CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/755,969, filed November 5, 2018, the disclosure of which is incorporated herein by reference m its entirety.

FIELD

[0002] The present disclosure relates generally to media and methods for culturing yeast cells to produce one or more NAD+-related and precursor compounds. The present disclosure also relates to compositions comprising yeast ceils that produce one or more NAD+-related and precursor compounds and methods of using the compositions, which may provide benefits for the treatment and prevention of mitochondrial diseases and extending lifespan.

BACKGROUND

[0003] Nicotinamide adenine dinucleotide (NAD+ or NADH) has important biological functions m cellular metabolism. Decline of NAD+ levels has been attributed to the development of many diseases and conditions associated with metabolic dysfunction.

Compounds such as nicotinamide (NAM), nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid mononucleotide (NAMN), nicotinamide mononucleotide (NMN), and nicotinic acid riboside (NAR) are involved in NAD+ biosynthesis and metabolism; thus, they can be used to modulate NAD+ levels. Additionally, NAD+ can be further converted to nicotinamide adenine dinucleotide phosphate (NADP+ or NADPH), which also play a role in cellular metabolism.

[0004] Provided herein are media and methods for culturing yeast cells to produce NAD+-related and precursor compounds. Also provided herein are compositions comprising these yeast or extracts thereof comprising one or more of the NAD+-related and precursor compounds, which may provide benefits for the treatment and prevention of mitochondrial diseases and extending

BRIEF SUMMARY

[0005] Provided herein is a medium for culturing yeast cells, comprising at least about 6 mM nicotinic acid (NA) or a salt thereof. In some embodiments, the medium comprises about 6 mM - 20 mM NA. In some embodiments, the medium further comprises nicotinamide (NAM).

In some embodiments, the medium comprises at least about 6 mM NAM. In some embodiments, the medium comprises about 6 mM - 20 mM NAM.

[0006] Also provided herein is a medium for culturing yeast cells, comprising at least about 6 mM nicotinamide (NAM) or a salt thereof. In some embodiments, the medium comprises about 6 mM - 20 mM N AM. In some embodiments, the medium further comprises NA. In some embodiments, the medium comprises at least about 6 mM of NA. In some embodiments, the medium comprises about 6 mM - 20 mM NA.

[0007] In some embodiments, the medium further comprises a flavonoid. In some embodiments, the flavonoid is fisetin, quercetin, apigenin, or iuteolin. In some embodiments, the medium comprises quercetin. In some embodiments, the medium comprises fisetin. In some embodiments, the medium further comprises a fruit or vegetable extract, a fruit or vegetable concentrate, or a fruit or vegetable powder. In some embodiments, the medium comprises further comprises piperlongumine.

[0008] In some embodiments, the medium is for culturing yeast cells that are not genetically modified organisms (GMOs). In some embodiments, the medium is for culturing yeast cells that are Saccharomyces cerevisiae.

[0009] Also provided herein is a method of screening a yeast strain, the method comprising (a) culturing yeast ceils from a yeast strain in a medium comprising 20 mM - 60 mM

NA and/or 20 mM - 60 mM NAM; and (b) determining if the yeast cells grow in the medium. In some embodiments, the method further comprises repeating step (a) and step (b) with a plurality of yeast strains. In some embodiments, the method further comprises (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucleotide (NAD+), nicotinic acid adenine dinucleotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast cells or the medium after step (a). In some embodiments, the method further comprises repeating step (c) with a plurality of yeast strains.

[001Q] In some embodiments, the method further comprises (d) selecting a yeast strain that grows in the medium and whose yeast cells produce one or more of NMN, NR, NAR,

NAD+, NAAD, or NAMN at a desired level after step (a). In some embodiments, the method further comprises (d) selecting a yeast strain that grows in the medium and whose yeast cells produce one or more of NMN, NR, NAR, NAD+, NAAD, NA, NAM, NADH, NADP+,

NADPH, NAMN, MNA, trigonelline, or NAMN at a desired level after step (a). In some embodiments, the desired level is an amount sufficient to produce a dry composition comprising an extract of the yeast cells comprising at least 1% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w) produced by the yeast cells. In some embodiments, the desired level is an amount sufficient to produce a dr composition comprising an extract of the yeast cells comprising one or more of: (a) 0.05% - 1% NAM, (b) 0.5% - 1.2% NA, (c) 0.05% - 0.23%

NAD+, (d) 0.002% - 0.02% NADH, (e) 0.02% - 0.08% NADP+, (f) 0.01% - 0.2% NADPH, (g) 0.02% - 0.07% NMN, (h) 0.01% - 0.2% NAMN, (i) 0.001% - 0.009% NR, (j) 0.002%-0.015% NAR, (k) 0.0005%-0.0Q4% MNA, and (1) 0.0Ql%-0.02% trigonelline (w/w). In some embodiments, the extract was produced by mechanically lysing the yeast ceils. In some embodiments, the extract was produced without the addition of methanol, ethanol, or hazardous chemicals. Also provided herein is a yeast cell from a yeast strain selected in a method described herein.

[0011] Also provided herein is a method of culturing yeast cells, the method comprising culturing yeast cells m a medium described herein. Also provided herein is a method of preparing NMN and/or NR, comprising culturing yeast cells in a medium described herein. In some embodiments, the method further comprises extracting NMN and/or NR from the yeast cells.

[0012] In some embodiments, the yeast cells are a plurality of a yeast cell from a yeast strain selected in a method described herein. In some embodiments, the yeast cells are

Saccharomyces cerevisiae. In some embodiments, the yeast cells are not genetically modified organisms (GMOs).

[0013] In some embodiments, the method comprises culturing the yeast cells for at least 2 hours. In some embodiments, the method comprises culturing the yeast cells for at least 30 hours. In some embodiments, the culturing comprises stirring and/or aerating the medium. In some embodiments, the culturing further comprises adding one or more of yeast nutrient, soya peptone, or a sugar after at least 2 hours of culturing. In some embodiments, the temperature of the medium is about 75° F - about 85° F during culturing.

[0014] In some embodiments, the method further comprises inactivating the yeast cells. In some embodiments, the inactivating comprises sonicating, microwaving, or pasteurizing the yeast cells. In some embodiments, the method further comprises concentrating the yeast cells. In some embodiments, the concentrating comprises centrifuging or filtering the yeast cells. In some embodiments, the method does not comprise adding methanol, ethanol, or hazardous chemicals to the yeast cells or an extract thereof.

[0015] In some embodiments, the method further comprises lysing the yeast cells to produce an extract. In some embodiments, the lysing is mechanical lysing. In some embodiments, the method further comprises centrifuging or filtering the extract. In some embodiments, the method further comprises freeze drying the extract.

[0016] Also provided herein is a composition comprising the yeast cells produced by a method described herein, or an extract thereof, or an extract produced by a method described herein. Also provided herein is a composition comprising yeast cells or an extract thereof comprising at least 500 mM NR and/or at least 500 mM NMN. Also provided herein is a composition comprising yeast cells or an extract thereof comprising at least 0.4% NR (w/w) and/or at least 0.4% NMN (w/w). In some embodiments, the composition comprises at least 0.4% NR (w/w) and at least 0.4% NMN (w/w). In some embodiments, the yeast cells produced at least 50% of the NR and/or NMN in the composition. Also provided herein is a composition comprising yeast cells or an extract thereof comprising one or more of: (a) 0.05% - 1% NAM, (b) 0.5% - 1.2% NA, (c) 0.05% - 0.23% NAD+, (d) 0.002% - 0.02% NADH, (e) 0.02% - 0.08% NADP+, (f) 0 01 % - 0.2% NADPH, (g) 0.02% - 0.07% NMN, (h) 0.01% - 0.2% NAMN, (i) 0 001 % - 0.009% NR, (j) 0.002%-0.015% NAR, (k) 0.0005%-0.004% MNA, and (3) 0.001 %- 0.02% trigonelline (w/w). Also provided herein is a composition comprising yeast cells or an extract thereof comprising a total amount of Q.1%-0.5% (w/w) of NAD+, NMN, NAMN, NR, and NAR. Also provided herein is a composition comprising yeast cells or an extract thereof comprising a total amount of 0.5% -2.25% (w/w) of NAM, NA, NAD+, NMN, NAMN, NR, and NAR.

|Ό017] In some embodiments, the yeast cells in the composition are not genetically modified organisms (GMOs). In some embodiments, the yeast cells in the composition are Saccharomyces cerevisiae. In some embodiments, the yeast cells in the composition have been inactivated. In some embodiments, the yeast cells were inactivated by somcation, microwaving, or pasteurization.

[0018] In some embodiments, the composition comprises NAD+. In some embodiments, the composition comprises at least 0.25% NAD+ (w/w). In some embodiments, the yeast cells produced at least 50% of the NAD+. In some embodiments, the composition further comprises NA, NAR, NAAD, NAMN, or combinations thereof. In some embodiments, the composition comprises at least 1% NA (w/w). In some embodiments, the composition further comprises a compound or plurality of compounds that are capable of lowering total plasma homocysteine in a subject. In some embodiments, the composition further comprises serine, vitamin Be,, vitamin E>9, and vitamin Bn. In some embodiments, the composition further comprises a f!avonoid. In some embodiments, the flavonoid is fisetin, quercetin, apigenin, and luteolin. In some embodiments, the composition comprises quercetin, fisetin, or quercetin and fisetin. In some embodiments, the composition further comprises piperlongumine, vitamin B ₂ , JV-acetylcysteine, 5~adenosylmethionine (SAM), trimethylglycine, or combinations thereof. In some embodiments, the composition further comprises a fruit or vegetable extract, a fruit or vegetable concentrate, a fruit or vegetable powder, tuna or an extract thereof, salmon or an extract thereof, bulgur or an extract thereof, barely or an extract thereof, or a combination thereof.

[0019] In some embodiments, the composition is a paste or a powder. In some embodiments, the composition has been freeze dried.

[0020] Also provided herein is a pharmaceutical composition comprising the

composition described herein and a pharmaceutically acceptable excipient. Also provided herein is a dietary supplement, food product, or medical food comprising a composition described herein or a pharmaceutical composition described herein.

[0021] Also provided herein is a method of modulating blood NAD+ level comprising administering to a subject an effective amount of a composition described herein, a

pharmaceutical composition described herein, a dietary supplement described herein, a food product described herein, or a medical food described herein. Also provided herein is a method of extending lifespan of a subject comprising administering to the subject an effective amount of a composition described herein, a pharmaceutical composition described herein, a dietary- supplement described herein, a food product described herein, or a medical food described herein. Also provided herein is a method of improving healthspan of a subject comprising administering to the subject an effective amount of a composition described herein, a

pharmaceutical composition described herein, a dietary supplement described herein, a food product described herein, or a medical food described herein. Also provided herein is a method of enhancing or maintaining muscle growth or performance comprising administering to a subject an effective amount of a composition described herein, a pharmaceutical composition described herein, a dietary supplement described herein, a food product described herein, or a medical food described herein. Also provided herein is a method of treating or preventing a mitochondrial disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of a composition described herein, a pharmaceutical composition described herein, a dietary supplement described herein, a food product described herein, or a medical food described herein.

DETAILED DESCRIPTION

[0022] NAD+ has important functions in cellular bioenergetics and adaptive stress responses. Decreasing NAD+ levels have been associated with metabolism-related diseases, including neurodegenerative diseases, cardiovascular diseases, muscle atrophy, and age-related conditions. Maintenance of NAD+ levels is especially important for cells with higher energy demands. Therefore, molecules involved in NAD+ biosynthesis and consumption are of particular interest in modulating NAD+ level as a way to combat these diseases and conditions. Nicotinamide (NAM) and nicotinic acid (NA) can be produced in large quantities; however production of NAD+ and other NAD+ precursors (such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN)) is more difficult and expensive, as well as can result in potentially toxic byproducts.

[0023] Previous attempts to use microorganisms to produce NAD+ and/or NAD+ precursors other than NAM or NA nearly always required the use of genetically modified organisms (GMOs) (Marinescu et al Nature Scientific Reports, 2018, 8: 12278; US2009202680). The use of GMOs, especially in food products, has been contentious and is problematic for a large number of people.

[0024] There is a need to produce large quantities of NAD+ and NAD+ precursors, such as NMN and NR, in a safe and/or natural way.

/ [0025] The following description is presented to enable a person of ordinary' skill in the art to make and use the various embodiments. Descriptions of specific compositions, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary' ^· skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

[0027] As used herein and in the appended claims, the singular forms“a,”“an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as“solely,”“only” and the like m connection with the recitation of claim elements, or use of a“negative” limitation.

[0028] As used herein, the terms“including,”“containing,” and“comprising” are used in their open, non-limiting sense. It is understood that embodiments of the invention described herein include“consisting” and/or“consisting essentially of’ embodiments. The term“consists essentially of’ means excluding other materials that contribute to function, unless otherwise defined herein. Nonetheless, such other materials may be present, collectively or individually, in trace amounts. [0029] To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term“about” It is understood that, whether the term“about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, description referring to“about X” includes description of“X”.

[0030] As used herein, the term“treat” or“treatment” refers to an approach for obtaining a beneficial or desired result, including clinical results. For purposes of this disclosure, beneficial or desired results include, but are not limited to: reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition, alleviating a symptom and/or diminishing the extent of a symptom and/or preventing a worsening of a symptom associated with a condition, arresting the development of a disease, symptom, or condition, relieving the disease, symptom, or condition, causing regression of the disease, disorders, conditions, or symptom (in terms of severity or frequency of negative symptoms), or stopping the symptoms of the disease or condition. Beneficial or desired results can also be slowing, halting, or reversing the progressive course of a disease or condition.

[0031]“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease or condition in a subject that may be predisposed to the disease or condition but has not yet been diagnosed with the disease or condition. In some embodiments, the provided compositions are used to delay development of a disease or condition or to slow the progression of a disease or condition.

[0032] The terms“nicotinic acid,”“niacin,”“NA,” and“vitamin Bs” are used interchangeably herein. Similarly , the terms“nicotinamide,”“NAM,” and“niacinamide” are used interchangeably herein. [0033] Except as otherwise noted, the methods and techniques of the present embodiments are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specificati on.

[0034] Except as otherwise noted, percentages described herein are % weight/weight (%w/w).

[0035] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described m the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to particular method steps, reagents, or conditions are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed.

[0036] Where any compound recited herein may also exist as a corresponding salt, solvate or hydrate, the use of such salts, solvates or hydrates in place of or in addition to the recited compound is also encompassed by the present invention.

[0037] A“pharmaceutically acceptable salt” is a salt form that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See generally Berge et al. (1977) J Pharm. Sci. 66, 1 -19. Particular pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response.

[0038] A“hazardous chemical” is a chemical that is categorized as a health hazard by the Globally Harmonized System of Classification and Labelling of Chemicals and/or the United States Occupational Safety and Health Administration standards. [0039] The skilled person understands that in the context of compositions for administration to the human or animal body, for minerals such as magnesium, potassium, calcium and strontium, stated dosages and weights refer to the quantity of elemental metal, not to the weight of the compound containing the metal. Herein, where a weight or dosage of such a mineral is specified, this refers to the weight or dosage of the particular element and not the compound which contains it, unless the context clearly implies otherwise, or unless explicitly stated otherwise.

[0040] NAD+ has important functions in cellular bioenergetics and adaptive stress responses. Decreasing NAD+ levels have been associated with metabolism-related diseases, including neurodegenerative diseases, cardiovascular diseases, muscle atrophy, and age-related conditions. Maintenance of NAD+ levels is especially important for cells with higher energy demands. Therefore, molecules involved in NAD+ biosynthesis and consumption are of particular interest in modulating NAD+ level as a way to combat these diseases and conditions.

Nicotinamide adenine dinucleotide (NAD+)

[0041] NAD+ can be synthesized from a variety of sources. Major precursors for NAD+ biosynthesis include, but are not limited to, nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), nicotinamide (NAM), nicotinic acid adenine dinucleotide, nicotinic acid mononucleotide, trigonelline, 1 -methylnicotinamide (MNA) and nicotinic acid riboside ( NAR)

[0042] Depending on the bioavailability of these precursors, NAD+ can be

biosynthesized from NA in the Preiss-Handler pathway; or it can be produced from NAM, NR, and NMN in the salvage pathway. In the Preiss-Handler pathway, NA can be converted to nicotinic acid mononucleotide, and then to nicotinic acid adenine dinucleotide, which can produce NAD+ enzymatically. In the salvage pathway, NR and NAM can be converted to NMN which can then produce NAD+ enzymatically. Fang et al. (2017) Trends Mol Med. 23, 899. NAR can also be generated from NA through NMN formation and is an integral part of NAD metabolism. Kulikova et al (2015) ,/. Bio. Chern. 290, 27124. NAD+ can be further converted to NADPH and NADP, another two major reagents and coenzymes in metabolism, especially in a multitude of redox reactions.

Nicotinic acid (NA); Nicotinamide riboside (NR); Nicotinamide mononucleotide (NMN);

Nicotinamide (NAM); Nicotinic acid riboside (NAR); Nicotinamide adenine dinucleotide phosphate (NADP)

Nicotinic acid adenine dinis eotide (NAAD); Nicotinic acid mononucleotide (NAMN); Trigonelline; l-meihylnicotinamide (MNA).

[0043] NMN is a direct precursor of NAD+. In common biosynthesis pathways, NR, NAM, and NA need to be converted to NMN in order to produce NAD+. As such, NMN may be a more potent NAD+ modulator compared to the other NAD+ precursors. In some cases, NMN is extracellularly degraded to Nicotinamide Riboside prior to cellular active transpor uptake.

See e.g., Nikiforov et al (2011) J. Biol. ( ^’ hem. 286, 21767; Grozio et al (2013). In some cases, NMN may act as a sustained-release prodrug formulation for Nicotinamide Riboside, extending its pharmacokinetic life and pharmacodynamics activity over time. See e.g., J. Biol. Chem. 288, 25938; Social! et al . (2016) Oncotarget 7, 2968. After NMN supplementation, NMN can make its way through the liver intact and bloodstream into muscle and is metabolized to NAD+ within 30 minutes. Mills et al. (2016) Cell Me tab. 24, 795. And NMN was shown to be retained m the body for longer than NAM Kawamura et al (2016) J. Nutr. Sci. Vitaminol 62, 272.

[0044] In some aspects, provided herein are media for culturing yeast cells. In some embodiments, provided herein are media for use in a method described herein. In some embodiments, the medium comprises nicotinic acid (NA) or a salt thereof. In some embodiments, the medium comprises at least about 1 mM NA, such as at least about 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, or 18 mM NA. In some embodiments, the medium comprises at least about 6 mM NA. In some embodiments, the medium comprises about 1 mM - 60 mM NA, such as about 2 mM - 60 mM, 2 5 mM - 60 mM, 3 mM - 60 mM, 4 mM - 60 mM, 5 mM - 60 mM, 6 mM - 60 mM, 8 mM - 60 mM, 10 - 60 mM, 20 - 60 mM, 30 - 60 mM, or 40 - 60 mM NA. In some embodiments, the medium comprises 6 mM - 60 mM NA. In some embodiments, the medium comprises 15 mM - 20 mM NA. In some embodiments, the medium comprises about 1 mM - 20 mM NA, such as about 2 mM - 20 mM, 2, 5 mM - 20 mM, 3 mM - 20 mM, 4 mM - 20 mM, 5 mM - 20 mM, 6 mM - 20 mM, 8 mM - 20 mM, or 10 - 20 mM NA. In some embodiments, the medium comprises 6 mM - 20 mM NA.

[0045] In some embodiments, the medium comprises nicotinamide (NAM) or a salt thereof. In some embodiments, the medium comprises at least about 1 mM NAM, such as at least about 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, or 18 mM NAM. In some embodiments, the medium comprises at least about 6 mM NAM. In some embodiments, the medium comprises about 1 mM - 60 mM NANI, such as about 2 mM - 60 mM, 2, 5 mM - 60 mM, 3 mM - 60 mM, 4 mM - 60 mM, 5 mM - 60 mM, 6 mM - 60 mM, 8 mM - 60 mM, 10 - 60 mM, 20 - 60 mM, 30 - 60 mM, or 40 - 60 mM NAM.

In some embodiments, the medium comprises 6 mM - 60 mM NAM. In some embodiments, the medium comprises 15 mM - 20 mM NAM. In some embodiments, the medium comprises about 1 mM - 20 mM NAM, such as about 2 mM - 20 mM, 2.5 mM - 20 mM, 3 mM - 20 mM, 4 mM - 20 mM, 5 mM - 20 mM, 6 mM - 20 mM, 8 mM - 20 mM, or 10 - 20 mM NAM. In some embodiments, the medium comprises 6 mM - 20 mM NAM.

[0046] In some embodiments, the medium comprises nicotinic acid (NA) or a salt thereof and nicotinamide (NAM) or a salt thereof. In some embodiments, the medium comprises at least about 1 mM NA, such as at least about 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 niM, 16 mM, or 18 mM NA. In some embodiments, the medium comprises at least about 6 mM NA. In some embodiments, the medium comprises about 1 mM - 60 mM NA, such as about 2 mM - 60 mM, 2.5 mM - 60 mM, 3 mM - 60 mM, 4 mM - 60 mM,

5 mM— 60 mM, 6 mM— 60 mM, 8 mM— 60 mM, 10 - 60 mM, 20 - 60 mM, 30 - 60 mM, or 40 - 60 mM NA. In some embodiments, the medium comprises 6 rnM - 60 mM NA. In some embodiments, the medium comprises 15 mM - 20 mM NA. In some embodiments, the medium comprises about 1 mM - 20 mM NA, such as about 2 mM - 20 mM, 2.5 rnM - 20 mM, 3 mM - 20 mM, 4 mM - 20 mM, 5 mM - 20 mM, 6 rnM - 20 mM, 8 mM - 20 mM, or 10 - 20 mM NA. In some embodiments, the medium comprises 6 rnM - 20 mM NA. In some embodiments, the medium comprises at least about 1 mM NAM, such as at least about 1.5 rnM, 2 rnM, 2,5 rnM, 3 rnM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, or 18 mM NAM. In some embodiments, the medium comprises at least about 6 mM NAM. In some embodiments, the medium comprises about 1 mM - 60 mM NAM, such as about 2 mM - 60 mM, 2.5 mM - 60 mM, 3 mM - 60 rnM, 4 mM - 60 mM, 5 mM - 60 rnM, 6 mM - 60 mM, 8 mM - 60 rnM, 10 - 60 mM, 20 - 60 mM, 30 - 60 mM, or 40 - 60 mM NAM. In some embodiments, the medium comprises 6 mM - 60 mM NAM. In some embodiments, the medium comprises 15 mM - 20 mM NAM. In some embodiments, the medium comprises about 1 mM - 20 mM NAM, such as about 2 mM - 20 mM, 2.5 mM - 20 mM, 3 mM - 20 mM, 4 mM - 20 mM, 5 mM - 20 mM, 6 mM - 20 mM, 8 mM - 20 mM, or 10 - 20 mM NAM. In some embodiments, the medium comprises 6 mM - 20 mM NAM.

[0047] In some embodiments, the medium comprises at least about 1 mM NA, such as at least about 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, or 18 mM NA and at least about 1 mM NAM, such as at least about 1.5 mM, 2 mM, 2.5 mM, 3 M, 4 M, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, or 18 mM NAM. In some embodiments, the medium comprises at least about 6 mM NA and at least about

6 mM NAM. In some embodiments, the medium comprises about 1 mM - 60 mM NA, such as about 2 mM - 60 mM, 2.5 mM - 60 mM, 3 mM - 60 mM, 4 mM - 60 mM, 5 mM - 60 mM, 6 about 1 mM - 60 mM NAM, such as about 2 mM - 60 mM, 2.5 mM - 60 mM, 3 mM - 60 mM,

4 mM— 60 mM, 5 mM— 60 mM, 6 mM— 60 mM, 8 mM— 60 mM, 10— 60 mM, 20— 60 mM,

30 - 60 mM, or 40 - 60 mM NAM. In some embodiments, the medium comprises 6 mM - 60 mM NA and comprises 6 mM - 60 mM NAM. In some embodiments, the medium comprises 15 mM --- 20 mM NA and comprises 15 mM --- 20 mM NAM. In some embodiments, the medium comprises about 1 mM - 20 mM NA, such as about 2 mM - 20 mM, 2.5 mM - 20 mM, 3 rnM - 20 mM, 4 mM - 20 mM, 5 rnM - 20 mM, 6 mM - 20 mM, 8 rnM - 20 mM, or 10 - 20 mM NA and comprises about 1 mM - 20 mM NAM, such as about 2 rnM --- 20 rnM, 2.5 rnM --- 20 rnM, 3 rnM - 20 rnM, 4 mM - 20 mM, 5 rnM - 20 rnM, 6 mM - 20 mM, 8 rnM - 20 rnM, or 10 - 20 mM NAM. In some embodiments, the medium comprises 6 mM - 20 mM NA and comprises 6 rnM - 20 mM NAM.

[0048] In some embodiments, the yeast cells are any of the yeast cells described herein.

In some embodiments, the yeast cells are not genetically modified organisms (GMOs). A GMO is an organism that has had its DNA altered or modified in some way through genetic engineering. For example, a GMO may comprise a heterologous nucleotide sequence, or may have had a gene deleted, silenced, or mutated using genetic engineering techniques such as recombinant DNA or CRISPER. A GMO is not an organism developed purely by traditional breeding techniques of selecting an organism with a desired phenoty pe.

[0049] In some embodiments, the medium comprises a sugar. The sugar can act as an energy source for the yeast to grow. In some embodiments, the sugar is sucrose, glucose, fructose, lactose, maltose, dextrose, ribose, galactose, or combinations thereof. In some embodiments, the sugar is sucrose, glucose, fructose, lactose, maltose, or galactose. In some embodiments, the sugar is sucrose. In some embodiments, the medium comprises molasses. In some embodiments, the medium comprises syrup, such as corn syrup. In some embodiments, the medium comprises dry malt extract. In some embodiments the medium comprises 1 g/L - 150 g/L, dry malt extract, such as 50 g/L - 100 g/L, 100 g/L - 150 g/L, 1 g/L - 50 g/L, 10 g/L - 40 g/L, 15 g/L - 30 g/L, 15 g/L - 25 g/L, 50 g/L, 100 g/L, 120 g/L, or 20 g/L dry malt extract. In some embodiments, the medium comprises sugarcane or beet extract. In some embodiments, the medium has at least 3° Brix, such as at least 4°, 5°, 6°, 7°, 8°, 9°, or 10°. In some embodiments, the medium has 3°~2Q° Brix, such as 4°-20°, 4°-15°, 4°-10°, or 8°-12° Brix. One degree“Brix” is 1 gram of sucrose in 100 grams of solution.

[0050] In some embodiments, the medium comprises dextrose, rihose, or dextrose and ribose. In some embodiments, the medium comprises 1 g/L - 50 g/L dextrose, such as 10 g/L - 40 g/L, 15 g/L - 30 g/L, 15 g/L - 25 g/L, or 20 g/L dextrose. In some embodiments, the medium comprises 1 g/L - 50 g/L ribose, such as 5 g/L - 40 g/L, 5 g/L - 30 g/L, 5 g/L - 20 g/L, 5 g/L· - 15 g/L or 10 g/L, ribose

[0051] In some embodiments, the medium comprises a yeast nutrient. Yeast nutrient may promote healthy growth of the yeast cells and may include, but is not limited to, diammomum phosphate (DAP); amino acids and/or proteins; vitamins and/or minerals such as biotin, pantothenic acid, calcium, magnesium, potassium, and zinc; yeast ghosts, yeast hulls, and/or inactivated yeast; or combinations thereof. In some embodiments, the yeast nutrient is a combination of diammomum phosphate, essential vitamins and co-factors, nitrogen (amino acids, proteins, and peptides) and minerals. In some embodiments, the medium comprises 100 pg/L - 500 pg/L yeast nutrient, such as 100 pg/L --- 400 pg/L, 150 pg/L --- 350 pg/L, 200 pg/L - 300 pg/L, 250 pg/L - 300 pg/L or 264 pg/L In some embodiments, the medium comprises a peptone. In some embodiments, the peptone is a soya peptone. In some embodiments, the medium comprises at least 1 g/L peptone, such as 1.1 g/L, 1.2 g/L, or 1.3 g/L peptone. In some embodiments, the medium comprises 1 g/L - 50 g/L peptone, such as 10 g/L - 40 g/L, 15 g/L - 30 g/L, 15 g/L - 25 g/L, or 20 g/L peptone. In some embodiments, the medium comprises biotin. In some embodiments, the medium comprises at least 100 pg/L biotin, such as at least 200 pg/L or 250 pg/L biotin. In some embodiments, the medium comprises between 250 pg/L and 300 pg/L biotin. [0052] In some embodiments, the medium comprise a flavonoid or a combination of flavonoids, such as 2 or 3 flavonoids. A“flavonoid” is a compound having the general structure of a i 5-carbon skeleton, which consists of two phenyl rings and a heterocyclic ring. Examples of flavonoids include, but are not limited to, anthocyanidms, such as malvidin, pelargondin, peoidin, and cyanidin; flavanols, such as catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, proanthocyanidins, theaflavins, thearubigins, apiforol, and luteoforol; flavones, such as luteolin, apigenin, tangeritin, chrysin, and 6-hydroxyflavone; flavonols, such as quercetin and kaempferol, 3-hydroxyflavone, azaleatin, fisetm, galangin, gossypetm,

kaempferide, isorhamnetin, morin, myricetm, natsudaidain, pachypodol, rhamnazin, rhamnetin; flavonones or flavanones, such as hesperetm, eriodictyol, and narmgenin; and isoflavones, such as genistein, giycitem, orobol, and daidzein. In some embodiments, the flavonoid is fisetin, quercetin, apigenin, and luteolin. In some embodiments, the medium comprises quercetin. In some embodiments, the medium comprises fisetin. In some embodiments, the medium comprises quercetin and fisetin.

[0053] In some embodiments, the medium comprises at least about 0.05 g/L, such as at least about 1 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, or 4 g/L of a flavonoid or a combination of flavonoids. In some embodiments, the medium comprises at least about 4 g/L of a

combination of flavonoids. In some embodiments, the medium comprises 0.05 g/L - 10 g/L, such as 2 g/L - 8 g/L, 3 g/L- 7 g/L, 4 g/L-6 g/L of a flavonoid or a combination of flavonoids. In some embodiments, the medium comprises 10 mg/L - 100 mg/L fisetin, such as 20 mg/L - 90 mg/L, 25 mg/L - 80 mg/L, 30 mg/L - 70 mg/L, 30 mg/L - 50 mg/L, 50 mg/L- 70 mg/L, 30 mg/L, or 65 mg/L - 70 mg/L fisetin. In some embodiments, the medium comprises 10 mg/L - 100 mg/L quercetin, such as 20 mg/L - 90 mg/L, 25 mg/L - 80 mg/L, 30 mg/L - 70 mg/L, 30 mg/L - SO mg/L, 50 mg/L- 70 mg/L, 30 mg/L, or 65 mg/L - 70 mg/L quercetin.

[0054] In some embodiments, the medium comprises a plant extract, a plant concentrate, or a plant powder. In some embodiments, the medium comprises a fruit or vegetable extract, a fruit or vegetable concentrate, or a fruit or vegetable powder. [0055] In some embodiments, the medium comprises ellagic acid, tryptophan, adenine, benfotiamine, thiamin, resveratroi, piperlongumine, vitamin B ₂ , A-acetylcysteine, S~

adenosylmethionine (SAM), trimethylglycine, or combinations thereof. In some embodiments, the medium comprises ellagic acid, tryptophan, adenine, benfotiamine, thiamin, resveratroi, or combinations thereof. In some embodiments, the medium comprises tryptophan, adenine, benfotiamine, thiamin, resveratroi, or combinations thereof. In some embodiments, the medium comprises piperlongumine, vitamin Eh, /V-acetyleysteine, Ladenosylmethionine (SAM), trimethylglycine, or combinations thereof. In some embodiments, the medium comprises piperlongumine.

[0056] In some embodiments, the medium comprises adenine. In some embodiments, the medium comprises 500 pg/L, - 500 rng/L adenine, such as 750 pg/L - 400 mg/L, 1 mg/L - 300 mg/L, 1 rng/L - 200 mg/L, 4 mg/L - 150 mg/L, 4 mg/ L - 70 mg/L, or 65 rng/L --- 133 mg/L adenine.

[0057] In some embodiments, the medium comprises benfotiamine. In some

embodiments, the medium comprises 500 pg/L -1.5 mg/L benfotiamine, such as 600 pgL - 1.25 mg/L, 700 pg/L - 1 rng/L, 750 pg/L - 900 pg/L, or 800 pg/L benfotiamine.

[0058] In some embodiments, the medium comprises thiamin. In some embodiments, the medium comprises 500 pg/L -500 mg/L thiamin, such as 1 rng/L --- 20 mg/L, 2 mg/L - 15 mg/L,

3 mg/L - 10 mg/L, 5 mg/L - 10 mg/L, or 5 mg/L thiamin

[0059] In some embodiments, the medium comprises ellagic acid. In some

embodiments, the medium comprises 1 rng/L - 30 mg/L ellagic acid, such as 1 mg/L - 20 rng/L, 2 mg/L - 15 rng/L, 2 mg/L - 10 mg/L, 3 mg/L --- 5 mg/L, or 3 mg/L ellagic acid.

[0060] In some embodiments, the medium comprises resveratroi. In some embodiments, the medium comprises 10 mg/L - 1 g/L resveratroi, such as 30 mg/L - 800 mg/L, 40 rng/L - 600 mg/L, 50 mg/L - 500 mg/L, 60 mg/L - 450 mg/L, 65 mg/ L - 70 mg/L, or 350 mg/L - 450 mg/L resveratrol.

[0061] In some embodiments, the medium comprises tryptophan. In some embodiments, the medium comprises 1 mg/L - 30 mg/L thiamin, such as 3 mg/L - 20 mg/L, 5 mg/1, - 15 mg/L, 10 mg/L - 15 mg/L, 10 mg/L - 11 mg/L, or 10.2 mg/L tryptophan.

[0062] In some embodiments, the medium comprises a pH stabilizer. In some embodiments, the medium does not comprise a pH stabilizer. In some embodiments, the medium comprises 500 mg/L -1.5 g/L pH stabilizer, such as 600 mg/L - 1.25 g/L, 600 mg/L - 1 g/L, 600 mg/L - 800 mg/L, or 700 mg/L pH stabilizer.

[0063] Also provided herein are methods of culturing yeast cells. In some embodiments, the method comprising culturing yeast cells, such as any of the yeast cells described herein, in a medium described herein, such as a medium comprising NA and/or NAM.

[0064] Also provided herein are methods of preparing NMN, methods of preparing NR, methods of preparing NAD+, or methods of preparing combinations of NMN, NR, and NAD+. In some embodiments, the method comprises culturing yeast cells m a medium described herein. In some embodiments, the method comprises culturing yeast cells in a medium described herein and extracting one or more of NMN, NR, or NAD+ from the yeast cells.

[0065] In some embodiments, the method comprises culturing the yeast cells at room temperature. In some embodiments, the temperature of the medium during culturing is about 75° F - about 85° F. In some embodiments, the method comprises culturing the yeast cells for at least about 1 hour, such as at least about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 21 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, or 60 hours. In some embodiments, the method comprises culturing the yeast cells for 1 hour-3 days, such as 1 hour-2 days, 2 hours-2 days, 2 hours- 1 day, or 3 hours- 1 day. In some embodiments, the culturing comprises stirring or aerating the medium.

[0066] In some embodiments, the method further comprises adding one or more of yeast nutrient, soya peptone, or a sugar after at least 2 hours of culturing, such as after at least 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours or 18 hours. In some embodiments, the method further comprises adding one or more of yeast nutrient, soya peptone, or a sugar 2 hours - 20 hours after the culturing began, such as 3 hours - 20 hours, 4 hours - 20 hours, 5 hours - 20 hours, 6 hours - 20 hours, 7 hours - 20 hours, 8 hours - 20 hours, 9 hours --- 20 hours, 10 hours - 20 hours, 11 hours - 20 hours, or 12 hours - 20 hours after the culturing began

[0067] In some embodiments, the method further comprises inactivating the yeast cells. In some embodiments, the inactivating comprises sonicating, microwaving, or pasteurizing the yeast cells. In some embodiments, the inactivating comprises sonicating the yeast cells. In some embodiments, the inactivating comprises sonicating the yeast cells using power ultrasound. Power ultrasound has been successfully used to inactivate microorganisms in fruit juices (Adekunte et a!., Food Chem, 2010, 122(3): 500-507). In some embodiments, the inactivating comprises heating the yeast cells to at least a temperature of 71.5 °C (160 7 °F) for at least 15 seconds. In some embodiments, the inactivating comprises heating the yeast cells to at least 135 °C (275 °F) for at least 1 second. In some embodiments, the inactivation comprises freezing with liquid nitrogen or freeze-drying.

[0068] In some embodiments, the method further comprises concentrating the yeast cells. In some embodiments, the concentrating comprises filtering the yeast cells. The filters used to concentrate the yeast cells can include, but are not limited to size-exclusion filters, such as ultrafiltration filters. In some embodiments, the concentrating comprises centrifuging the yeast cells. In some embodiments, the method further comprises drying the yeast cells. In some embodiments, the method further comprises freeze drying the yeast cells. [0069] In some embodiments, the method further comprises lysing the yeast cells to produce an extract. In some embodiments, the lysing is chemical or mechanical lysing. In some embodiments, the lysing is mechanical lysing. In some embodiments, the mechanical lysing is performed by grinding the yeast cells with rotating blades. In some embodiments, the lysing is homogenization or sonication.

[007Q] In some embodiments, the method further comprises purifying the extract. In some embodiments, the pH of the extract is less than about 7, such as less than about 6.5, 6, 5.5, or 5. In some embodiments, the method further comprises centrifuging or filtering the extract.

In some embodiments, the purifying does not comprise chemically purifying the extract. For example, the purifying does not comprise the addition of methanol, ethanol, or hazardous chemicals. In some embodiments, the method further comprises freeze drying the extract. In some embodiments, the method further comprises freeze drying the liquid portion of the extract.

[0071] Accordingly, in some embodiments, the method comprises; (a) culturing yeast cells, such as any of the yeast cells described herein, in a medium described herein, such as a medium comprising NA and/or NAM; (b) concentrating the yeast cells of (a); (c) lysing the yeast cells of (b) to form an extract; (d) centrifuging or filtering the extract of (c); and (e) freeze drying the liquid portion of the extract of (d). Other orders and combinations of method steps described herein are also contemplated.

[0072] Also provided herein are methods of screening a yeast strain. In some

embodiments, the method comprises (a) culturing yeast cells from a yeast strain in a medium comprising at least about 9 raM (e.g., at least about 20 tiiM) NA and/or at least about 9 rnM (e.g., at least about 20 mM) NAM; and (b) determining if the yeast cells grow in the medium. In some embodiments, the method comprises fa) culturing yeast cells from a yeast strain in a medium comprising 9 mM - 60 mM NA (e.g., 20 mM - 60 mM) NA and/or 9 mM - 60 mM NAM (e.g., 20 mM - 60 mM) NAM; and (b) determining if the yeast cells grow in the medium. In some embodiments, the method further comprises repeating step fa) and step (b) with a plurality of yeast strains. In some embodiments, step (a) and step (b) are repeated 1 , 2, 3, 4, 5, 10, 15, 20,

25, 30, 40, 50, 100, 500, or 1000 times.

[0073] In some embodiments, the medium comprises at least about 9 mM NA, such as at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM NA. In some embodiments, the medium comprises about 9 mM - 60 mM NA, such as about 20 mM - 60 mM, 25 mM— 60 mM, 30 mM— 60 mM, 35 mM— 60 mM, 40 mM— 60 mM, 45 mM - 60 mM, or 50 mM - 60 mM, NA. In some embodiments, the medium comprises 9 mM - 30 mM NA. In some embodiments, the medium comprises 20 mM - 60 mM NA. In some embodiments, the medium comprises at least about 9 mM NAM, such as at least about 10 mM,

15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM NAM. In some embodiments, the medium comprises 9 mM - 30 mM NAM. In some embodiments, the medium comprises about 9 mM - 60 mM NAM, such as about 20 mM - 60 mM, 25 mM - 60 mM, 30 mM - 60 mM, 35 mM - 60 mM, 40 mM - 60 mM, 45 mM - 60 mM, or 50 mM - 60 mM, NAM. In some embodiments, the medium comprises 20 mM - 60 mM NA and 20 mM - 60 mM NAM. In some embodiments, the medium comprises 9 mM - 30 mM NA and 9 mM - 30 mM NAM

[0074] In some embodiments, the method comprises (a) culturing yeast ceils from a yeast strain in a medium comprising 20 mM - 60 mM NA and/or 20 mM - 60 mM NAM; (b) determining if the yeast cells grow in the medium; and (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucleotide (NAD+), nicotinic acid adenine dinucleotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast cells or the medium after step (a). In some embodiments, the method comprises repeating step (c) with a plurality of yeast strains. In some embodiments, step (c) is repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 500, or 1000 times. In some embodiments, step (c) comprises determining the concentration of NMN, NR, or NMN and NR in the yeast cells or the medium after step (a). [0075] In some embodiments, the method comprises (a) culturing yeast cells from a yeast strain in a medium comprising 9 mM - 30 mM NA and/or 9 mM - 30 mM NAM; (h) determining if the yeast cells grow in the medium; and (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucieotide (NAD+), nicotinic acid adenine dinucleotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast cells or the medium after step (a). In some embodiments, the method comprises repeating step (c) with a plurality of yeast strains. In some embodiments, step (c) is repeated 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 500, or 1000 times. In some embodiments, step (c) comprises determining the concentration of NMN, NR, or NMN and NR in the yeast cells or the medium after step (a).

[0076] In some embodiments, the method comprises (a) culturing yeast cells from a yeast strain in a medium comprising 20 mM - 60 mM NA and/or 20 mM --- 60 mM NAM; (b) determining if the yeast cells grow in the medium; (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucieotide (NAD+), nicotinic acid adenine dinucieotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast celis or the medium after step (a); and (d) selecting a yeast strain that grows m the medium and whose yeast celis produce one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN at a desired level after step (a). In some embodiments, step (d) comprises selecting a yeast strain that grows in the medium and whose yeast cells produce NMN, NR, or NMN and NR at a desired level after step (a). In some embodiments, the desired level is an amount sufficient to produce a dry composition comprising an extract of the yeast cells comprising at least 0.1%, such as at least 0.2%, 0.5%, 0.8%, 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w). In some embodiments, the desired level is an amount sufficient to produce a diy composition comprising the yeast ceils and 1 %- 10% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w), such as 1%-10% NMN, 1%-10% NR (w/w), or 1%- 10% NMN (w/w) and 1%-10% NR (w/w). [0077] In some embodiments, the method comprises (a) culturing yeast cells from a yeast strain in a medium comprising 9 mM - 30 mM NA and/or 9 mM - 30 mM NAM; (h) determining if the yeast cells grow in the medium; (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucleotide (NAD+), nicotinic acid adenine dinucleotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast cells or the medium after step (a); and (d) selecting a yeast strain that grows in the medium and whose yeast cells produce one or more of NMN, NR, NAR, NAD+, NAAD, or N AMN at a desired level after step (a). In some embodiments, step (d) comprises selecting a yeast strain that grows in the medium and whose yeast cells produce NMN, NR, or NMN and NR at a desired level after step (a). In some embodiments, the desired level is an amount sufficient to produce a dry composition comprising an extract of the yeast cells comprising at least 0.1%, such as at least 0.2%, 0.5%, 0.8%, 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w). In some embodiments, the desired level is an amount sufficient to produce a dry composition comprising the yeast ceils and 1 %- 10% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w), such as 1%-10% NMN, 1%-10% NR (w/w), or S %- 10% NMN (w/w) and 1 %- 10% NR (w/w).

|Ό078] Also provided herein are yeast cells. In some embodiments, the yeast cell is produced in a method described herein, such as a yeast cell produced in method of culturing yeast cells. In some embodiments, the yeast cell is from a yeast strain selected in a method of screening yeast cells discloses herein.

[0079] In some embodiments, the yeast cells described herein are yeast cells that produce NMN and/or NR when cultured in a medium comprising NA and/or NAM. In some

embodiments, the yeast ceils described herein are yeast cells that produce NAR, NAD+, NAAD, NAMN, or combinations thereof when cultured m a medium comprising NA and/or NAM. In some embodiments, the yeast cells described herein are yeast cells that produce NMN and/or NR in an amount sufficient to produce a dry' composition compri sing an extract of the yeast cells comprising 0.5%-10% NMN and/or NR (w/w) when cultured in a medium comprising NA and/or NAM.

[008Q] In some embodiments, the yeast cells are not genetically modified organisms (GMOs). A GMO is an organism that has had its DNA altered or modified in some way through genetic engineering. For example, a GMO may comprise a heterologous nucleotide sequence, or may have had a gene deleted, silenced, or mutated using genetic engineering techniques such as recombinant DNA or CRISPER. A GMO is not an organism developed purely by traditional breeding techniques of selecting an organism with a desired phenotype.

[0081] In some embodiments, the yeast cells are Saccharomyces (e.g., Saccharomyces cerevisiae). In some embodiments, the yeast cells are Kluyveromyces . In some embodiments, the yeast cells are Candida. In some embodiments, the yeast cells are not Candida. In some embodiments, the yeast cells are Pichia.

COMPOSITIONS

[0082] Also provided herein are compositions comprising yeast cells or extracts thereof. In some embodiments, the yeast cells are any of the yeast cell disclosed herein, such as a yeast cell produced by a method described herein. In some embodiments, the yeast cells have been inactivated. In some embodiments, the yeast cells have been inactivated by sonication, microwaving, or pasteurization. In some embodiments, the composition has been dried. In some embodiments, the composition has been freeze dried. In some embodiments, the composition is a paste or powder

[0083] In some embodiments, the composition is sterile. Sterile pharmaceutical formulations are compounded or manufactured according to pharmaceutical-grade sterilization standards (United States Pharmacopeia Chapters 797, 1072, and 1211; California Business & Professions Code 4127 7; 16 California Code of Regulations 1751, 21 Code of Federal

Regulations 211) known to those of skill in the art. [0084] In some embodiments, the composition comprises at least 500 mM NR, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NR. In some embodiments, the composition comprises at least 500 mM NMN, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NMN. In some embodiments, the composition comprises at least 500 mM NAD+, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NAD+. In some embodiments, the composition comprises at least 500 mM NR, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NR and at least 500 mM NMN, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NMN. In some embodiments, the composition comprises at least 500 m.M NR, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NR and at least 500 mM NAD+, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NAD+. In some embodiments, the composition comprises at least 500 mM NMN, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM,

5 mM, or 6 mM NMN and at least 500 mM NAD+, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NAD+. In some embodiments, the composition comprises at least 500 mM NR, such as at least 600 mM, 700 mM, 800 mM, 900 mM,

1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NR; at least 500 mM NMN, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NMN; and at least 500 mM NAD+, such as at least 600 mM, 700 mM, 800 mM, 900 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM NAD+.

[0085] In some embodiments, the composition comprises one or more of: (a) 0.05% - 1% NAM, (b) 0.5% - 1.2% NA, (c) 0.05% - 0.23% NAD+, (d) 0.002% - 0.02% NADH, (e) 0.02% - 0.08% NADP+, (f) 0.01% - 0.2% NADPH, (g) 0.02% - 0.07% NMN, (h) 0.01% - 0.2% NAMN, (i) 0.001% - 0.009% NR, (j) 0.002%-0.Q15% NAR, (k) 0.0005%-0.004% MNA, and (1) 0.001%-0.02% tngonellme (w/w). In some embodiments, the composition comprises (a) 0.05% - 1% NAM, such as 0.06% - 1% NAM, 0.07% - 1% NAM, 0.08% - 1% NAM, or 0.09% - 1% NAM. In some embodiments, the composition comprises (b) 0.5% - 1.2% NA, such as 0.6% - 1.2% NA, 0.7% - 1.2% NA, 0 8% - 1.2% NA, 0.9% - 1.2% NA, 1 % - 1.2% NA, or 1.1% - 1.2% NA. In some embodiments, the composition comprises (c) 0.05% - 0.23% NAD+, such as 0.08% - 0.23% NAD+, 0 1% - 0.23% NAD+, 0.12% - 0.23% NAD+, 0.14% - 0 23% NAD+, 0.16% - 0.23% NAD+, or 0.2% - 0.23% NAD+. In some embodiments, the composition composes (d) 0.002% - 0 02% NADH, such as 0 005% - 0.02% NADH, 0.01% - 0.02% NADH, or 0.015% - 0.02% NADH. In some embodiments, the composition comprises (e) 0.02% - 0.08% NADP+, such as 0.02% - 0.08% NADP+, 0.04% - 0.08% NADP+, 0.06% - 0.08% NADP+, or 0 07% - 0.08% NADP+. In some embodiments, the composition comprises (f) 0.01% - 0.2% NADPH, such as 0.03% - 0.2% NADPH, 0.06% - 0.2% NADPH, 0.09% - 0.2% NADPH, 0.12% - 0.2% NADPH, or 0.15% - 0 2% NADPH. In some embodiments, the composition comprises (g) 0.02% - 0.07% NMN, such as 0.03% - 0.07% NMN, 0.04% - 0.07% NMN, 0.05% - 0.07% NMN, or 0.06% - 0.07% NMN. In some embodiments, the composition composes (h) 0.01% - 0.2% NAMN, such as 0.03% - 0.2% NAMN, 0.06% - 0.2% NAMN, 0.1% - 0.2% NAMN, or 0.1 5% - 0.2% NAMN. In some embodiments, the composition comprises (i) 0.001% - 0.009% N such as 0.003% - 0.009% NR, 0.005% - 0.009% NR. 0.007% - 0.009% NR, or 0.008% - 0.009% NR. In some embodiments, the composition comprises (]) 0.002%-0.015% NAR, such as 0.005%-0.015% NAR. Q.008%-0.015% NAR, 0.01%-0.015% NAR, or 0.012%-0.015% NAR In some embodiments, the composition comprises (k) 0.0005%-0.004% IVINA, such as 0.001%- 0.004% AINA, 0.002%-0.004% MNA, or 0.003%-0.004% AINA. In some embodiments, the composition comprises (1) 0.001%-0.02% trigonelline, such as 0.005%-0.02% trigonelline, 0.0l%-0.02% trigonelline, or 0.015%-0.02% trigonelline.

[0086] In some embodiments, the composition comprises a total amount of 0.1%-0.5% (w/ ^' w) of NAD+, NMN, N MN, NR, and NAR. In some embodiments, the composition comprises a total amount of 0.5%-2.25% (w/w) of NAM, NA, NAD+, NMN, NAMN, NR, and NAR. In some embodiments, the composition comprises a total amount of 0. l%-0.6% (w/ ^' w) of NAD+, NADH, NADP+, NADPH, NMN, NAMN, NR NAR, MNA, and trigonelline. In some embodiments, the composition comprises a total amount of 0.5%-2.26% (w/w) of NAM, NA,

NAD+, NADH, NADP+, NADPH, NMN, NAMN, NR, NAR, AINA, and trigonelline. [0087] In some embodiments, the composition comprises at least 0.005% NR (w/w). In some embodiments, the composition comprises at least 0.5% NR (w/w) and/or at least 0.05 (e.g., 0.5%) NMN (w/w). In some embodiments, the composition comprises at least 0.005% NR (w/w) (e.g., 0.05% or 0.5% NR (w/w)), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NR (w/w). In some embodiments, the composition comprises at least 0 05 (e.g., 0 5%) NMN (w/w), such as at least 1%, 1.5%, 1 8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NMN (w/w). In some embodiments, the composition comprises at least 0.005% NR (w/w) (e.g., 0 05% or 0.5% NR (w/w)), such as at least 1%, 1.5%, 1 8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NR and comprises at least 0 05 (e.g., 0 5%) NMN (w/w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NMN (w/w). In some embodiments, the composition comprises at least 0.5% NAR (w/w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NAR (w/w). In some embodiments, the composition comprises at least 0.5% NAD+ (w/w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NAD+ (w/w). In some embodiments, the composition comprises at least 0.5% NAAD (w/w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NAAD (w/w). In some embodiments, the composition comprises at least 0.5% NAMN (w/w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NAMN (w/w). In some embodiments, the composition comprises 0.5%-10% NAMN (w/w). In some embodiments, the composition comprises at least 1% NR (w/w) and at least 1% NMN (w/w). In some embodiments, the composition comprises at least 0.005% NR (w/w) (e.g., 0.05% NR (w/w)) and at least 1% NMN (w/w).

[0088] In some embodiments, the composition comprises 0.01%-0.1% or 0.5%-10% NMN (w/w). In some embodiments, the composition comprises 0.001-0.0% or 0.5%-l0% NR (w/w). In some embodiments, the composition comprises 0.5%~ 10% NAR (w/w). In some embodiments, the composition comprises Q.5%-10% NAD+ (w/w). In some embodiments, the composition comprises 0.5%-10% NAAD (w/w). In some embodiments, the composition comprises 0.5%-10% NAMN (w/w).

90 [0089] In some embodiments, the composition comprises NR and the yeast cells produced at least 50% of the NR, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the NR In some embodiments, the compositi on comprises NR and the yeast cells produced 95% of the NR. In some embodiments, the composition does not comprise chemically synthesized NR. In some embodiments, 100% of the NR in the composition was either produced by the yeast cells or was produced by one or more other organisms, such as bacteria, plants, or animals. In some embodiments, 100% of the NR in the composition was either produced by the yeast cells or was produced by one or more other non-GMOs, such as bacteria, plants, or animals. In some embodiments, the composition comprises NR and the yeast cells produced 100% of the NR.

[0090] In some embodiments, the composition comprises NMN and the yeast ceils produced at least 50% of the NMN, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%,

97%, 99%, or 100% of the NMN. In some embodiments, the composition comprises NMN and the yeast cells produced 95% of the NMN . In some embodiments, the composition does not comprise chemically synthesized NMN. In some embodiments, 100% of the NMN in the composition was either produced by the yeast cells or was produced by one or more other organisms, such as bacteria, plants, or animals. In some embodiments, 100% of the NMN in the composition was either produced by the yeast cells or was produced by one or more other non- GMOs, such as bacteria, plants, or animals. In some embodiments, the composition comprises NMN and the yeast cells produced 100% of the NMN.

[0091] In some embodiments, the composition composes NAD+ and the yeast ceils produced at least 50% of the NAD+, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the NAD+. In some embodiments, the composition comprises NAD+ and the yeast cells produced 95% of the NAD+. In some embodiments, the composition does not comprise chemically synthesized NAD+. In some embodiments, 100% of the NAD+ m the composition was either produced by the yeast cells or was produced by one or more other organisms, such as bacteria, plants, or animals. In some embodiments, 100% of the NAD+ in the composition was either produced by the yeast cells or was produced by one or more other non-GMOs, such as bacteria, plants, or animals. In some embodiments, the composition comprises NAD+ and the yeast cells produced 100% of the NAD+.

[0092] In some embodiments, the composition comprises NAAD and the yeast cells produced at least 50% of the NAAD, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the NA AD. In some embodiments, the composition comprises NAAD and the yeast cells produced 95% of the NAAD. In some embodiments, the composition does not comprise chemically synthesized NA AD. In some embodiments, 100% of the NA AD in the composition was either produced by the yeast cells or was produced by one or more other organisms, such as bacteria, plants, or animals. In some embodiments, 100% of the N AAD in the composition was either produced by the yeast cells or was produced by one or more other non-GMOs, such as bacteria, plants, or animals. In some embodiments, the composition comprises NAAD and the yeast cells produced 100% of the NAAD.

[0093] In some embodiments, the composition further comprises NA. In some embodiments, the composition comprises at least 0.5% NA (w/'w), such as at least 1%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% NA (w/w). In some embodiments, the composition comprises 0.5%-10% NA (w/w).

[0094] In some embodiments, the composition further comprises a compound or plurality of compounds that are capable of lowering total plasma homocysteine in a subject. Examples of such compounds include, but are not limited to, serine, vitamin Be, vitamin B9, vitamin B12, and combinations thereof.

[0095] In some embodiments, the composition comprises a flavonoid or a combination of f!avonoids, such as 2 or 3 flavonoids. A“flavonoid” is a compound having the general structure of a 1 5-carbon skeleton, which consists of two phenyl rings and a heterocyclic ring. Examples of flavonoids include, but are not limited to, anthoeyamdins, such as malvidm, pelargondin, peoidm, and cyanidin; flavanols, such as cateehm, epicatechm gal late, epigal!ocatechin, epigallocatechm gall ate, proanthocyanidins, theaflavins, thearubigms, apiforol, and luteoforol; fiavones, such as luteoim, apigenin, tangeritin, chrysin, and 6-hydroxyflavone; flavonols, such as quercetin and kaempferol, 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, rhamnazin, rhamnetin; flavonones or f!avanonoes, such as hesperetin, eriodictyol, and naringenin; and isof!avones, such as genistein, giycitein, orobol, and daidzein. In some embodiments, the flavonoid is fisetin, quercetin, apigenin, and luteolin. In some embodiments, the composition comprises quercetin.

In some embodiments, the composition comprises fisetin. In some embodiments, the

composition comprises quercetin and fisetin.

[0096] In some embodiments, the composition comprises at least 0.5% of a flavonoid or a combination of a flavonoid (w/w), such as at least 1 %, 1 .5%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% of a flavonoid or a combination of a flavonoid (w/w). In some embodiments, the composition comprises 0.5%-10% of a flavonoid or a combination of a flavonoid (w/w).

[0097] In some embodiments, the composition further comprises a plant extract, a plant concentrate, or a plant powder. In some embodiments, the plant is bulgur, barely, rice, or combinations thereof. In some embodiments, the plant is tea. In some embodiments, the plant is green or black tea. In some embodiments, the medium comprises a fruit or vegetable extract, a fruit or vegetable concentrate, or a fruit or vegetable powder. In some embodiments, the fruit or vegetable is a broccoli, a cabbage, a cucumber, an edamame, an avocado, a tomato, a mushroom, a peanut, a pea, a squash, an asparagus, a potato, a lentil, a lima bean, or a combination thereof.

[0098] In some embodiments, the composition further comprises tuna or an extract thereof, salmon or an extract thereof, bulgur or an extract thereof, barely or an extract thereof, or a combination thereof.

[0099] In some embodiments, the composition comprises piperlongumme, vitamin B2, N- acetyleysteme, A-adenosylmethiomne (SAM), trimethylglycine, or combinations thereof. [0100] Also provided herein is a composition comprising NA, wherein the NA was produced by yeast cells described herein. Also provided herein is a composition comprising NA isolated from yeast cells described herein. In some embodiments, the composition is at least 50% pure NA, such as at least 60%, 70%, 80%, 90%, 95%, or 99% pure NA.

[0101] Also provided herein is a composition comprising NMN, wherein the NMN was produced by yeast cells described herein. Also provided herein is a composition comprising NMN isolated from yeast cells described herein. In some embodiments, the composition is at least 50% pure NMN, such as at least 60%, 70%, 80%, 90%, 95%, or 99% pure NMN.

[0102] Also provided herein is a composition comprising NAD+, wherein the NAD+ was produced by yeast cells described herein. Also provided herein is a composition comprising NAD+ isolated from y¾ast cells described herein. In some embodiments, the composition is at least 50% pure NAD+, such as at least 60%, 70%, 80%, 90%, 95%, or 99% pure NAD+.

Pharmaceutical Compositions

[0103] The present disclosure also relates to pharmaceutical compositions comprising the composition as described above and a pharmaceutically acceptable carrier, excipient, binder, or diluent.

[0104] In some embodiments, the pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients. A pharmaceutically-acceptable excipient is a substance that is non-toxic and otherwise biologically suitable for administration to a subject. Such excipients facilitate administration of the compositions described herein and are compatible with the active ingredient. Examples of pharmaceutically-acceptable excipients include stabilizers, lubricants, surfactants, diluents, anti-oxidants, binders, coloring agents, bulking agents, emulsifiers, or taste-modifying agents. In some embodiments, pharmaceutical compositions according to the embodiments are sterile compositions. Pharmaceutical compositions may be prepared using compounding techniques known or that become available to those skilled in the art. Sterile compositions are also contemplated by the embodiments, including compositions that are in accord with national and local regulations governing such compositions.

[0105] The pharmaceutical compositions and compositions described herein may be formulated as solutions, emulsions, suspensions, dispersions, or inclusion complexes such as eyc!odextrins m suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, bars, suppositories, sachets, dragees, granules, powders, powders for reconstitution, or capsules along with solid carriers according to conventional methods known m the art for preparation of various dosage forms. Pharmaceutical compositions provided herein may be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, or topical route, or by inhalation. In some embodiments, the compositions are formulated for intravenous or oral administration.

[0106] For oral administration, the pharmaceutical composition may be provided in a solid form, such as a tablet or capsule, or as a solution, emulsion, or suspension. To prepare the oral compositions, the pharmaceutical composition may be formulated to yield a dosage of the composition, e.g., from about 0.01 to about 20 mg/kg daily, or from about 20 to about 50 mg/kg daily or from about 50 to about 200 mg/kg daily. Oral tablets may include the active

ingredient(s) mixed with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvmyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and algmic acid are exemplar disintegrating agents.

Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid, or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating. [0107] Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, active ingredients) may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the active ingredient with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.

[0108] Liquids for oral administration may be in the form of suspensions, solutions, emulsions, or syrups, or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.

[0109] The compositions described herein may be formulated for rectal administration as a suppository. For parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, the agents provided herein may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit-dose form such as ampoules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation.

Illustrative infusion doses range from about 1 to 2000 pg/kg/minute of the composition admixed with a pharmaceutical carrier over a period ranging from several minutes to several days. [0110] For nasal, inhaled, or oral administration, the compositions or pharmaceutical compositions described herein may be administered using, for example, a spray formulation also containing a suitable carrier

[0111] In some embodiments, for topical applications, the compositions of the present embodiments are formulated as creams or ointments or a similar vehicle suitable for topical administration. For topical administration, the pharmaceutical compositions described herein may be mixed with a pharmaceutical carrier at a concentration of about 0.1% to 1%, 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 0.1 % to 0 5%, 0 5% to 1%, 1% to 1.5%, 1.5% to 2%, 2% to 2 5%, 2.5% to 5%, 5% to 7 5%, or 7 5% to 10% of drug to vehicle. Another mode of administering the compositions provided herein may utilize a patch formulation to affect transdermal delivery.

[0112] In another aspect, the pharmaceutical composition further comprises a matrix. In some embodiments, the matrix is a hydrophilic matrix, including, without limitation, non-ionic soluble cellulose ether (e.g., hydroxypropylmethylcellulose, hydroxypropylcellulose, and hydroxy lethylcellulose), non-ionic homopolymers of ethylene oxide (e.g., poly(ethylene oxide)), water soluble natural gums of polysaccharides (e.g., xanthum gum, alginate, and locust bean gum), water swellable, but insoluble, high molecular weight homopolymers and copolymers of acrylic acid optionally crosslinked with polyalkenyl alcohols, polyvinyl acetate, povidone mixture, cross-linked high amylose starch, and ionic methacrylate copolymers. In some embodiments, the matrix is a hydrophobic matrix, including, without limitation, fatty acids, fatty acid esters, fatty alcohols, waxes of natural and synthetic origins with differing melting points, and hydrophobic polymers. In some embodiments, the hydrophobic matrix comprises stearic acid, lauryl, cetyl or cetostearyl alcohol, carnauba wax, beeswax, eandehlla wax,

microcrystalline wax, low molecular weight polyethylene, ammoniomethacrylate copolymers, ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate or latex dispersions of insoluble polymers. In some embodiments, the matrix is a lipid type matrix, biodegradable type matrix, or mineral type matrix. In some embodiments, the matrix is part of the outer layer of a tablet. In some embodiments, the wax matrix comprises a polymer. In some embodiments, the polymer is selected from acrylic polymer.

[0113] In some embodiments, the pharmaceutical composition is formulated for extended release or slow release. In some embodiments, the pharmaceutical composition is formulated with a matrix. In some embodiments, the pharmaceutical composition is formulated with a wax matrix. In some embodiments, the wax matrix is vegetable-based. In some embodiments, the wax matrix comprises an acrylic polymer.

[0114] The present disclosure also relates to compositions in the form of a food product, a medical food product, or a dietary supplement.

[0115] A food product comprises a substance that can be used or prepared for use as food. A food product may be in a solid or a liquid (e.g., beverage) form. A food product may contain fruits, plants, vegetables, nuts, seeds, or juice, extracts, jam, concentrate, wheat, or alcohol thereof. A food product may also contain milk, yogurt, meat, fish, or processed products thereof. A food product may be flowers, leaves or bark of a plant. In some embodiments, the food product is kombucha. A food product may be a product prepared from a natural food.

[0116] A food product may be a medical food, a functional food, a food additive or a nutritional food. A medical food comprises foods that are specially formulated and intended for dietary management of a disease or condition that has distinctive nutritional needs that cannot be met by normal diet alone. Medical foods can be for oral ingestion or tube feeding. A functional food comprises foods that have a potentially positive effect on health beyond basic nutrition. A food additive comprises any substance added to food and its intended use results or may reasonably be expected to result, directly or indirectly, in its becoming a component or otherwise affecting the characteristics of any food. The addition of food additives may be during production, processing, treatment, packaging, or transportation or storage of food. A nutritional food comprises foods that provide a high amount of nutrients. In some embodiments, the nutritional foods also comprise few calories.

[0117] A dietary supplement comprises a manufactured product intended to supplement the diet. A dietary supplement can be synthetic or natural. A dietary supplement can comprise one component or more than one component in combination. A dietary supplement may comprise vitamins, amino acids, probiotics, minerals, fiber, fatty acids, pigments, polyphenols, lipids, or proteins. In some embodiments, the dietary supplement may be formulated as a pharmaceutical composition as discussed herein. In some embodiments, the dietary' supplement is intended to be taken by mouth as a pill, capsule, tablet, or liquid. In some embodiments, the dietary supplement comprises a label as being a dietary supplement. In some embodiments, the dietary supplement comprises non-dietary ingredients such as fillers, artificial colors, sweeteners, flavors, or binders.

[0118] In some embodiments, the food product or dietary supplement comprises an effective amount of the composition for the treatment or prevention of a condition that can benefit from increased NAD+ level. In some embodiments, the condition is muscle performance deficiency, muscle growth deficiency, or mitochondrial disease. In some embodiments, the condition is aging.

[0119] Provided herein are kits comprising a plurality of yeast cells, a composition, a pharmaceutical composition, a dietary supplement, a food product, or a medical food described herein. In some embodiments, the kit is for use in a method described herein. In some embodiments, the kits comprises one or more containers comprising a plurality of yeast cells, a composition, a pharmaceutical composition, a dietary supplement, a food product, or a medical food described herein. [0120] In some embodiments, the kit and/or components of the kits are packaged, such as in packaging containing paper (e.g cardboard), plastic or other suitable material. In certain embodiments, the kits are in suitable packaging. Suitable packaging include, but is not limited to, vials, botles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. In some embodiments, the components of the kit are contained in one or more containers. In some embodiments, the kit further comprises instructions for use in accordance with any of the methods described herein. Instructions supplied in the kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

VI L METHODS OF USE

[0121] The present disclosure also relates to a method of modulating blood NAD+ level in a subject in need thereof. The method comprises administering to the subject an effective amount of a composition, a pharmaceutical composition, a dietary supplement, a food product, or a medical food described herein.

[0122] The present disclosure also relates to a method of treatment or prevention of a disease or condition that can benefit from increased NAD+ levels, an anticancer or anti- inflammatory treatment, or an extend lifespan. The method comprises administering to a subject in need thereof a therapeutically effective amount of a composition, a pharmaceutical composition, a dietary supplement, a food product, or a medical food described herein.

[0123] In some embodiments, a composition, a pharmaceutical composition, a dietary supplement, a food product, or a medical food described herein is administered to a subject in need thereof. In some embodiments, the subject is a mammalian patient. In some embodiments, the subject is a human or an animal (e.g., cat, dog, cow, rat, mouse, horse, sheep, pig, goat, buffalo, chicken, duck, goose or other domesticated mammal). In some embodiments, the subject is a human patient. [0124] In some embodiments, a composition, a pharmaceutical composition, a dietary' supplement, a food product, or a medical food described herein is administered to an

experimental subject for research purposes. In some embodiments, the subject is a mammalian patient. In some embodiments, the subject is a human or an animal (e.g., mouse, rat, monkey, ape, worm, fly, fruit fly, fish, Zebrafish, frog, Xenopus, cat, dog, cow, pig, horse, sheep, goat, buffalo, chicken, duck, goose or other domesticated mammal).

[0125] In another aspect, the disease or condition to be treated or prevented using any of the compositions or methods described herein includes deficiency in muscle growth or muscle performance or a mitochondrial disease, including, but not limited to, aging, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis; neurodegenerative disorders, such as Huntington’s disease, AIDS dementia complex, adrenoleukodystrophy, Alexander disease, Alper’s disease, Batten disease, Bovine spongiform encephalopathy, ataxia

telangiectasia, Canavan disease, corticobasal degeneration, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, Lyme disease, and multiple sclerosis; metabolic disorders, such as diabetes, obesity, insulin resistance, and a metabolic syndrome, or any combination thereof.

[0126] In some embodiments, the mitochondrial disease or disorder is a neuromuscular disorder, a disorder of neuronal instability, a neurodegenerative disorder, or a mitochondrial myopathy. In yet further embodiments, the mitochondrial disease or disorder is Friedrich’s Ataxia, muscular dystrophy, multiple sclerosis, seizure disorders, migraine, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, ischemia, renal tubular acidosis, age-related neurodegeneration and cognitive decline, chemotherapy fatigue, frailty, age-related or chemotherapy-induced menopause or irregularities of menstrual cycling or ovulation, mitochondrial myopathies, mitochondrial damage (e.g., calcium accumulation, excitotoxicity, nitric oxide exposure, drug induced toxic damage or hypoxia), mitochondrial deregulation, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, multiple system atrophy, Huntington’s disease, Kennedy’s disease, frontotemporal lobar degeneration, Machado- Joseph disease, Krabbe disease, neuroacanthocytosis, Pick’s disease, Niemann-Pick disease, progressive supranuclear palsy, primary lateral sclerosis, Sandhoff disease, diffuse

myeimoclastic sclerosis, Refsum disease, spinocerebellar ataxia, tabes dorsalis, subacute combined degeneration of spinal cord, Tay-Sachs disease, transmissible spongiform

encephalopathy, wobbly hedgehog syndrome, toxic encephalopathy, Barth syndrome, beta- oxidation defects, carnitme-acyl- carnitine deficiency, carnitine deficiency, creatine deficiency syndrome, co-enzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency, complex V deficiency, chronic progressive external ophthalmoplegia syndrome, CPT I deficiency, CPT IT deficiency, Kearns-Sayre syndrome, lactic acidosis, leukodystrophy, Leigh disease, Luft disease, mitochondrial encephalomyopathy lactic acidosis and strokelike episodes (MELAS), progeria, Cockayne syndrome, myoclonic epilepsy and ragged-red fiber disease (MERRF), mitochondrial recessive ataxia syndrome, mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy,

myoneurogastrointestinal disorder and encephalopathy, neuropathy, ataxia, and retinitis pigmentosa, Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, POLG2 mutations, encephalopathy and possibly liver disease or cardiomyopathy, acyl-CoA dehydrogenase deficiency, chrome traumatic encephalopathy (CTE), cancer, cachexia, infections caused by Hepatitis A, B, and/or C Virus, Human Immunodeficiency Virus (HIV), Human Papilloma Virus, human T-cell leukemia-lymphoma viruses (HTLV) or any combination thereof or any of their medical sequelae.

[0127] In some embodiments, the disease is a disease associated with dementia. In certain embodiments, the disease associated with dementia is selected from the group consisting of Alzheimer’s disease, vascular dementia, dementia with Lewy bodies (DLB), Parkinson’s disease, frontotemporal dementia, Creutzfeldt-Jakob disease, Normal pressure hydrocephalus, Huntington’s disease and Wernicke-Korsakoff syndrome, and any combination thereof.

[0128] In some embodiments, the disease is a disease associated with deficient cognitive performance. In some embodiments, the disease is anxiety, depression, memory' loss, or PTSD. [0129] In some embodiments, the present disclosure provides methods for maintaining or enhancing muscle performance or muscle growth in a subject. Muscle performance may include, without limitation, the capacity of a muscle or a group of muscles to generate forces to produce, maintain, sustain and modify postures and movements that are prerequisite to functional activity. In some embodiments, the method maintains or enhances muscle strength, muscle endurance, speed, muscle power, maximum muscle length, or oxygen level in muscle. In some

embodiments, the method maintains muscle performance after or during physical labor or exercise. In some embodiments, the method reduces muscle fatigue, muscle sourness, or muscle tension after physical labor or exercise. In some embodiments, the method reduces the muscle reaction time after stimulation. In some embodiments, the method increases muscle mass or mitochondrial mass m muscle. In some embodiments, the method maintains or increases the number of myofibrils and rate of protein synthesis. In some embodiments, the method reduces inflammator response or oxidative stress m muscle. In some embodiments, the subject has a deficiency in muscle performance or muscle growth. In some embodiments, the subject does not have a deficiency in muscle performance or muscle growth.

[0130] The present disclosure is also directed to a method of extending lifespan. In certain embodiments, the method maintains or enhances the healthspan in a subject with respect to body-mass index, physical performance, cardiac health, sexual performance, mental health, diet, or substance uses. Physical performance may include athletic performance and general fitness. Physical performance may refer to the capacity to complete daily round of activities with enough energy left for recreation and relaxation. Cardiac health may include health of the heart and blood vessels. Mental health may include emotional, psychological, and social well-being.

1. A medium for culturing yeast cells, comprising at least about 6 mM nicotinic acid (NA) or a salt thereof.

2. The medium of embodiment 1 , comprising about 6 mM - 20 mM NA.

3. The medium of embodiments 1 or 2, further comprising nicotinamide (NAM). 4. The medium of embodiment 3, comprising at least about 6 mM NAM.

5. The medium of embodiment 3 or 4, comprising about 6 mM - 20 mM NAM.

6. A medium for culturing yeast cells, comprising at least about 6 mM nicotinamide (NAM) or a salt thereof.

7. The medium of embodiment 6, comprising about 6 mM - 20 mM NAM.

8. The medium of any one of embodiments 6 or 7, further comprising NA.

9. The medium of embodiment 8, comprising at least about 6 mM of NA.

10. The medium of embodiments 8 or 9, comprising about 6 mM - 20 mM NA.

11. The medium of any one of embodiments 1 -10, further comprising a flavonoid.

12. The medium of embodiment 1 1, wherein the flavonoid is fisetin, quercetin, apigenin, or fuieolin.

13. The medium of embodiments 1 1 or 12, wherein the medium comprises quercetin.

14. The medium of any one of embodiments 11-13, wherein the medium comprises fisetin.

15. The medium of any one of embodiments 1-14, further comprising a fruit or vegetable extract, a fruit or vegetable concentrate, or a fruit or vegetable powder.

16. The medium of any one of embodiments 1-15, further comprising piperlongumine.

17. The medium of any one of embodiment 1-16, wherein the yeast cells are not genetically modified organisms (GMOs).

18. The medium of any one of embodiment 1-17, wherein the yeast cells are

Saceharomyces cerevisiae.

19. A method of screening a yeast strain, the method comprising (a) culturing yeast cells from a yeast strain in a medium comprising 20 mM - 60 mM NA and/or 20 mM - 60 mM NAM; and (b) determining if the yeast cells grow in the medium.

20. The method of embodiment 19, further comprising repeating step (a) and step (b) with a plurality of yeast strains.

21. The method of embodiments 19 or 20, further comprising (c) determining the concentration of one or more of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid riboside (NAR), nicotinamide adenine dinucleotide (NAD+), nicotinic acid adenine dinucleotide (NAAD), or nicotinic acid mononucleotide (NAMN) in the yeast cells or the medium after step (a).

22. The method of embodiment 21 , further comprising repeating step (c) with a plurality of yeast strains.

23. The method of embodiments 21 or 22, further comprising (d) selecting a yeast strain that grows in the medium and whose yeast cells produce one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN at a desired level after step (a).

24. The method of embodiment 23, wherein the desired level is an amount sufficient to produce a dry composition comprising an extract of the yeast cells comprising at least 1% of one or more of NMN, NR, NAR, NAD+, NAAD, or NAMN (w/w) produced by the yeast cells.

25. The method of embodiment 24, wherein the extract was produced by mechanically lysing the yeast cells.

26. The method of embodiments 24 or 25, wherein the extract was produced without the addition of methanol, ethanol, or hazardous chemicals.

27. A yeast cell from the yeast strain selected in any one of embodiments 23-26.

28. A method of culturing yeast cells, the method comprising culturing yeast cells in the medium of any one of embodiments 1-18.

29. A method of preparing NMN and/or NR, comprising culturing yeast cells in the medium of any one of embodiments 1-18.

30. The method of embodiment 29, further comprising extracting NMN and/or NR from the yeast cells.

31. The method of any one of embodiments 28-30, wherein the yeast cells are a plurality of the yeast cell of embodiment 27.

32. The method of any one of embodiments 19-26 or 28-31 , wherein the yeast cells are Saecharomyees cerevisiae

33. The method of any one of embodiments 19-26 or 28-32, wherein the yeast cells are not genetically modified organisms (GMOs). 34. The method of any one of embodiments 19-26 or 28-33, wherein the method comprises culturing the yeast ceils for at least 2 hours.

35. The method of any one of embodiments 19-26 or 28-34, wherein the method comprises culturing the yeast cells for at least 12 hours.

36. The method of any one of embodiments 19-26 or 28-35, wherein the culturing comprises stirring and/or aerating the medium.

37. The method of any one of embodiments 19-26 or 28-36, wherein the culturing further comprises adding one or more of yeast nutrient, soya peptone, or a sugar after at least 2 hours of culturmg.

38. The method of any one of embodiments 19-26 or 28-37, wherein the temperature of the medium is about 75 °F - about 85 °F during culturmg.

39. The method of any one of embodiments 19-26 or 28-38, further composing inactivating the yeast cells.

40. The method of embodiment 39, wherein the inactivating comprises sonicating, microwaving, or pasteurizing the yeast cells.

41. The method of any one of embodiments 19-26 or 28-40, further comprising concentrating the yeast cells.

42. The method of embodiment 41, wherein the concentrating comprises centrifuging or filtering the yeast cells.

43. The method of any one of embodiments 19-26 or 28-42, further comprising lysing the yeast cells to produce an extract.

44. The method of embodiment 43, wherein the lysing is mechanical lysing.

45. The method of any one of embodiments 19-26 or 28-42, wherein the method does not comprise adding methanol, ethanol, or hazardous chemicals to the yeast cells or an extract thereof.

46. The method of embodiments 43 or 44, wherein the method does not comprise adding methanol, ethanol, or hazardous chemicals to the extract. 47. The method of any one of embodiments 43-46, further comprising centrifuging or filtering the extract.

48. The method of any one of embodiments 43-47, further comprising freeze drying the extract.

49. A composition comprising the yeast cells produced by the method of any one of embodiments 28-42 or 45, or an extract thereof, or the extract of any one of embodiments 43 or 46-48.

50. A composition comprising yeast cells or an extract thereof comprising at least 500 mM NR and/or at least 500 m.M NMN.

51. A composition comprising yeast cells or an extract thereof comprising at least 0.4% NR (w/w) and/or at least 0.4% NMN (w/w).

52. The composition of embodiment 51, comprising at least 0.4% NR (w/w) and at least 0.4% NMN (w/w).

53. The composition of embodiments 51 or 52, wherein the yeast cells produced at least 50% of the NR and/or NMN.

54. The composition of any one of embodiments 50-53, wherein the yeast cells are not genetically modified organisms (GMOs).

55. The composition of any one of embodiments 50-54, wherein the yeast cells are Saccharomyces cerevisiae.

56. The composition of any one of embodiments 50-55, further comprising NAD+.

57. The composition of embodiment 56, wherein the composition comprises at least 0.25% NAD+.

58. The composition of embodiments 56 or 57, wherein the yeast cells produced at least 50% of the NAD+.

59. The composition of any one of embodiments 50-58, further comprising NA, NAR, NAAD, NAMN, or combinations thereof.

60. The composition of embodiment 59, wherein the composition comprises at least 1% NA (w/w). 61. The composition of any one of embodiments 50-60, further comprising a compound or plurality of compounds that are capable of lowering total plasma homocysteine in a subject.

62. The composition of any one of embodiments 50-60, further comprising serine, vitamin B6, vitamin B9, and vitamin B12.

63. The composition of any one of embodiments 50-62, further comprising a f!avonoid.

64. The composition of embodiment 63, wherein the flavonoid is fisetin, quercetin, apigemn, and luteolin.

65. The composition of embodiments 63 or 64, comprising quercetin, fisetin, or quercetin and fisetin

66. The composition of any one of embodiments 50-65, further comprising

piperlongumine, vitamin B2, N-acetylcysteine, S-adenosylmethionine (SAM), trimethylglycine, or combinations thereof.

67. The composition of any one of embodiments 50-66, further comprising a fruit or vegetable extract, a fruit or vegetable concentrate, a fruit or vegetable powder, tuna or an extract thereof, salmon or an extract thereof, bulgur or an extract thereof, barely or an extract thereof, or a combination thereof.

68. The composition of any one of embodiments 50-67, wherein the yeast cells have been inactivated.

69. The composition of embodiment 68, wherein the yeast cells were inactivated by sonication, microwaving, or pasteurization.

70. The composition of any one of embodiments 50-69, wherein the composition is a paste or a powder.

71. The composition of any one of embodiments 50-70, wherein the composition has been freeze dried.

72. A pharmaceutical composition comprising the composition of any one of embodiments 50-71 and a pharmaceutically acceptable excipient.

73. A dietary' supplement, food product, or medical food comprising the composition of any one of embodiments 50-71 or the pharmaceutical composition of embodiment 72. 74. A method of modulating blood NAD+ level comprising administering to a subject an effective amount of the composition of any of embodiments 50-71 , the pharmaceutical composition of embodiment 72, or the dietary supplement, food product, or medical food of embodiment 73.

75. A method of extending lifespan of a subject comprising administering to the subject an effective amount of the composition of any of embodiments 50-71, the pharmaceutical composition of embodiment 72, or the dietary supplement, food product, or medical food of embodiment 73

76. A method of improving healthspan of a subject comprising administering to the subject an effective amount of the composition of any of embodiments 50-71 , the pharmaceutical composition of embodiment 72, or the dietary supplement, food product, or medical food of embodiment 73.

77. A method of enhancing or maintaining muscle growth or performance comprising administering to a subject an effective amount of the composition of any of embodiments 50-71, the pharmaceutical composition of embodiment 72, or the dietary supplement, food product, or medical food of embodiment 73.

78. A method of treating or preventing a mitochondrial disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of embodiments 50-71, the pharmaceutical composition of embodiment 72, or the dietary supplement, food product, or medical food of embodiment 73.

EXAMPLES

[0131] The following examples are offered to illustrate but not to limit the invention. One of skill in the art will recognize that the following procedures may be modified using methods known to one of ordinary skill in the art.

Example 1: Culture yeast in a medium comprising nicotinic acid [0132] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of nicotinic acid

Methods:

[0133] 1 gallon (-3.8 L) distilled water, lib (-435.6 g) dry malt extract (DME), 1.5 g FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammomum phosphate (DAP)), 0.5g Yeast Nutrient (WHITE LABS® lot# 1320028262, comprises diammonium phosphate, essential vitamins and co-factors, nitrogen (ammo acids, proteins, and peptides) and minerals), 3 g nicotinic acid, and 1000 pg biotin were added to a 5000 ml flask. The flask was covered with aluminum foil to allow' aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 mins. The pH was measured at 5.5, the Brix was 10°, the measured specific gravity' (SG) was 1.04 and the temperature was 79 °F (~26.1 °C), yielding a calibrated SG of 1.0423.

[0134] Saccharomyces cerevisiae (1 PUREPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate with aeration at a rate of 600 rpm for 3 hours. Cells were counted using a hemocytometer. A sample of the medium with yeast cells was diluted 8 fold with 1 % methylene blue. The final pH was 5.5 and the average cell count was 14. After an additional 2.5 hours of culturing, a sample was again taken. The pH was 4 and the average cell count was 29.7. The sample comprised a mixture of intact (blue) and living

(transparent) cells.

[0135] After a 21.5 total hours of culturing, the medium was removed and the yeast cells (45 oz. (1.32 L)) were blended in a VITAMIX® for 5 min at power level 10. The temperature prior to mixing was 46 °F (-7.8 °C) and after mixing was 144 °F (-62.6 °C). The mass appeared light gray or white and had a yogurt consistency.

[0136] The sample was then heated to 160 °F (-71.1 °C) for 15 minutes to inactivate the yeast. The sample was then cooled in a refrigerator 2 hrs and then centrifuged at 3000 rpm for 10 min. The liquid (4.5 cups or ~IL) was then freeze dried. The final pH was 3 and the procedure yielded 28.4 g of freeze dried extract with a crysta!ly, sticky, and fluffy texture. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN and NR. The final product was determined to contain 2.044% NMN and 0.53% NR.

[0137] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of nicotinic acid, quercetin, and fisetin.

Methods:

[0138] 1 gallon (-3.8 L) distilled water, lib (-435.6 g) dry malt extract (DME), 1.5 g FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammomum phosphate (DAP)), 0.5g Yeast Nutrient (WHITE LABS® lot# 1320028262), 3 g nicotinic acid, 1000 pg biotin, 15 g quercetin, and 2 g fisetin were added to a 5000 mL flask. The flask was covered with aluminum foil to allow aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 mins. The pH was measured at 5.5, the Brix was 10°, the measured specific gravity (SG) was 1.04 and the temperature was 79 °F (-26.1 °C), yielding a calibrated SG of 1.0423.

[0139] Saccharomyces cerevisiae (1 PUREPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate with aeration at a rate of 600 rpm for 3 hours. Cells were counted using a hemocytometer. A sample of the medium with yeast cells was diluted 8 fold with 1% methylene blue. The final pH was 5.5 and the average cell count was 29. After an additional 2.5 hours of culturing, a sample was again taken. The pH was 5 and the average cell count was 46.7. The sample comprised yellow string/crystalline rods and the cells did not stain blue. [0140] After a 21.5 total hours of culturing, the medium was removed and the yeast cells (50 oz. (1.44 L)) were blended in a VITAMIX® for 5 min at power level 10. The temperature prior to mixing was 48 °F (—8.9 °C) and after mixing was 110 °F (~43.3 °C). The mass appeared yellow and was very foamy with stiff peaks.

[0141] The sample was then heated to 160 °F (-71.1 °C) for 15 minutes to inactivate the yeast. The sample was then cooled in a refrigerator 2 hrs and then centrifuged at 3000 rpm for 10 min. The liquid (4 cups or -0.95 L) was then freeze dried. The final pH was 3 and the procedure yielded 35.2 g of freeze dried extract that was yellow with a flaky and crystally texture. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN and NR. The final product was determined to contain 1.460% NMN and 3.008% NR

[0142] Purpose: to produce M AD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of nicotinic acid, sugar, and molasses.

Methods:

[0143] 1 gallon (-3.8 L) distilled water, 15tbs THE NATURAL CITIZEN™ organic protein (plant protein), 35 g nutritional yeast, 40 g organic sugar (sucrose), 40 g organic molasses, 1.5 g FERMAID® O (an auto!yzed yeast nutrient with a high content of nitrogen and no diammonium phosphate (DAP)), 0.5g Yeast Nutrient (WHITE LABS® lot# 1320028262), 3 g nicotinic acid, and 1000 pg biotin were added to a 5000 ml flask. The flask was covered with aluminum foil to allow aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 mins. The pH was measured at 6, the Brix was 4°, the measured specific gravity (SG) was 1.015 and the temperature was 79 °F (-26.1 °C), yielding a calibrated SG of 1.0173. [0144] Saccharomyces cerevisiae (1 PUREPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate at a rate of 600 rpm for 3 hours. Cells were counted using a hemocytometer. A sample of the medium with yeast cells was diluted 8 fold with 1% methylene blue. The final pH was 6 and the average cell count was 63.3. After an additional 2,5 hours of culturing, a sample was again taken. The pH was 5.75 and the average cell count was 102.33. The sample comprised cells that formed large irregular clumps and all cells were stained blue.

[0145] After a 21 5 total hours of culturing, the medium was removed and the yeast cells (45 oz. (1.32 L)) were blended in a VITAMIX® for 5 min at power level 10. The temperature prior to mixing was 44 °F (-6.7 °C) and after mixing was 83 °F (-28.3 °C). The mass appeared tan with dark specs and grams were present before and after blending

[0146] The sample was then heated to 160 °F (-71.1 °C) for 15 minutes to inactivate the yeast. The sample was then cooled in a refrigerator 2 hrs and then centrifuged at 3000 rpm for 10 min. The liquid (3 cups or -0.7 L) was then freeze dried. The final pH was 5.5 and the procedure yielded 5.1 g of freeze dried extract that was extremely sticky and had a caramel texture. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN and NR The final product was determined to contain 1.635% NMN and 1 096% NR.

Example 4: Culture yeast in a medium comprising nicotinic acid, nicotinamide, quercetin, and fisetin

[0147] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of nicotinic acid, nicotinamide, quercetin, and fisetin.

Methods:

[0148] 1 gallon (-3 8 L) distilled water, 11b (-435.6 g) dry malt extract (DIME), 1.5 g FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammonium phosphate (DAP)), 0 5g Yeast Nutrient (WHITE LABS® lot# 1320028262), 3 g nicotinic acid, 3 g nicotinamide, 1000 pg biotin, 3 g quercetin, 3 g fisetin, and 5 g soya peptone were added to a 5000 mL flask. The flask was covered with aluminum foil to allow' aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 rams.

[0149] Saccharomyces cerevisiae (1 PUREPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate at a rate of 600 rpm for 12 hours. 0.5 g yeast nutrient, 5 g soya peptone, and 10 g sugar (sucrose) were added and the culture was stirred and aerated for an additional 6.5 hours. When the additional yeast nutrient, soya peptone, and sugar were added, the culture began to form and was still foaming when the culturing was completed and the culture was placed in a refrigerator to cool overnight.

[0150] The culture was removed from the refrigerator. The foam from the top of the culture was removed and the broth was poured off. The yeast sediment and foam were combined and were blended in a VITAMIX® for 5 min at power level 10. The sample was then heated to 160 °F (~7l .1 °C) for 1 5 minutes to inactivate the yeast. The sample was then cooled in a freezer for 2 hrs and then centrifuged at 3000 rpm for 10 mm. The liquid was then freeze dried the procedure yielded 55 g of freeze dried extract. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN, NR, N R, NAD+, and NAMN. The final product was determined to contain 0.498% NMN, 2.00% NR, and 0.517% NAD+. NAR and NAMN were not detected in the final product.

Example 5: Culture yeast in a medium comprising nicotinic add, nicotinamide, quercetin, and fisetin

[0151] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of fisetin and higher concentrations of nicotinic acid, nicotinamide, and quercetin. Methods:

[0152] 1 gallon (-3.8 L) distilled water, lib (-435.6 g) dry malt extract (DME), 1.5 g FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammonium phosphate (DAP)), 0.5g Yeast Nutrient (WHITE, LABS® lot# 1320028262), 9 g nicotinic acid, 9 g nicotinamide, 1000 _ug biotin, 9 g quercetin, 3 g fisetin, and 5 g soya peptone were added to a 5000 mL flask. The flask was covered with aluminum foil to allow aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 mms.

[0153] Saccharomyces cerevisiae (1 PUKEPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate at a rate of 600 rpm for 12 hours. 0.5 g yeast nutrient, 5 g soya peptone, and 10 g sugar (sucrose) were added and the culture was stirred and aerated for an additional 6.5 hours. The culture was placed in a refrigerator to cool overnight.

[0154] The culture was removed from the refrigerator. Any foam on the top of the culture was removed and the broth was poured off. The yeast sediment and foam were combined and were blended in a VITAMIN® for 5 min at power level 10. The sample was then heated to 160 °F (-71.1 °C) for 15 minutes to inactivate the yeast. The sample was then cooled in a freezer for 2 hrs and then centrifuged at 3000 rpm for 10 min. The liquid was then freeze dried the procedure yielded 54 g of freeze dried extract. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN, NR, NAR, NAD+, and NAMN. The final product was determined to contain 0.460% NMN, 4.960% NR, and 0.549% NAD+. NAR and NAMN were not detected in the final product. The final product was a yellow flaky and fluffy powder. The lighter colored areas were sweet to the taste and darker brown areas were more bitter.

[0155] Purpose: to produce NAD+ precursors such as NMN and N from yeast by culturing the yeast in the presence of higher concentrations of nicotinic acid and nicotinamide.

Methods:

[0156] 1 gallon (-3.8 L) distilled water, lib (-435.6 g) dry malt extract (DME), 1.5 g FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammomum phosphate (DAP)), 0.5g Yeast Nutrient (WHITE LABS® lot# 1320028262), 9 g nicotinic acid, 9 g nicotinamide, 1000 _ug biotin, 9 g quercetin, 3 g fisetin, and 5 g soya peptone were added to a 5000 mL flask. The flask was covered with aluminum foil to allow aeration and prevent contamination. The flask was aerated on a stir plate at a rate of approximately 1000 rpm for 30 mms.

[0157] Saccharomyces cerevisiae (1 PUREPITCH® yeast culture (lot# 1059235)) was added to the medium and was mixed on a stir plate at a rate of 600 rpm for 12 hours. 0.5 g yeast nutrient, 5 g soya peptone, and 10 g sugar (sucrose) were added and the culture was stirred and aerated for an additional 6.5 hours. The culture was placed m a refrigerator to cool overnight.

[0158] The culture was removed from the refrigerator. Any foam on the top of the culture was removed and the broth was poured off ^’ . The yeast sediment and foam were combined and were blended in a VET AMEX® for 5 min at power level 10. The sample was then heated to 160 °F (-71.1 °C) for 15 minutes to inactivate the yeast. The sample was then cooled in a freezer for 2 hrs and then centrifuged at 3000 rpm for 10 min. The liquid was then freeze dried. The procedure yielded 50.5 g of freeze dried extract. The freeze dried extract was then analyzed by HPLC to determine the percent concentration of NMN, NR, NAR, NAD+, and NAMN. The final product was determined to contain 0.679% NMN, 5.890% NR, and 0.419% NAD+. NAR and NAMN were not detected in the final product. The final product was a yellow flaky and fluffy powder. Example 7: Culture yeast in different media

[0159] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast with or without niacin and nicotinamide, and with or without ellagic acid, tryptophan, and/or ribose.

Methods:

[0160] Yeast extract, peptone, dextrose (YPD) base media was prepared by mixing distilled H ₂ 0, FERMAID® O (an autolyzed yeast nutrient with a high content of nitrogen and no diammonium phosphate (DAP)), nutritional yeast powder (STARWEST BOTANIC ALS®, product number 210205-51), peptone, dextrose, and pH stabilizer to create a final solution comprising 5 g/L FERMAID® O, 5 g/L nutritional yeast powder, 20 g/L peptone, 20 g/L dextrose, and 0.7 g/L pH stabilizer. Dry malt extract-Ferm-0 (DME-FO) base media was prepared by mixing distilled H2O, dry malt extract (DME), and FERMAID® O to create a final solution comprising 120 g/L dry malt extract and 0.40 g/L FERMAID® O.

[0161] 700 ml YPD base media or 700 ml DME-FO base media were added to a total of 10 experimental flasks. The flasks were autoclaved at 121 °C for 15 min and allowed to cool to room temperature. Yeast nutrient (0.2g; WHITE LABS® lot#! 320028262, biotin (200 gg), niacin (0.89 g), nicotinamide (0.89 g), ellagic acid (2.25 g), tryptophan (7.65 g), and/or ribose (7.5 g) were added to the flasks. The 10 experimental flasks (F!-FIO) are summarized in Table 1 , below.

[0162] Once the additional components were added to the experimental flasks as summarized in the preceding table, the flasks were warmed in a 27 °C shaker at 150 rpm.

Separately, an inoculant was prepared in either YPD base media or DME-FO base media by adding Saccharomyces cerevisiae (1 packet per 250 ml of base media of WLP001

CALIFORNIA ALE YEAST®. The inoculant media was shaken to homogeneity. Flasks F1 -F7 were inoculated with 50 ml of the YPD inoculant (to yield 750 ml total media) and flasks F8-F10 were inoculated with 50 ml of the DME-FO inoculant (to yield 750 ml total media).

[0163] Experimental Flasks F1-F10 were then incubated at 27 °C with shaking (150 rpm) for 18 hours. Each flask was covered with aluminum foil to allow aeration and prevent contamination. After incubation, the flasks were allowed to settle for 3 hours at 4 °C. After the yeast had settled to the bottom of each flask, the media was poured off. As a media control, the supernatant from experiment flask FI was saved (FI -broth). Distilled water was added to each experimental flask and the pellets were agitated by gentle swirling. The flasks were allowed to settle again for one hour at 4 °C, The supernatant was poured off and discarded. The wash step was repeated and the supernatant again discarded. The yeast pellets were then resuspended m 100 ml of distilled water.

[0164] The final yeast slurries were placed into silicone mold trays and placed in a -80 °C freezer. After freezing, the silicone molds were placed onto racks in a freeze dryer. The samples were dried over 18 hours. After drying, the samples were prepared for mass

spectrometry analysis to determine the percent by weight (%/w) of each of NAD, NADP, 1- methylnicotinamide (AINA), trigonelline, NR, NAIL NMN, NAMN, NA, and NAM. The results for the YPD media are summarized in Table 2, below. The results for the supernatant of the FI flask (i.e., the YPD media removed after allowing the yeast to settle, designated FI -broth) is summarized in Table 2, as w ^r ell.

7

0165] The results for the DME-FO media are summarized in Table 3, below. [0166] Conditions corresponding to experimental flasks F3, F6, and F8 produced the highest total % of the tested compounds, not counting niacin and niacinamide. Accordingly, biotin, niacin, and niacinamide were found to have positive effects on metabolite yield in this culture experiment. In contrast, e!iagic acid and tryptophan did not have a positive effect on metabolite yield. Ribose may have a positive effect on metabolite yield.

[0167] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast and harvesting with centrifugation.

Methods:

[0168] Based on the results from the experimental conditions corresponding to experimental flasks F6 and F8 from the preceding Example 7, an experiment was designed to test the influence of harvesting the yeast by centrifugation with a YPD culture (designated F6-11) and a DME-FO culture (designated F8-11). The media were prepared for inoculation.

Specifically, for YPD base media, yeast extract, dextrose, and soya peptone were diluted in distilled H20 to a final concentration of 20 g/L dextrose, 20 g/L peptone, and 10 g/L yeast extract in a total of 2000 ml. DME-FO base media was prepared by mixing H20 with dry malt extract and FERMAID® O to a final concentration of 120 g/L DME and 0 4 g/L FERMAID® O in a total of 2000 ml. The base media were sterilized by autoclaving at 121 °C for 15 minutes.

[0169] After autoclaving, the base media were allowed to cool and then placed in a 27 °C incubator. Biotin (264 gg/L), niacin (1.19 g/L), nicotinamide (1 19 g/L), and ribose (10 g/L) were added to the YPD base media (flask F6-1 1). Yeast nutrient (0.26 g/L), biotin (264 gg/L), and nicotinamide (1.19 g/L.) were added to the DME-FO base media (flask F8-11).

[0170] 100 ml of YPD and 100 ml of DME-FO base media were removed from each of the 2000 ml media flasks (F6-1 1 and F8-11) and combined in a smaller flask. The smaller mixture of combined base media was then mixed with Saccharomyces cerevisiae (WLP001 CALIFORNIA ALE YEAST® (LN 1075337)) and shaken to homogeneity (1 packet per 200 ml). After mixing, 100 ml of the yeast slurry was added to the 1900 ml of YPD base media, and 100 ml of the yeast slurry was added to the 1900 ml of DME-FO base media, giving a volume of 2000 ml for each. A cell count revealed the inoculated YPD media contained approximately 1 .31 x 10 ⁷ total cells, and the DME-FO media contained approximately 1.23 x 10 ⁷ total cells.

[0171] After inoculation, the media were incubated at 27 °C for 18 hours with shaking (150 rpm). After incubation, the samples were each distributed into three centrifuge bottles and the yeast were pelleted at 4000 rpm for 3 minutes. The supernatant was removed from each bottle. For each sample, one centrifuge bottle was used to test a no wash condition, one centnfuge bottle was used to test a single wash condition, and one centrifuge bottle was used to test a double wash condition. For the no wash condition, the pellet was resuspended in 100 ml of distilled water. For the single (one) wash condition, the pellet was resuspended in 250 ml of distilled water, vortexed for 30 seconds, then pelleted at 4000 rpm for 3 minutes. The one wash pellet was then resuspended in 100 ml of distilled water. For the double (two) wash condition, the pellet was resuspended in 250 ml of distilled water, vortexed for 30 seconds, then pelleted at 4000 rpm for 3 minutes followed by a repeat of the wash step with 250 ml of new distilled water. After pelleting again, the double wash pellet was then resuspended in 100 ml of distilled water.

[0172] All six resuspended pellets (100 ml for each of the 0 wash, 1 wash, and 2 wash conditions for each of the F6-11 and F8-1 1) were transferred to silicone mold trays and flash frozen by submersion in liquid nitrogen. The silicone molds were then stored at -80 °C. After storage, the molds were placed m a freeze dry er for a total of about 20 hours. The final product was homogenized by mechanical mixing with a seoopula.

[0173] After freeze drying, the samples were prepared for mass spectrometry analysis to determine the percent by w¾ight (%/w) of each of NAM, NA, NAD, NMN, NAMN, NR, NAIL and MN A. The results are summarized in Table 4, below. The samples are summarized for 0 washes (F6-11-0 and F8-1 1 -0), with one wash (F6-11-1 and F8- 11-1), and with two washes (F6- 11-2 and F8-1 1 -2).

[0174] Comparing F8-1 1-0 to F8 from Example 7, the NAMN, NMN, and total % (not counting niacin or nicotinamide) was significantly higher in the F8-1 1 -0 condition compared to the F8 condition. Accordingly, the harvesting method, including centrifugation, may be important for increasing yield of these products. Increasing washing was correlated with decreased metabolite yield, with the exception of NR.

Example 9: Yeast culture growth conditions

[0175] Purpose: to determine the ideal inoculation concentration to observe exponential growth curves.

Methods:

[0176] A yeast culture medium was prepared according to the method described for F6- 11 in the preceding Example 8. The prepared media were distributed into four flasks, and inoculated 1 :20 with inoculant at 10 ', lO ⁵ , or 10 ² cells/ml. For the 10 ⁵ cells/ml condition, the incubation temperature was also adjusted from 27 °C to 30 °C for one experiment. The experimental conditions (conditions 1 -4) are summarized in Table 5, below.

[0177] The samples were incubated under the indicated conditions. Samples were collected at various time points and yeast cells w ^r ere counted. The cell counts are summarized m Table 6, below.

[0178] Based on the cell count results, it was found that a i 0 ⁵ inoculant was preferred for exponential growth. The 27 °C and 30 °C incubation temperatures were not significantly different.

Example 10: Yeast culture with media additions [0179] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by testing the effect of media additions, including fisetin, quercetin, thiamin, adenine, and henfotiamine.

Methods:

[018Q] YPD base media was prepared as discussed in Example 8. After autoclaving, the YPD base media was supplemented with 264 pg/L biotin, 1.19 g/L niacin, 1.19 g/L

nicotinamide, 10 g/L ribose, and 3.57 g/L pH stabilizer in a total volume of 750 ml. The supplemented YPD base media was then distributed into 6 flasks. The first flask, 13.1, was not further supplemented. Flasks 13 2-13.6 were each with one of fisetin (0.07 g/L; flask 13.2), adenine (0 004 g/L; flask 13.3), benfotiamine (0.8 mg/L; flask 13.4), thiamin (0 005 g/L; flask 13.5), or quercetin (0.03 g/L; flask 13.6). The flasks were inoculated with inoculant containing Saccharomyces cerevisiae (WLP001 CALIFORNIA ALE YEAST®).

[0181] Samples were incubated at 27 °C for 18 hours with shaking (150 rprn). After incubation, samples were distributed into centrifuge bottles, pelleted at 4000 rpm for 3 minutes, washed with 250 ml of water by resuspension, repelleted at 4000 rprn for 3 minutes, and finally- resuspended in 50 ml distilled water. Resuspended pellets were frozen and freeze dried as described in the preceding examples.

[0182] Samples were prepared for mass spectrometry to measure the influence of benfotiamine, adenine, thiamin, fisetin, and quercetin on the levels of NAM, NA. NAD. NMN, NAMN, NR, NAR, and MNA. The results (%/w) are summarized in Table 7, below.

[0183] Adenine addition at 0.004 g/L was the only condition that increased the yield (not counting niacin and niacinamide).

[0184] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast with resveratrol, low glucose, lower niacin/nicotinamide, or without pH stabilizer.

Methods:

[0185] Five experimental flasks (14.1-14.5) were prepared. YPD base media was prepared as discussed in Example 8 for experimental flasks 14.1 , 14.2, 14.4, and 14.5. For flask 14.3, 7.5 g yeast extract, 15 g peptone, and 3.75 g dextrose ware added to 700 mL H2O and the mixture was autoclaved. After autoclaving, each flask was supplemented with the supplements as indicated in Table 8, below.

[0186] The experimental flasks were inoculated with IQ ³ cells/mL Saccharomyces cerevisiae (WLP001 CALIFORNIA ALE YEAST®; 1 packet per 250 ml of inoculant). The flasks w¾re then incubated at 27 °C for 18 hours with shaking (150 rpm). [0187] After incubation, samples were pelleted at 4000 rpm for 3 minutes, washed with 250 ml of water, repelleted at 4000 rpm for 3 minutes, and then resuspended in 50 ml of distilled water. Samples were frozen and freeze dried as discussed in the preceding examples.

[0188] Samples were prepared for mass spectrometry and analyze for their content of NAM, NA, NAD, NMN, NAMN, NR, NAR, and MNA. The results are summarized in Table 9, below.

niacin and niacinamide) than with pH stabilizer, indicating pH stabilizer may be omitted.

Resveratrol significantly increased precursors (not counting niacin and niacinamide), indicating resveratrol has a positive effect on metabolite yield. The conditions associated with experimental flasks 14.4 and 14.5 both had significantly increased metabolite levels, indicating that lower levels of glucose, niacin, and niacinamide may lead to higher metabolite yield.

[0190] Purpose; to produce NAD+ precursors such as NMN and NR from yeast by- culturing the yeast and observing the effect of different incubation lengths.

Methods: [0191] YPD base media was prepared as discussed in Example 8 and, after autoclaving. supplemented with 264 pg/ml biotin, 1.19 g/L niacin, 1.19 g/L nicotinamide, 10 g/L ribose, and

0.7 g/L pH stabilizer. The supplemented YPD base media was inoculated with Saccharomyces cerevisiae. The inoculated sample was incubated at 27 °C with shaking (150 rpm).

[0192] At 12, 15, 18, 24, 30, and 48 hours samples were collected and assayed for concentration of NAM, NA, NAD, NADH, NADP, NADPH, NMN, NAMN, NR, NAR, MNA, and trigonelline, as summarized (by %/w) in Table 10, below.

[0193] The precursors, excluding niacin and niacinamide, were significantly higher at the 30 hour incubation time. NR was negatively correlated with incubation time (p=0.03) whereas total % precursors (p=0.0097) and % niacin (p=0.014) were positively correlated.

Example 13: Yeast culture growth patters*

[0194] Purpose: to produce NAD+ precursors such as NMN and NR from yeast by culturing the yeast in the presence of resveratrol, adenine, quercetin, fisetin, benfotiamine, and/or thiamin.

Methods: [0195] F1-F9) were prepared. YPD base media was prepared as discussed in Example 8 for experimental flasks F1-F8. For flask F9, 7.5 g yeast extract, 15 g peptone, and 3.75 g dextrose were added to 700 mL H2O and the mixture was autoclaved. After autoclaving, the flasks were supplemented with the supplements as indicated in Table 1 1 , below.

[0196] 100 ml of YPD was mixed with Saccharomyces cerevisiae (WLP001

CALIFORNIA ALE YEAST® (LN 1075337)) and shaken to homogeneity (1 packet per 200 ml). After mixing, a cell count revealed the inoculated YPD media contained approximately 5.34 x 10 ⁸ total cells. 1 mL of the inoculated YTD media was added to each condition.

[0197] After inoculation, the media were incubated at 27 °C for 18 hours with shaking (150 rpm). After incubation, 5 mL was removed for cell count and pH measurements and the samples were each distributed into three centrifuge bottles and the yeast were pelleted at 4000 rpm for 3 minutes. The supernatant was removed from each bottle. For each sample, the pellet was resuspended in 50 ml of distilled water.

[0198] The samples were transferred to silicone mold trays and flash frozen by submersion in liquid nitrogen. The silicone molds were then stored at -80 °C. After storage, the molds were placed in a freeze drier for a total of about 18 hours. The final product was homogenized by mechanical mixing with a scoopuia. [0199] After freeze drying, the samples were prepared for mass spectrometry analysis to determine the percent by weight (%/w) of each of NAM, NA, NAD, NADH, NADP, NADPH, NMN, NAMN, NR, NAR, MNA, and trigonelline (trig ). The results are summarized in Table 12, below.

[0200] Adenine concentration in growth media was positively correlated with % nicotinamide riboside (p=0.03), % NADH ί r 0.02). and % total without NA & NAM (p=0.047). Adenine concentration in growth media was negatively correlated with % NAM (p=0.02) and % total (p=0.057). These observations lead us to conclude that the addition of adenine stimulates yeast consumption of NAM and yeast production of NAD metabolites m a dose responsive manner.