ６．０、ｐＨ６．５、ｐＨ７．０、ｐＨ７．５にそれぞれ調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各ｐＨでのβ－ＮＭＮの生成率は、図５に示すようになった。反応ｐＨが４．５～６．０付近の範囲、特にｐＨ５．０付近で最も高いβ－ＮＭＮ生成率を示した。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 4>
(Examination of optimum enzyme reaction pH)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was 54 ° C., the reaction pH was 9 N HCl or 9 N NaOH, pH 4.0, pH 4.5, pH 5.0, pH 5.5, The pH was adjusted to 6.0, pH 6.5, pH 7.0, and pH 7.5, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae was added, and the enzyme reaction was performed for 1 hour. The production rate of β-NMN at each pH as measured by LC-MS was as shown in FIG. The highest β-NMN production rate was exhibited when the reaction pH was in the range of 4.5 to 6.0, particularly around pH 5.0. The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
（至適酵素反応pHの検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５４℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ４．０、ｐＨ４．５、ｐＨ５．０、ｐＨ５．５、ｐＨ６．０、ｐＨ６．５、ｐＨ７．０、ｐＨ７．５にそれぞれ調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各ｐＨでのβ－ＮＭＮの生成率は、図５に示すようになった。反応ｐＨが４．５～６．０付近の範囲、特にｐＨ５．０付近で最も高いβ－ＮＭＮ生成率を示した。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 4>
(Examination of optimum enzyme reaction pH)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was 54 ° C., the reaction pH was 9 N HCl or 9 N NaOH, pH 4.0, pH 4.5, pH 5.0, pH 5.5, The pH was adjusted to 6.0, pH 6.5, pH 7.0, and pH 7.5, and 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae was added, and the enzyme reaction was performed for 1 hour. The production rate of β-NMN at each pH as measured by LC-MS was as shown in FIG. The highest β-NMN production rate was exhibited when the reaction pH was in the range of 4.5 to 6.0, particularly around pH 5.0. The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
＜実施例５＞
（酵素の金属イオン要求性の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、最終濃度１００ ｍＭになるように、塩化マンガン (MnCl2・4H2O)、塩化亜鉛 (ZnCl2)、塩化銅 (CuSO4・5H2O)、塩化マグネシウム (MgCl2・6H2O)、塩化カルシウム (CaCl2・2H2O)、塩化第二鉄 (FeCl3・6H2O)、エチレンジアミン四酢酸 (EDTA・2Na)をそれぞれ加えた。さらに、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各金属イオン存在下でのβ－ＮＭＮの生成率は、図６に示すようになった。反応液中のZnイオン、Cuイオン、Feイオンの存在はβ－NMNの生成が阻害されることが示された。さらに、反応液中にキレ－ト剤であるEDTAが存在している際にもβ－ＮＭＮの生成活性を示すことから、本酵素反応には金属化合物の添加は必要としない。β－ＮＭＮの生成率は、金属化合物未添加の酵素反応後の液中のβ－ＮＭＮのイオン強度を１００とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。
<Example 5>
(Examination of metal ion requirement of enzyme)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the final concentration was 100 mM. Manganese chloride (MnCl 2・ 4H 2 O), Zinc chloride (ZnCl 2) , Copper chloride (CuSO 4・ 5H 2 O), Magnesium chloride (MgCl 2・ 6H 2 O), Calcium chloride (CaCl 2・ 2H 2 O) , Ferric chloride (FeCl 3 · 6H 2 O), and ethylenediaminetetraacetic acid (EDTA · 2Na) were added. Furthermore, after adding 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae, the enzyme reaction was performed for 1 hour. The production rate of β-NMN in the presence of each metal ion, as measured by LC-MS, was as shown in FIG. The presence of Zn ions, Cu ions, and Fe ions in the reaction solution was shown to inhibit the formation of β-NMN. Furthermore, since EDTA, which is a chelating agent, is present in the reaction solution, it exhibits β-NMN-forming activity, so that the addition of a metal compound is not necessary for this enzyme reaction. The production rate of β-NMN is a relative value when the ionic strength of β-NMN in the solution after the enzyme reaction without addition of a metal compound is taken as 100. The ionic strength was measured by LC-MS.
（酵素の金属イオン要求性の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、最終濃度１００ ｍＭになるように、塩化マンガン (MnCl2・4H2O)、塩化亜鉛 (ZnCl2)、塩化銅 (CuSO4・5H2O)、塩化マグネシウム (MgCl2・6H2O)、塩化カルシウム (CaCl2・2H2O)、塩化第二鉄 (FeCl3・6H2O)、エチレンジアミン四酢酸 (EDTA・2Na)をそれぞれ加えた。さらに、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、1時間の酵素反応を行った。ＬＣ－ＭＳを用いた測定による、各金属イオン存在下でのβ－ＮＭＮの生成率は、図６に示すようになった。反応液中のZnイオン、Cuイオン、Feイオンの存在はβ－NMNの生成が阻害されることが示された。さらに、反応液中にキレ－ト剤であるEDTAが存在している際にもβ－ＮＭＮの生成活性を示すことから、本酵素反応には金属化合物の添加は必要としない。β－ＮＭＮの生成率は、金属化合物未添加の酵素反応後の液中のβ－ＮＭＮのイオン強度を１００とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。
<Example 5>
(Examination of metal ion requirement of enzyme)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the final concentration was 100 mM. Manganese chloride (MnCl 2・ 4H 2 O), Zinc chloride (ZnCl 2) , Copper chloride (CuSO 4・ 5H 2 O), Magnesium chloride (MgCl 2・ 6H 2 O), Calcium chloride (CaCl 2・ 2H 2 O) , Ferric chloride (FeCl 3 · 6H 2 O), and ethylenediaminetetraacetic acid (EDTA · 2Na) were added. Furthermore, after adding 0.1% (w / v) of the crude enzyme prepared from Rhizopus oryzae, the enzyme reaction was performed for 1 hour. The production rate of β-NMN in the presence of each metal ion, as measured by LC-MS, was as shown in FIG. The presence of Zn ions, Cu ions, and Fe ions in the reaction solution was shown to inhibit the formation of β-NMN. Furthermore, since EDTA, which is a chelating agent, is present in the reaction solution, it exhibits β-NMN-forming activity, so that the addition of a metal compound is not necessary for this enzyme reaction. The production rate of β-NMN is a relative value when the ionic strength of β-NMN in the solution after the enzyme reaction without addition of a metal compound is taken as 100. The ionic strength was measured by LC-MS.
＜実施例６＞
（至適酵素添加量、酵素反応時間の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、Rhizopus oryzaeから調製した粗酵素を０．０１０％（ｗ/ｖ）、０．０２５％（ｗ/ｖ）、０．０５０％（ｗ/ｖ）、０．１０％（ｗ/ｖ）、０．２５％（ｗ/ｖ）、０．５０％（ｗ/ｖ）、０．７５％（ｗ/ｖ）、１．０％（ｗ/ｖ）それぞれ添加量後、５時間の酵素反応を行った。反応中は３０分、１時間、２時間、３時間、４時間、５時間と１時間毎にサンプリングを行い、それぞれのβ－ＮＭＮ生成の継時変化を調べた。ＬＣ－ＭＳを用いた測定による、各添加量ならびに反応時間における酵母エキス中のβ－ＮＭＮの生成率は、図７に示すようになった。粗酵素の添加量が０．２５％(w/v)以上となると生成されたβ－ＮＭＮは30分～1時間付近で分解されていくことが示された。０．０１０％（ｗ/ｖ）と０．０２５％（ｗ/ｖ）の添加では緩やか且つリニアにβ－ＮＭＮの生成を示した。最もβ－ＮＭＮの生成率が高かったのが０．１０％（ｗ/ｖ）添加の３時間の酵素反応であった。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 6>
(Examination of optimal enzyme addition amount and enzyme reaction time)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the crude enzyme prepared from Rhizopus oryzae was adjusted to 0. .010% (w / v), 0.025% (w / v), 0.050% (w / v), 0.10% (w / v), 0.25% (w / v), 0 Enzymatic reaction was carried out for 5 hours after addition of 50% (w / v), 0.75% (w / v) and 1.0% (w / v) respectively. During the reaction, sampling was performed every 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 1 hour, and changes over time in the production of each β-NMN were examined. The production rate of β-NMN in the yeast extract at each addition amount and reaction time as measured by LC-MS was as shown in FIG. It was shown that when the amount of the crude enzyme added was 0.25% (w / v) or more, the produced β-NMN was degraded in the vicinity of 30 minutes to 1 hour. Addition of 0.010% (w / v) and 0.025% (w / v) showed the formation of β-NMN slowly and linearly. The highest β-NMN production rate was the enzyme reaction for 3 hours with the addition of 0.10% (w / v). The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
（至適酵素添加量、酵素反応時間の検討）
実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、Rhizopus oryzaeから調製した粗酵素を０．０１０％（ｗ/ｖ）、０．０２５％（ｗ/ｖ）、０．０５０％（ｗ/ｖ）、０．１０％（ｗ/ｖ）、０．２５％（ｗ/ｖ）、０．５０％（ｗ/ｖ）、０．７５％（ｗ/ｖ）、１．０％（ｗ/ｖ）それぞれ添加量後、５時間の酵素反応を行った。反応中は３０分、１時間、２時間、３時間、４時間、５時間と１時間毎にサンプリングを行い、それぞれのβ－ＮＭＮ生成の継時変化を調べた。ＬＣ－ＭＳを用いた測定による、各添加量ならびに反応時間における酵母エキス中のβ－ＮＭＮの生成率は、図７に示すようになった。粗酵素の添加量が０．２５％(w/v)以上となると生成されたβ－ＮＭＮは30分～1時間付近で分解されていくことが示された。０．０１０％（ｗ/ｖ）と０．０２５％（ｗ/ｖ）の添加では緩やか且つリニアにβ－ＮＭＮの生成を示した。最もβ－ＮＭＮの生成率が高かったのが０．１０％（ｗ/ｖ）添加の３時間の酵素反応であった。β－ＮＭＮ生成率は、酵素未反応の抽出液中のβ－ＮＭＮのイオン強度を１とした際の相対値を示す。イオン強度はＬＣ－ＭＳによって測定した。 <Example 6>
(Examination of optimal enzyme addition amount and enzyme reaction time)
Yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and the crude enzyme prepared from Rhizopus oryzae was adjusted to 0. .010% (w / v), 0.025% (w / v), 0.050% (w / v), 0.10% (w / v), 0.25% (w / v), 0 Enzymatic reaction was carried out for 5 hours after addition of 50% (w / v), 0.75% (w / v) and 1.0% (w / v) respectively. During the reaction, sampling was performed every 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 1 hour, and changes over time in the production of each β-NMN were examined. The production rate of β-NMN in the yeast extract at each addition amount and reaction time as measured by LC-MS was as shown in FIG. It was shown that when the amount of the crude enzyme added was 0.25% (w / v) or more, the produced β-NMN was degraded in the vicinity of 30 minutes to 1 hour. Addition of 0.010% (w / v) and 0.025% (w / v) showed the formation of β-NMN slowly and linearly. The highest β-NMN production rate was the enzyme reaction for 3 hours with the addition of 0.10% (w / v). The β-NMN production rate indicates a relative value when the ionic strength of β-NMN in the extract not reacted with the enzyme is 1. The ionic strength was measured by LC-MS.
＜実施例７＞
（至適酵素反応条件でのβ－ＮＭＮ含量の測定）
Candida utilis IAM 4264を用いて、実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、３時間の至適反応を行った。その後、乾燥工程によりβ－ＮＭＮを含む酵母エキスの乾燥物を得た。乾燥物は前記の測定条件で定量分析を行った。クロマトグラムに関しては、図９に示すようになった。至適酵素反応条件における酵母エキス中のβ－ＮＭＮの含量は、図８の検量線粗用いてβ－ＮＭＮを定量した所、図１０に示すように乾燥固形分あたり２．１５％（ｗ/ｗ）であった。反応前では、ＮＡＤが乾燥固形分あたり２．２９％（ｗ／ｗ）含まれており、反応後にはＮＡＤが乾燥固形分あたり０．１８％（ｗ／ｗ）と減少するとともに、β－ＮＭＮが生成されている。このことから、本酵素反応によるβ－ＮＭＮの生成は、図１１に示すような機構が予測される。 <Example 7>
(Measurement of β-NMN content under optimal enzyme reaction conditions)
Using Candida utilis IAM 4264, yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and Rhizopus After adding 0.1% (w / v) of crude enzyme prepared from oryzae, an optimal reaction was performed for 3 hours. Thereafter, a dried yeast extract containing β-NMN was obtained by a drying step. The dried product was quantitatively analyzed under the above measurement conditions. The chromatogram is as shown in FIG. The β-NMN content in the yeast extract under the optimal enzyme reaction conditions was determined by quantifying β-NMN using the calibration curve crude of FIG. 8, and as shown in FIG. w). Before the reaction, NAD was contained at 2.29% (w / w) per dry solid content, and after the reaction, NAD decreased to 0.18% (w / w) per dry solid content, and β-NMN Has been generated. From this, the mechanism as shown in FIG. 11 is predicted for the production of β-NMN by this enzyme reaction.
（至適酵素反応条件でのβ－ＮＭＮ含量の測定）
Candida utilis IAM 4264を用いて、実施例１と同様に酵母を培養、抽出処理し、酵母抽出液の温度を５５℃、反応ｐＨを９ Ｎ ＨＣｌまたは９ Ｎ ＮａＯＨでｐＨ５．０に調整し、Rhizopus oryzaeから調製した粗酵素を０．１％（ｗ/ｖ）添加量後、３時間の至適反応を行った。その後、乾燥工程によりβ－ＮＭＮを含む酵母エキスの乾燥物を得た。乾燥物は前記の測定条件で定量分析を行った。クロマトグラムに関しては、図９に示すようになった。至適酵素反応条件における酵母エキス中のβ－ＮＭＮの含量は、図８の検量線粗用いてβ－ＮＭＮを定量した所、図１０に示すように乾燥固形分あたり２．１５％（ｗ/ｗ）であった。反応前では、ＮＡＤが乾燥固形分あたり２．２９％（ｗ／ｗ）含まれており、反応後にはＮＡＤが乾燥固形分あたり０．１８％（ｗ／ｗ）と減少するとともに、β－ＮＭＮが生成されている。このことから、本酵素反応によるβ－ＮＭＮの生成は、図１１に示すような機構が予測される。 <Example 7>
(Measurement of β-NMN content under optimal enzyme reaction conditions)
Using Candida utilis IAM 4264, yeast was cultured and extracted in the same manner as in Example 1, the temperature of the yeast extract was adjusted to 55 ° C., the reaction pH was adjusted to pH 5.0 with 9 N HCl or 9 N NaOH, and Rhizopus After adding 0.1% (w / v) of crude enzyme prepared from oryzae, an optimal reaction was performed for 3 hours. Thereafter, a dried yeast extract containing β-NMN was obtained by a drying step. The dried product was quantitatively analyzed under the above measurement conditions. The chromatogram is as shown in FIG. The β-NMN content in the yeast extract under the optimal enzyme reaction conditions was determined by quantifying β-NMN using the calibration curve crude of FIG. 8, and as shown in FIG. w). Before the reaction, NAD was contained at 2.29% (w / w) per dry solid content, and after the reaction, NAD decreased to 0.18% (w / w) per dry solid content, and β-NMN Has been generated. From this, the mechanism as shown in FIG. 11 is predicted for the production of β-NMN by this enzyme reaction.
＜実施例８＞
市販されているRhizopus属の酵素を用いて、β－ＮＭＮの生成を確認した。実施例１と同様に、酵母抽出液を作成し、市販酵素「リリパーゼA-10D」（ナガセケムテックス社製）を０．１％（ｗ/ｖ）添加量後、３時間の至適反応を行った。反応条件は、ｐＨ５．０、温度５５℃で行った。その結果、β－ＮＭＮを２．０１％（ｗ／ｗ）含む酵母エキスを得ることができた。 <Example 8>
Production of β-NMN was confirmed using a commercially available enzyme belonging to the genus Rhizopus. In the same manner as in Example 1, a yeast extract was prepared, and 0.1% (w / v) of commercially available enzyme “Lipase A-10D” (manufactured by Nagase ChemteX) was added for 3 hours for optimal reaction. went. The reaction conditions were pH 5.0 andtemperature 55 ° C. As a result, a yeast extract containing 2.01% (w / w) of β-NMN could be obtained.
市販されているRhizopus属の酵素を用いて、β－ＮＭＮの生成を確認した。実施例１と同様に、酵母抽出液を作成し、市販酵素「リリパーゼA-10D」（ナガセケムテックス社製）を０．１％（ｗ/ｖ）添加量後、３時間の至適反応を行った。反応条件は、ｐＨ５．０、温度５５℃で行った。その結果、β－ＮＭＮを２．０１％（ｗ／ｗ）含む酵母エキスを得ることができた。 <Example 8>
Production of β-NMN was confirmed using a commercially available enzyme belonging to the genus Rhizopus. In the same manner as in Example 1, a yeast extract was prepared, and 0.1% (w / v) of commercially available enzyme “Lipase A-10D” (manufactured by Nagase ChemteX) was added for 3 hours for optimal reaction. went. The reaction conditions were pH 5.0 and
食用として安全な酵母からβ－ＮＭＮを得ることが出来、医薬品だけでなく、機能性食品、栄養補助食品としても摂取可能であり、本発明品の摂取により、β－ＮＭＮの有する機能性を得ることが出来る。
Β-NMN can be obtained from edible safe yeast and can be ingested not only as a pharmaceutical product, but also as a functional food and a dietary supplement. By ingesting the product of the present invention, the functionality possessed by β-NMN is obtained. I can do it.
Β-NMN can be obtained from edible safe yeast and can be ingested not only as a pharmaceutical product, but also as a functional food and a dietary supplement. By ingesting the product of the present invention, the functionality possessed by β-NMN is obtained. I can do it.
Claims (6)
- 次の理化学的性質を有する酵素を用いて反応させる工程を含むβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。
（１）作用：ニコチンアミドアデニンジヌクレオチドをニコチンアミドモノヌクレオチドに加水分解する。
（２）至適ｐＨ：ｐＨ４．５～６．０。
（３）至適温度：４５℃～６０℃。
（４）由来：Rhizopus 属に属する微生物 A method for producing a β-nicotinamide mononucleotide-containing yeast extract comprising a step of reacting with an enzyme having the following physicochemical properties.
(1) Action: Hydrolyzes nicotinamide adenine dinucleotide to nicotinamide mononucleotide.
(2) Optimum pH: pH 4.5 to 6.0.
(3) Optimal temperature: 45 ° C to 60 ° C.
(4) Origin: Microorganisms belonging to the genus Rhizopus - 請求項１のβ‐ニコチンアミドモノヌクレオチド含有酵母エキスが、β‐ニコチンアミドモノヌクレオチドを乾燥固形分あたり2.0％(ｗ/ｖ)以上含有するβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。 The method for producing a β-nicotinamide mononucleotide-containing yeast extract, wherein the β-nicotinamide mononucleotide-containing yeast extract according to claim 1 contains 2.0% (w / v) or more of β-nicotinamide mononucleotide per dry solid content.
- 請求項１の製造方法において、使用する酵素が、Rhizopus 属に属する微生物から抽出したタンパク質であるβ‐ニコチンアミドモノヌクレオチド含有酵母エキスの製造方法。 The method for producing a yeast extract containing β-nicotinamide mononucleotide, wherein the enzyme used is a protein extracted from a microorganism belonging to the genus Rhizopus.
- 請求項１～３の製造方法により得られたβ‐ニコチンアミドモノヌクレオチド含有酵母エキスを含有する組成物。 A composition containing a β-nicotinamide mononucleotide-containing yeast extract obtained by the production method according to claims 1 to 3.
- 次の理化学的性質を有する酵素。
（１）作用：ニコチンアミドアデニンジヌクレオチドをニコチンアミドモノヌクレオチドに加水分解する。
（２）至適ｐＨ：ｐＨ４．５～６．０。
（３）至適温度：４５℃～６０℃。
（４）由来：Rhizopus 属に属する微生物 An enzyme with the following physicochemical properties:
(1) Action: Hydrolyzes nicotinamide adenine dinucleotide to nicotinamide mononucleotide.
(2) Optimum pH: pH 4.5 to 6.0.
(3) Optimal temperature: 45 ° C to 60 ° C.
(4) Origin: Microorganisms belonging to the genus Rhizopus - β‐ニコチンアミドモノヌクレオチドを乾燥固形分あたり2.0％(ｗ/ｖ)以上含有するβ‐ニコチンアミドモノヌクレオチド高含有酵母エキス。 A β-nicotinamide mononucleotide-rich yeast extract containing β-nicotinamide mononucleotide at 2.0% (w / v) or more per dry solid content.
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Cited By (6)
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|---|---|---|---|---|
| WO2019181961A1 (en) * | 2018-03-20 | 2019-09-26 | 三菱商事ライフサイエンス株式会社 | METHOD FOR PRODUCING β-NMN AND COMPOSITION CONTAINING SAME |
| WO2020129997A1 (en) | 2018-12-18 | 2020-06-25 | 帝人株式会社 | Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same |
| JP2021008447A (en) * | 2019-07-01 | 2021-01-28 | ベイジン サイエンスキュアキャンサー テクノロジー カンパニー リミテッド | Health keeping product composition applicable to adult female, elderly, and sub-healthy people |
| WO2021070829A1 (en) | 2019-10-11 | 2021-04-15 | 国立大学法人静岡大学 | Lactic acid bacteria that produce nicotinamide riboside, and lactic acid bacteria that produce nicotinamide mononucleotide and nicotinamide riboside |
| CN114099556A (en) * | 2020-08-25 | 2022-03-01 | 大江生医股份有限公司 | Yeast powder rich in nicotinamide mononucleotide, preparation method and application thereof |
| WO2022138656A1 (en) * | 2020-12-25 | 2022-06-30 | 株式会社大阪ソーダ | Method for producing nicotinamide mononucleotide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0856611A (en) * | 1994-08-29 | 1996-03-05 | Cosmo Shokuhin Kk | Production of yeast extract |
| WO2015069860A1 (en) * | 2013-11-06 | 2015-05-14 | President And Fellows Of Harvard College | Biological production of nad precursors and analogs |
-
2017
- 2017-05-18 JP JP2018518359A patent/JP6997080B2/en active Active
- 2017-05-18 WO PCT/JP2017/018709 patent/WO2017200050A1/en active Application Filing
- 2017-05-19 TW TW106116539A patent/TW201742924A/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0856611A (en) * | 1994-08-29 | 1996-03-05 | Cosmo Shokuhin Kk | Production of yeast extract |
| WO2015069860A1 (en) * | 2013-11-06 | 2015-05-14 | President And Fellows Of Harvard College | Biological production of nad precursors and analogs |
Non-Patent Citations (1)
| Title |
|---|
| MAGNI GIULIO ET AL.: "ENZYMOLOGY OF NAD+ SYNTHESIS", ADVANCES IN ENZYMOLOGY AND RELATED AREAS OF MOLECULAR BIOLOGY, vol. 73, 22 November 2006 (2006-11-22), pages l35 - 182 * |
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| EP3770268A4 (en) * | 2018-03-20 | 2021-12-22 | Mitsubishi Corporation Life Sciences Limited | METHOD FOR PRODUCING ß-NMN AND COMPOSITION CONTAINING SAME |
| CN111954718A (en) * | 2018-03-20 | 2020-11-17 | 三菱商事生命科学株式会社 | Method for producing beta-NMN and composition containing same |
| WO2019181961A1 (en) * | 2018-03-20 | 2019-09-26 | 三菱商事ライフサイエンス株式会社 | METHOD FOR PRODUCING β-NMN AND COMPOSITION CONTAINING SAME |
| JPWO2019181961A1 (en) * | 2018-03-20 | 2021-04-01 | 三菱商事ライフサイエンス株式会社 | Method for producing β-NMN and its contained composition |
| JP7416684B2 (en) | 2018-03-20 | 2024-01-17 | 三菱商事ライフサイエンス株式会社 | Method for producing β-NMN and composition containing it |
| WO2020129997A1 (en) | 2018-12-18 | 2020-06-25 | 帝人株式会社 | Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same |
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| JP2021008447A (en) * | 2019-07-01 | 2021-01-28 | ベイジン サイエンスキュアキャンサー テクノロジー カンパニー リミテッド | Health keeping product composition applicable to adult female, elderly, and sub-healthy people |
| WO2021070829A1 (en) | 2019-10-11 | 2021-04-15 | 国立大学法人静岡大学 | Lactic acid bacteria that produce nicotinamide riboside, and lactic acid bacteria that produce nicotinamide mononucleotide and nicotinamide riboside |
| CN114099556A (en) * | 2020-08-25 | 2022-03-01 | 大江生医股份有限公司 | Yeast powder rich in nicotinamide mononucleotide, preparation method and application thereof |
| WO2022042615A1 (en) * | 2020-08-25 | 2022-03-03 | 大江生医股份有限公司 | Yeast powder rich in nicotinamide mononucleotide, preparation method therefor, and application thereof |
| US11833116B2 (en) | 2020-08-25 | 2023-12-05 | Tci Co., Ltd. | Preparation method of yeast powder rich in nicotinamide mononucleotide, yeast powder, and methods for improving skin condition, hair health, antiinflammation, cardiovascular health, antioxidation, antiaging and/or relieving body fatigue |
| WO2022138656A1 (en) * | 2020-12-25 | 2022-06-30 | 株式会社大阪ソーダ | Method for producing nicotinamide mononucleotide |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201742924A (en) | 2017-12-16 |
| JP6997080B2 (en) | 2022-02-03 |
| JPWO2017200050A1 (en) | 2019-04-11 |
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