Yeast powder rich in nicotinamide mononucleotide, its production method and use

Abstract

The present invention discloses a yeast powder enriched in nicotinamide mononucleotide, as well as its production method and use. The production method includes the steps of preparing a first medium, a second medium, and a third medium; inoculating yeast into the first medium and fermenting it to obtain a first fermentation broth; inoculating the first fermentation broth into a second medium and fermenting it to obtain a second fermentation broth; inoculating the second fermentation broth into a third medium and fermenting it to obtain a third fermentation broth; centrifuging the third fermentation broth to obtain a fermented product; and drying the fermented product to obtain a yeast powder. The first, second, and third media contain nicotinamide, tryptophan, and nicotinic acid. The nicotinamide mononucleotide content of the yeast powder is at least greater than 5,000 ppm.

Description

[Technical field]

[0001] The present invention relates to a yeast powder, and in particular to a method for producing yeast powder that is rich in nicotinamide mononucleotide and has a variety of uses, and to the yeast powder produced by this method. [Background technology]

[0002] Research has shown that nicotinamide adenine dinucleotide (NAD + ) is involved in anti-aging and NAD + is a component whose major physiological functions include generating more than 95% of the energy for vital activities, repairing damaged DNA, priming sirtuins, etc., and maintaining mitochondrial activity in the body.

[0003] However, dietary NAD + cannot be obtained directly, and as humans age, NAD is naturally produced in the body. + Therefore, the NAD content in the body is reduced. + To replenish and maintain NAD levels + By ingesting the precursor of NAD + Studies have shown that the content can be increased.

[0004] For example, when nicotinamide mononucleotide (NMN) is ingested, it is transported to the bloodstream and converted into NAD + and promotes the removal of aging substances accumulated in the body. Summary of the Invention

[0005] The content of natural nicotinamide mononucleotide in plants (such as broccoli, avocado, etc.) is very low and difficult to extract, so currently, most of the commercially available nicotinamide mononucleotide is a chemically synthesized formula. However, the process of chemically synthesizing nicotinamide mononucleotide may involve the generation of by-products present in nicotinamide mononucleotide products, and when such nicotinamide mononucleotide products are ingested by the human body, these by-products are prone to become a metabolic burden on the human liver.

[0006] In view of this, the present invention provides a method for producing yeast powder rich in nicotinamide mononucleotide and the yeast powder produced by this production method, and also provides uses of the yeast powder produced by this production method for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue level, etc.

[0007] In some embodiments, a method for producing yeast powder rich in nicotinamide mononucleotide includes the steps of preparing a first medium, a second medium and a third medium, inoculating yeast into the first medium and fermenting to obtain a first fermentation liquid, inoculating the first fermentation liquid into a second medium and fermenting to obtain a second fermentation liquid, inoculating the second fermentation liquid into a third medium and fermenting to obtain a third fermentation liquid, centrifuging the third fermentation liquid to obtain a fermented product, and drying the fermented product to obtain a yeast powder. The composition components of the first medium, the second medium and the third medium include nicotinamide (NAM), tryptophan and nicotinic acid. The content of nicotinamide mononucleotide (NMN) contained in the yeast powder is at least greater than 5000 ppm.

[0008] In some embodiments, the yeast is inoculated into a first medium at an inoculation rate of 5% by volume, the first fermentation broth is inoculated into a second medium at an inoculation rate of 6% by volume, and the second fermentation broth is inoculated into a third medium at an inoculation rate of 10% by volume.

[0009] In some embodiments, the concentration of nicotinamide is between 0.01% and 0.3% by weight, the concentration of tryptophan is between 0.1% and 0.5% by weight, and the concentration of nicotinic acid is between 0.01% and 0.06% by weight.

[0010] In some embodiments, the concentration of nicotinamide is 0.1% by weight, the concentration of tryptophan is 0.2% by weight, and the concentration of nicotinic acid is 0.0369% by weight.

[0011] In some embodiments, the volume ratio of the first medium, the second medium and the third medium is 3:50:500.

[0012] In some embodiments, the compositional components of the first medium, the second medium, and the third medium further comprise any one of the following extracts: banana peel extract, chocolate berry extract, and black tomato extract.

[0013] In some embodiments, the yeast powder is produced by the method of claim 1, and the content of nicotinamide mononucleotide (NMN) contained in the yeast powder is at least 5000 ppm.

[0014] In some embodiments, the use of yeast powder for producing a composition for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging, and / or improving physical fatigue levels comprises the yeast powder produced by the production method of claim 1, and the content of nicotinamide mononucleotide (NMN) contained in the yeast powder is at least 5000 ppm.

[0015] In some aspects, the improvement in skin tone is increased skin firmness, reduced wrinkles, reduced roughness, or a combination thereof.

[0016] In some aspects, hair care is reducing hair loss or lessening the extent of hair thinning.

[0017] In some embodiments, the yeast powder is 10 It further contains biosynthetic proteins (COQ proteins).

[0018] In some aspects, the yeast powder is for regulating blood lipids.

[0019] In some embodiments, the yeast powder is for reducing the expression of C-reactive protein (CRP) in a subject.

[0020] In some aspects, the yeast powder achieves anti-aging by regulating the expression levels of anti-aging related genes, activating mitochondria, or reducing brain age.

[0021] In some embodiments, the anti-aging related genes include a CCT gene, a PARP2 gene, a Parkin gene, an Atg gene, a FOXO gene, a SIRT1 gene, a NADSYN gene, a MRPS5 gene, a SOD3 gene, or a combination thereof.

[0022] In some embodiments, the yeast powder achieves its antioxidant function by reducing damage caused by reactive oxygen species (ROS).

[0023] In some embodiments, the daily dose of yeast powder is 100 mg.

[0024] From the above, according to the method for producing yeast powder rich in nicotinamide mononucleotide of any one of the embodiments, yeast powder rich in nicotinamide mononucleotide can be produced, and the content of nicotinamide mononucleotide contained in this yeast powder is at least 5000 ppm. The problem that conventional nicotinamide mononucleotide can only be obtained by chemical synthesis and harmful by-products are generated is solved, and the technical bottleneck that conventional nicotinamide mononucleotide cannot be eaten and can only be used externally is also solved. And, the yeast powder produced by the method for producing yeast powder rich in nicotinamide mononucleotide of any one of the embodiments has functions such as improving skin condition (e.g., functions such as enhancing skin firmness, reducing wrinkles, and reducing rough skin), hair care (e.g., functions such as reducing hair loss or reducing the degree of thinning hair), anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue, and can be used to produce a composition having the above functions. In some embodiments, the yeast powder is Q 10 Further comprising biosynthetic protein (COQ protein). In some embodiments, the yeast powder has one or any combination of the following functions: regulating blood lipids, lowering the expression level of C-reactive protein (CRP) in a specimen, regulating the expression level of anti-aging related genes (e.g., CCT gene, PARP2 gene, Parkin gene, Atg gene, FOXO gene, SIRT1 gene, NADSYN gene, MRPS5 gene, SOD3 gene, or a combination thereof), achieving anti-aging by activating mitochondria or reducing brain age, achieving antioxidant function by reducing damage caused by reactive oxygen species (ROS), etc.

[0025] The present invention will now be described in detail with reference to the drawings and specific examples, which are not intended to limit the present invention. [Brief description of the drawings]

[0026] [Figure 1] 1 is a flow chart for the production of nicotinamide mononucleotide enriched yeast powder. [Diagram 2] FIG. 1 shows the experimental results of an analysis of NMN yields from different bacterial species. [Diagram 3] FIG. 1 shows the experimental results of NMN yield analysis using different fermentation medium components. [Figure 4] FIG. 1 shows the experimental results of NMN yield analysis using plant extracts and compounds. [Diagram 5] FIG. 1 shows experimental results of relative ROS production. [Figure 6] FIG. 1 shows experimental results of a mitochondrial activity test. [Figure 7] FIG. 1 shows the experimental results of the relative expression level of the PARP2 gene. [Figure 8] FIG. 1 shows experimental results of relative expression levels of CCT-related genes. [Figure 9] FIG. 1 shows experimental results of relative expression levels of anti-aging-related genes. [Figure 10] FIG. 1 shows the experimental results of the relative expression levels of the NADSYN gene and the MRPS5 gene. [Figure 11] FIG. 1 shows experimental results of relative expression levels of anti-aging-related genes at 0 and 8 weeks. [Figure 12] FIG. 1 shows the experimental results of C-reactive protein content at 0 and 8 weeks. [Figure 13] FIG. 1 shows experimental results of low-density cholesterol content at 0 and 8 weeks. [Figure 14] FIG. 1 shows experimental results of low-density cholesterol / high-density cholesterol ratio at 0 and 8 weeks. [Figure 15] FIG. 13 is a graph showing experimental results of wrinkle percentage at 0 and 8 weeks. [Figure 16] FIG. 13 shows experimental results of skin texture percentage at 0 and 8 weeks. [Figure 17] FIG. 1 shows experimental results of brain age at 0 and 8 weeks. [Figure 18] Experimental results of the overall physical condition evaluation at weeks 0 and 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In the following description of the embodiments, unless otherwise specified, the symbol "%" means percent by weight, and the symbol "volume %" means volume percent concentration. In addition, the following number terms such as "first", "second", "third", ..., etc. are intended to distinguish the components shown, and are not intended to order the components shown or limit the differences between them, and are not intended to limit the scope of the present invention.

[0028] Referring to Fig. 1, first, second and third culture media are prepared (step S100). Here, the first, second and third culture media have the same compositional components, and contain components such as a basal medium, nicotinamide (NAM), tryptophan and nicotinic acid (Niacin). For example, the first medium, the second medium, and the third medium are fermentation media for carrying out fermentation, and their components include a basal medium consisting of yeast peptone, yeast extract, potassium dihydrogen phosphate (KH2PO4), potassium hydrogen phosphate (K2HPO4), magnesium sulphate-7hydrate (MgSO4·7H2O), glucose, citric acid, sodium acetate, manganese gluconate, cysteine, deionized distilled water (ddH2O) and an antifoaming agent, and other components such as nicotinamide, tryptophan, and nicotinic acid. In some embodiments, glucose is a component of the basal medium, but is sterilized separately from the other components before inoculating the fermentation medium with the strain, and is mixed with the other components to form the sterilized fermentation medium before inoculating the strain.

[0029] In some embodiments, the compositional components of the first medium, the second medium, and the third medium contain 0.01% to 0.3% nicotinamide, 0.1% to 0.5% tryptophan, and 0.001% to 0.06% nicotinic acid in addition to the basal medium. Preferably, the compositional components of the first medium, the second medium, and the third medium contain 0.1% nicotinamide, 0.2% tryptophan, and 0.0369% nicotinic acid in addition to the basal medium. Based on this, when the fermentation medium contains nicotinamide, tryptophan, and nicotinic acid, which are precursors for the synthesis of nicotinamide mononucleotide, it is advantageous for the synthesis of nicotinamide mononucleotide by yeast.

[0030] In some embodiments, the compositional components of the first medium, the second medium and the third medium further include one or more of the following extracts: Musa extract, Aronia extract, and Solanum extract. Here, the Musa extract may be banana peel extract, the Aronia extract may be chocolate berry extract, and the Solanum extract may be black tomato extract. For example, when the compositional components of the fermentation medium are supplemented with 0.05% banana peel extract, 0.085% chocolate berry extract or 0.05% black tomato extract in addition to the basal medium, nicotinamide, tryptophan and nicotinic acid, all of these are advantageous for improving the nicotinamide mononucleotide content in the fermentation liquid.

[0031] In some embodiments, for the banana peel extract, the peel and pulp of bananas (Musa spp.) may be separated, and the banana peel may be treated as follows to provide a banana peel extract: 1. Mix the banana peel with an extraction solvent (the volume ratio of banana peel to extraction solvent is 1:6, and the extraction solvent system is provided in a volume ratio dosage of citric acid:water=1:100), and extract at 85°C for 0.5 hours to provide a crude extract; 2. Centrifuge the crude extract of step 1 at a rotation speed of 5000 rpm. After removing the supernatant for 10 minutes, filter through a 400 mesh filter to provide a filtrate; 3. Concentrate the filtrate of step 2 under reduced pressure at 55±5°C to provide a concentrated extract; 4. Freeze-dry the concentrated extract of step 3 to provide a dry product (i.e., the banana peel extract described above). In some embodiments, the chokeberry extract is a concentrated juice extracted from the fruit of black chokeberry (also known as chocoberry, scientific name Aronia melanocarpa, English name Black chokeberry). In some embodiments, the black tomato extract is obtained by cutting black tomatoes (Solanum lycopersicum 'Kumato') into blocks and grinding them homogeneously to form a black tomato homogenate, and then extracting the black tomato homogenate with water at 40°C to 60°C for 0.5 to 2 hours, with a liquid-solid ratio of water to black tomato homogenate of 5-20:1-5.

[0032] In some embodiments, the volume ratio of the first medium, the second medium, and the third medium is 3:50:500.

[0033] Continuing from step S100. The yeast is inoculated on the first medium and fermented to obtain a first fermentation liquid (step S200). Here, the yeast used may be Saccharomyces cerevisiae. For example, the Saccharomyces cerevisiae may be a commercially available Saccharomyces cerevisiae or Saccharomyces cerevisiae TCI907. Saccharomyces cerevisiae TCI907 is a strain of Saccharomyces cerevisiae isolated from draft beer. Saccharomyces cerevisiae TCI907 is deposited at the Food Industry Development Institute Foundation under the deposit number BCRC 920118 and at the German Microorganism Collection Center under the deposit number DSZ33480. Saccharomyces cerevisiae TCI907 is an aerobic yeast and is ovoid. The colonies of Saccharomyces cerevisiae TCI907 are opaque milky white, with smooth surfaces and regular edges. The growth temperature of the budding yeast TCI907 is 28°C to 37°C. The budding yeast TCI907 can survive in an environment with an acid-base value (pH value) of 3 to 7. In some embodiments, the budding yeast TCI907 has the ability to resist gastric acid bile salts. For example, the budding yeast TCI907 has a survival rate of 99.4% in a stomach simulation environment (pH value 3 to 4) and a survival rate of 99.8% in an intestine simulation environment (pH value 7).

[0034] In some embodiments, the yeast is inoculated into the first medium at a seeding amount of 5% by volume. For example, before seeding the yeast, 3 L of the first medium is first prepared and sterilized at high temperature at 121° C. for 30 minutes. After it is determined that the components contained in the first medium are completely dissolved and the temperature is lowered, 5% by volume (corresponding to 150 mL) of the yeast is further inoculated into the first medium for subsequent fermentation. Here, the OD of the yeast for seeding is 600 is 6~8.

[0035] In some embodiments, during fermentation of the first fermentation broth, the fermentation temperature is maintained at 30±1° C., the acid-base value (pH value) is maintained at 6.5±0.1, and the dissolved oxygen (DO) value is maintained at 40 mg / L to 50 mg / L. The pH value of the first fermentation broth is adjusted and controlled with 10 N sodium hydroxide (NaOH).

[0036] Then, the first fermentation liquid is fermented in the fermentation tank. For example, 3 L of the first medium and 150 mL of yeast can be fermented in a 5 L fermentation tank. In some embodiments, the aeration amount set during the fermentation of the first fermentation liquid is set to 3 L / min and the stirring speed is set to 250 rpm, thereby ensuring that the fermentation is carried out in an oxygen environment during fermentation. Also, before starting fermentation, the first medium is stirred with an aeration amount of 3 L / min and a stirring speed of 250 rpm, and the inoculation can be carried out only after the dissolved oxygen amount is greater than 40%.

[0037] In some embodiments, the fermentation time required for the first fermentation liquid is 4 to 5 hours, and the OD of the yeast in the first fermentation liquid after fermentation is completed is 600 is 4.0~5.0.

[0038] Continuing from step S200, the first fermentation broth is inoculated onto a second medium and fermented to obtain a second fermentation broth (step S300).

[0039] In some embodiments, the first fermentation broth is inoculated into the second medium at a seeding amount of 6% by volume. For example, before inoculating the first fermentation broth, 50 L of the second medium is first prepared and sterilized at high temperature for 25 to 30 minutes at 121°C. After it is determined that the components contained in the second medium are completely dissolved and the temperature is lowered, 6% by volume (corresponding to 3 L) of the first fermentation broth is further inoculated into the second medium to carry out the subsequent fermentation.

[0040] In some embodiments, during fermentation of the second fermentation liquor, the fermentation temperature is maintained at 30±1° C., the acid-base value (pH value) is maintained at 6.5±0.1, and the dissolved oxygen (DO) value is maintained at 40 mg / L to 50 mg / L, and the pH value of the second fermentation liquor is adjusted and controlled with 10 N sodium hydroxide (NaOH).

[0041] The second fermentation broth is then fermented in the fermenter. For example, 50 L of the second medium and 3 L of the first fermentation broth can be fermented in a 75 L fermenter. In some embodiments, the aeration rate set during the fermentation of the second fermentation broth is 50 L / min, the stirring speed is set at 200 rpm, and the pressure value of the fermenter internal pressure is 0.3±0.1 kg / cm. 2 This ensures that the fermentation takes place in an oxygen-rich environment during fermentation. Before starting fermentation, the second medium is first stirred at an aeration rate of 30 L / min and an agitation speed of 200 rpm, and the amount of dissolved oxygen in the medium is increased to more than 40% before inoculation.

[0042] In some embodiments, the fermentation time required for the second fermentation liquid is 4 to 5 hours, and the OD of the yeast in the second fermentation liquid after fermentation is completed is 600 is 4.0~5.0.

[0043] Continuing from step S300, the second fermentation broth is inoculated onto a third medium and fermented to obtain a third fermentation broth (step S400).

[0044] In some embodiments, the second fermentation broth is inoculated into the third medium at a seeding rate of 10% by volume. For example, 500 L of the third medium is first inoculated with the second fermentation broth. 3 The medium is prepared and sterilized at a high temperature of 121° C. for 30 minutes. After it is determined that the components contained in the third medium are completely dissolved and the temperature is lowered, 10% by volume (corresponding to 50 L) of the second fermentation liquid is seeded in the third medium to carry out the subsequent fermentation.

[0045] In some embodiments, during the fermentation of the third fermentation liquor, the fermentation temperature is maintained at 30±1° C., the acid-base value (pH value) is maintained at 6.5±0.1, and the dissolved oxygen (DO) value is maintained at 40 mg / L to 60 mg / L, and the pH value of the third fermentation liquor is adjusted and controlled with 10 N sodium hydroxide (NaOH).

[0046] The third fermentation broth is then fermented in a fermenter. For example, 500 L of the third medium and 50 L of the second fermentation broth can be fermented in a 750 L fermenter. In some embodiments, the aeration rate set during the fermentation of the third fermentation broth is 500 L / min, the stirring speed is set at 100 rpm, and the pressure value of the fermenter is 0.3±0.1 kg / cm. 2 This ensures that the fermentation takes place in an oxygen-rich environment during fermentation. Before starting fermentation, the third medium is first stirred at an aeration rate of 100 L / min and an agitation speed of 200 rpm, and the amount of dissolved oxygen in the medium is greater than 40% before inoculation.

[0047] In some embodiments, the fermentation time required for the third fermentation broth is 12 to 14 hours, and the OD of the yeast in the third fermentation broth after fermentation is completed is 600 is 48.0 to 53.0.

[0048] Continuing from step S400, the third fermentation broth is centrifuged to obtain a fermentate (step S500). In some embodiments, a centrifuge is used to separate the fermentate from the third fermentation broth, and the OD of the fermentate is 600 is 500, and the solid content is greater than 65% and less than 80%.

[0049] In some embodiments, the acid-base value (pH value) during centrifugation is 6.5±0.1. In some embodiments, the tank pressure of a 750 L centrifuge is 1.0 kg / cm 2 and the set flow rate is 0.3m 3 / h.

[0050] Here, the "fermented product" is a yeast fermentation product, and includes yeast cells, the yeast's own proteins (derived from a small number of yeast cells disrupted during the manufacturing process), and a fermentation liquid (including a medium) containing metabolic products of the yeast. The metabolic products of the yeast include substances secreted by the yeast into the fermentation medium during cultivation, such as nicotinamide mononucleotide (NMN) and coenzyme Q. 10 For example, the fermentation product contains nicotinamide mononucleotide (NMN), Q10 Biosynthetic proteins (COQ proteins) and coenzyme Q 10 The compounds include several compounds such as:

[0051] Continuing from step S500. After drying the fermentation product, yeast powder is obtained (step S600). The obtained yeast powder contains at least 1.0×10 9 CFU / g of yeast cells are included. Examples of drying methods include freeze-drying, low-temperature drying, dehydration drying, etc.

[0052] In some embodiments, the crude fermentate is freeze-dried and pulverized to obtain the fermentate, and a lyoprotectant is added during the freeze-drying step to protect the cells in the crude fermentate from destruction. Examples of lyoprotectants include skim milk powder, maltodextrin, sucrose, and glycerin.

[0053] Here, the content of nicotinamide mononucleotide (NMN) contained in the obtained yeast powder is at least greater than 5000 ppm, preferably 5000 ppm to 10000 ppm. For example, the content of nicotinamide mononucleotide contained in the yeast powder is 5264.15 ppm.

[0054] In some embodiments, the yeast powder contains 10 It also contains biosynthetic proteins (COQ proteins). 10 Biosynthetic proteins are synthesized by yeast 10 Q can aid in protein synthesis. 10 The increased content of biosynthetic proteins increases the amount of coenzyme Q synthesized in yeast. 10 Based on this, the yeast powder has the potential to increase the coenzyme Q content. 10 may be included.

[0055] In some embodiments, the yeast powder produced by the above production flow can have a nicotinamide mononucleotide content that is at least two-fold higher when fermented using a fermentation medium (i.e., the first medium, the second medium, and the third medium) containing components such as nicotinamide, tryptophan, and nicotinic acid, compared to a yeast powder produced using a fermentation medium that does not contain nicotinamide, tryptophan, or nicotinic acid.

[0056] In some embodiments, the nicotinamide (NAM) content in the fermentation medium (i.e., medium 1, medium 2, and medium 3) is 0.1% and the yeast has a favorable yield of nicotinamide mononucleotide, and when the nicotinamide content in the fermentation medium is greater than 1.1%, the yeast yield of nicotinamide mononucleotide decreases to or below the yield of nicotinamide mononucleotide in the medium without nicotinamide added.

[0057] In some embodiments, when one or more plant extracts of Solanum genus plant extract (e.g., black tomato extract), Aronia genus plant extract (e.g., chocolate berry extract), and Musa genus plant extract (e.g., banana peel extract) are added to the fermentation medium, it is advantageous to improve the nicotinamide mononucleotide (NMN) content in the produced yeast powder. For example, yeast powder produced in a fermentation medium further containing banana peel extract can improve the nicotinamide mononucleotide content in the yeast powder by at least 1.1 times. In some examples, yeast powder produced in a fermentation medium further containing chocolate berry extract can improve the nicotinamide mononucleotide content in the yeast powder by at least 1.47 times. In some embodiments, yeast powder produced in a fermentation medium further containing black tomato extract can improve the nicotinamide mononucleotide content in the yeast powder by at least 1.19 times.

[0058] Based on these, the yeast powder rich in nicotinamide mononucleotide (NMN) produced by the production method of any one of the embodiments not only reliably improves the content of nicotinamide mononucleotide, but the following several examples further show that the yeast powder rich in nicotinamide mononucleotide can be used for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue, and can also be used to produce a composition for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue.

[0059] In some embodiments, the yeast powder can be used to improve the skin condition of a subject by increasing skin firmness, reducing wrinkles, reducing rough skin of a subject, or a combination thereof. For example, when a subject takes yeast powder, it can reduce wrinkles and skin texture of the subject, increase the skin firmness of the subject, smooth and elasticize the skin of the subject, and improve the overall skin condition. Based on these, yeast powder has the function of improving skin condition.

[0060] In some embodiments, yeast powder can reduce the hair loss of a subject and / or reduce the degree of thinning hair of the subject to achieve the effect of hair care.For example, when a subject takes yeast powder, it can reduce the hair loss condition of the subject and reduce the degree of thinning hair of the subject.Based on this, yeast powder has the function of hair care.

[0061] In some embodiments, the yeast powder can reduce the content of reactive oxygen species (ROS) in cells. For example, when a subject takes yeast powder, it can make the subject's cells resist cellular oxidative stress and achieve an antioxidant effect.

[0062] In some embodiments, the yeast powder can activate mitochondrial activity. For example, when a subject ingests yeast powder, it can activate mitochondrial activity in the subject's cells, further achieving antioxidant and / or anti-aging effects.

[0063] In some embodiments, the yeast powder can achieve anti-aging by regulating the expression level of anti-aging-related genes in a specimen, activating mitochondria, or reducing brain age. For example, the anti-aging genes include CCT genes, PARP2 genes (Gene ID: 10038), Parkin genes (Gene ID: 5071), Atg genes, FOXO genes (Gene ID: 2308), SIRT1 genes (Gene ID: 23411), NADSYN genes (Gene ID: 55191), MRPS5 genes (Gene ID: 64969), SOD3 genes (Gene ID: 6649), or combinations thereof. Here, the CCT genes include CCT5 genes (Gene ID: 22948), CCT6A genes (Gene ID: 908), CCT7 genes (Gene ID: 10574), and CCT8 genes (Gene ID: 10694). The Atg genes include the Atg1 gene (Gene ID: 8408) and the Atg8 gene (Gene ID: 11345).

[0064] In some embodiments, when the subject takes yeast powder, the expression level of the CCT gene, the Parkin gene, the Atg gene, the FOXO gene, the SIRT1 gene, the NADSYN gene, the MRPS5 gene, the SOD3 gene, or a combination thereof can be improved, and the expression level of the PARP2 gene can be suppressed. For example, by improving the expression level of the CCT gene, which is a folded protein (Chaperonin), it is possible to promote the correction of misfolded proteins, and send unsuccessfully repaired proteins to the protease complex (Proteasome) for hydrolysis, thereby avoiding the decline of cell function and the accelerated aging and death of cells, and thus exerting an anti-aging cell regulation effect. By improving the expression level of the Parkin gene, it is possible to promote the recovery of mature cells to young cells. By suppressing the expression level of the PARP2 gene, it is possible to promote the activity of telomerase and the rejuvenation of cells, and ensure that the DNA ends are not deficient in telomerase during replication, thereby reducing the probability of cellular aging. By improving the expression levels of the Atg gene and the MRPS5 gene, it is possible to improve the anti-aging ability of cells and extend the lifespan of cells. By increasing the expression of FOXO genes, aged cells can be eliminated and young cells can be protected. By increasing the expression of SIRT1 genes, important metabolic functions of the body can be regulated. By increasing the expression of NADSYN genes, antioxidant effects can be achieved.

[0065] In some embodiments, the subject receives yeast powder and then supplements it with nicotinamide mononucleotide to increase nicotinamide adenine dinucleotide (NAD) levels in the subject's blood. + It has been shown that the expression levels of downstream regulatory genes (e.g., SIRT1 gene and SOD3 gene) of yeast serotonin A (SOD1) are improved, and that yeast powder can activate mitochondria in the test cells and has anti-aging properties.

[0066] In some embodiments, the yeast powder is for reducing the expression level of C-reactive protein (CRP) in a subject. The amount of C-reactive protein in blood can be increased in the situations of body tissue damage, chronic inflammation, etc., so C-reactive protein can be used as an indicator of inflammatory response and risk of cardiovascular disease. For example, it has been shown that when a subject takes yeast powder, the content of C-reactive protein in the blood can be reduced, and the risk of the subject suffering from cardiovascular disease can be reduced. Based on this, yeast powder has an anti-inflammatory function.

[0067] In some embodiments, yeast powder can be used to regulate blood lipids. Regulating blood lipids means suppressing the content of low-density cholesterol (LDL-C) and lowering the low-density cholesterol / high-density cholesterol ratio (T.CHOL / HDL). For example, when a subject takes yeast powder and then supplements with nicotinamide mononucleotide, the content of low-density cholesterol in the subject's blood is reduced, and the low-density cholesterol / high-density cholesterol ratio is reduced, which indicates that the subject's risk of suffering from coronary artery sclerosis is reduced. Based on this, yeast powder can be used to regulate blood lipids and has the function of cardiovascular health care.

[0068] In some embodiments, yeast powder can reduce brain age.For example, when a subject takes yeast powder, the detected brain age can be reduced by at least 10 years, and when the brain age is detected, the subject's mental clarity is improved, and the detected brain age is relatively young.Based on this, yeast powder has anti-aging function.

[0069] In some embodiments, the yeast powder can be used to improve the subject's physical fatigue level. For example, when a subject takes yeast powder, the subject's sense of physical fatigue can be effectively reduced.

[0070] In some embodiments, the yeast powder can be redissolved in a lysis buffer and reacted with DNase at a concentration of 1 μg / mL for 10 minutes to degrade the DNA of the yeast powder. It is then sterilized three times in a high-pressure sterilizer at pressure values ​​of 25 kilopounds per square inch (Kpsi), 30 Kpsi, and 32 Kpsi in sequence to form a yeast extract. The yeast extract contains at least 5000 ppm nicotinamide mononucleotide. In some embodiments, the yeast extract contains Q 10 Biosynthetic proteins (COQ proteins) and coenzyme Q 10 Since the yeast extract is made from yeast powder, the yeast extract also has the same functions as the yeast powder described above, such as improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging, and / or improving physical fatigue.

[0071] Here, the "yeast extract" refers to the self-proteins of yeast and substances contained in the raw material (yeast powder), and other substances contained in the raw material (yeast powder) may be metabolic products of yeast and culture medium.

[0072] Based on this, the nicotinamide mononucleotide-rich yeast powder produced by the production method of any one of the embodiments and / or the yeast extract produced with the yeast powder can be used to produce a composition for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue. The composition contains at least 5000 ppm nicotinamide mononucleotide. In some embodiments, the composition contains at least 5000 ppm nicotinamide mononucleotide. 10 It further contains biosynthetic proteins (COQ proteins).

[0073] In some embodiments, the composition may be a solid (e.g., a powder, tablet, capsule, etc.). In some embodiments, the daily dose of yeast powder is 100 mg. That is, the amount of the composition used is 100 mg of yeast powder per day.

[0074] In some embodiments, any one of the compositions described above may be a pharmaceutical product, i.e., the pharmaceutical product comprises an effective amount of yeast powder and / or a yeast extract prepared with yeast powder.

[0075] In some embodiments, the pharmaceutical agents described above can be prepared into dosage forms suitable for enteral, parenteral, oral, or topical administration using techniques well known to those skilled in the art.

[0076] In some embodiments, the enteral or oral dosage form may be, but is not limited to, a tablet, a troche, a lozenge, a pill, a capsule, a dispersible powder or granule, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, or the like. In some embodiments, the parenteral or topical dosage form may be, but is not limited to, an injection, a sterile powder, an external preparation, or the like. In some embodiments, the mode of administration of the injection may be a subcutaneous injection, an intraepidermal injection, an intradermal injection, or an intralesional injection.

[0077] In some embodiments, the pharmaceutical preparation may include a pharma- ceutical acceptable carrier commonly used in the pharmaceutical manufacturing art. In some embodiments, the pharma-ceutical acceptable carrier may be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The type and number of carriers selected is within the expertise and ordinary skill of the artisan. In some embodiments, the solvent for the pharma- ceutically acceptable carrier may be water, normal saline, phosphate buffered saline (PBS), or an aqueous solution containing alcohol.

[0078] In some embodiments, any one of the compositions described above may be an edible product. In other words, the edible product comprises a specific content of yeast powder and / or yeast extract made with yeast powder. In some embodiments, the edible product may be a general food, health food, or dietary supplement.

[0079] In some embodiments, the edible products described above can be manufactured into a suitable dosage form for oral administration using techniques well known to those skilled in the art. In some examples, the common food products described above can be the edible products themselves. In some examples, the common food products can be, but are not limited to, beverages, fermented foods, bakery products, or condiments.

[0080] In some embodiments, the obtained yeast powder and / or yeast extract produced from the yeast powder may further be used as a food additive for producing a food composition containing the yeast powder and / or yeast extract produced from the yeast powder. Here, the yeast powder of any one of the Examples may be added during the production of raw materials by conventional methods, or the yeast powder of any one of the Examples may be added during the production process of food, and then formulated together with any one of the edible materials as an edible product (i.e., a food composition) for consumption by humans and non-human animals.

[0081] Example 1 Fermentation Process - Production of Nicotinamide Mononucleotide (NMN)-Enriched Yeast Powder The fermentation medium is prepared and divided into the first medium, the second medium and the third medium according to the fermentation order. Here, the fermentation medium is composed of a basal medium, nicotinamide, tryptophan and nicotinic acid, and its composition is 2.0% yeast peptone (English product name: Yeast peptone GLS; manufacturer: Shangding Biotechnology Co., Ltd.), 2.0% yeast extract 902 (English product name: Angel yeast extract 902; manufacturer: Deon Seiyi Co., Ltd.), 0.5% potassium dihydrogen phosphate (manufacturer: Sigma-Aldrich Co., Ltd.), 0.5% dipotassium hydrogen phosphate (manufacturer: Kyomei Chemical Co., Ltd.), 0.05% magnesium sulfate (manufacturer: Jingren Industrial Co., Ltd.), 10% glucose (manufacturer: Yugen Kogyo Co., Ltd.), 0.17% citric acid (manufacturer: Shinwa Foods Co., Ltd.), 0.5% sodium acetate (manufacturer: Shinwa Foods Co., Ltd.), 0.01% sodium sulfate ... The mixture is 6% manganese gluconate (manufacturer: Mei Long Trading Co., Ltd.), 0.1% cysteine ​​(manufacturer: Yugen Kogyo Co., Ltd.), 0.05% antifoaming agent (manufacturer: Kakang Industrial Co., Ltd.), 0.1% nicotinamide (manufacturer: Sigma-Aldrich Co., Ltd.), 0.2% tryptophan (manufacturer: Sigma-Aldrich Co., Ltd.), and 0.0369% nicotinic acid (manufacturer: Sigma-Aldrich Co., Ltd.), and is made up to 100% with deionized distilled water. Glucose is sterilized separately from the other components. Here, the volume of the first medium is 3L, the volume of the second medium is 50L, and the volume of the third medium is 500L.

[0082] 3L of the sterilized first medium was placed in a 5L fermenter, and the subsequent inoculation could be performed only after the dissolved oxygen content of the first medium was greater than 40%. Then, Saccharomyces cerevisiae TCI907 reached its OD 600 After activation to a pH of 6-8, the bacteria is inoculated into a first medium having an oxygen content of more than 40% with a seeding amount of 5% by volume (i.e., 150 mL) and fermented for the first time to form a first fermentation broth, with a fermentation time of 4-5 hours. During the fermentation of the first fermentation broth, the fermentation temperature is maintained at 30±1°C, the acid-base value (pH value) is 6.5±0.1, and the dissolved oxygen value is maintained in the range of 40mg / L-50mg / L.

[0083] When the first fermentation is complete, its OD600 The oxygen concentration of the second medium should be 4.0-5.0. Next, the first fermentation broth is seeded in a 75L fermenter containing 50L of the second medium at a seeding amount of 6% by volume (i.e., 3L) and fermented a second time to form a second fermentation broth, and the fermentation time is 4-5 hours. The dissolved oxygen content of the second medium should be greater than 40%. During the fermentation of the second fermentation broth, the fermentation temperature is maintained at 30±1°C, the acid-base value (pH value) is 6.5±0.1, and the dissolved oxygen value is maintained in the range of 40mg / L-50mg / L.

[0084] When the second fermentation is complete, its OD 600 The pH value of the third fermentation broth is 4.0-5.0. Next, the second fermentation broth is seeded in a 750L fermenter containing 500L of the third medium at a seeding amount of 10% by volume (i.e. 50L) and fermented a third time to form the third fermentation broth, and the fermentation time is 12-14 hours. The dissolved oxygen content of the third medium must be greater than 40%. During the fermentation of the third fermentation broth, the fermentation temperature is 30±1°C, the acid-base value (pH value) is 6.5±0.1, and the dissolved oxygen value is maintained in the range of 40mg / L-50mg / L.

[0085] When the third fermentation is complete, its OD 600 The OD value is 48.0 to 53.0. After the third fermentation liquid is transferred to a centrifuge, the solid content is greater than 65% and less than 80%, and the OD value is 48.0 to 53.0. 600 A fermented product having a molecular weight of more than 500 is obtained. Then, 40 g of skim milk powder, 120 g of maltodextrin, 80 g of sucrose and 40 g of glycerin are added as freeze-drying protection agents to each kilogram of the fermented product, and after freeze-drying and pulverization, yeast powder is obtained.

[0086] Example 2 Fermentation process - Yeast species test Here, nicotinamide mononucleotide (NMN) contained in yeast powder is used as the judgment indicator.

[0087] The experimental group was the yeast powder produced in Example 1, and the yeast strain used was Saccharomyces cerevisiae TCI907.

[0088] The yeast powder of the control group was produced according to the production flow of Example 1 except for replacing the strain used in Example 1. That is, the difference between the production flow of the control group and the production flow of Example 1 is that the yeast strain used is Torula yeast (Cyberlindnera jadinii) (purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute, Taiwan, China, accession number BCRC20325).

[0089] Weigh out 1 gram of yeast powder for the experimental group and the control group, dissolve each in 9 grams of water to form a yeast solution, and shatter the yeast cells on ice with a cell disrupter (manufacturer: Shangwei Co., Ltd.). Next, place the yeast solutions and reaction solutions that have been subjected to cell disruption treatment for the experimental group and the control group into 96-well plates, react for 2 minutes on ice, add 88% formic acid (commercial product used), and further react for 10 minutes at 37°C to obtain a test solution. Here, the reaction solution contains 20% acetophenone (commercial product used) soluble in DMSO, and 2M potassium hydroxide (commercial product used).

[0090] Commercially available NMN (manufacturer: Wuxi Xima Biotechnology Co., Ltd.) is serially diluted to prepare NMN samples of different concentrations, such as 0.04 mM to 0.000625 mM, and 250 μL of each is taken and placed in a 96-well plate for the creation of the subsequent NMN concentration reference line.

[0091] Next, the test solutions of the experimental and control groups, as well as the above-mentioned NMN samples of different concentrations, are measured for UV radiation at 445 nm with an excitation wavelength of 382 nm by a microplate detector (Microplate Reader; manufacturer: Thermo Fisher Scientific) to obtain corresponding readings, and the NMN contents of the experimental and control groups are converted by interpolation using the NMN concentration reference line created by the NMN samples of different concentrations, as shown in Figure 2.

[0092] Please refer to Figure 2. The nicotinamide mononucleotide content of the experimental group is 0.00845mM, and the nicotinamide mononucleotide content of the control group is 0.00565mM. In other words, the nicotinamide mononucleotide content of the yeast powder produced by Saccharomyces cerevisiae in the experimental group is 1.5 times that of the control group.

[0093] As can be seen from this, fermentation with Saccharomyces cerevisiae can improve the nicotinamide mononucleotide content in yeast powder.

[0094] Example 3 Fermentation process - Fermentation medium testing Here, nicotinamide mononucleotide (NMN) contained in yeast powder is used as the judgment indicator.

[0095] The experimental group was the yeast powder produced in Example 1, and the fermentation medium used was composed of 99.6631% basal medium, 0.1% nicotinamide (NAM), 0.2% tryptophan, and 0.0369% nicotinic acid (Niacin). The composition of the basal medium is 2.0% yeast peptone GLS, 2.0% yeast extract 902, 0.5% potassium dihydrogen phosphate (KH2PO4), 0.5% dipotassium hydrogen phosphate (K2HPO4), 0.05% magnesium sulfate (MgSO4·7H2O), 10% glucose, 0.17% citric acid, 0.5% sodium acetate, 0.016% manganese gluconate, 0.1% cysteine, 0.05% antifoam agent, and deionized distilled water (diluted to 99.6631%).

[0096] The yeast powder of the control group was produced according to the production flow of Example 1 except that the fermentation medium used in Example 1 was replaced. That is, the difference between the production flow of the control group and the production flow of Example 1 is that the fermentation medium used was a basal medium, and the components of the basal medium were 2.0% yeast peptone GLS, 2.0% yeast extract 902, 0.5% potassium dihydrogen phosphate (KH2PO4), 0.5% dipotassium hydrogen phosphate (K2HPO4), 0.05% magnesium sulfate (MgSO4·7H2O), 10% glucose, 0.17% citric acid, 0.5% sodium acetate, 0.016% manganese gluconate, 0.1% cysteine, 0.05% antifoaming agent, and deionized distilled water (diluted to 100%).

[0097] Weigh out 1 gram of yeast powder for the experimental group and the control group, dissolve each in 9 grams of water to form a yeast solution, and shatter the yeast cells on ice with a cell disrupter (manufacturer: Shangwei Co., Ltd.). Next, place the yeast solutions and reaction solutions that have been subjected to cell disruption treatment for the experimental group and the control group into 96-well plates, react for 2 minutes on ice, add 88% formic acid (commercial product), and further react for 10 minutes at 37°C to obtain the test solution. The reaction solution contains 20% acetophenone (commercial product) soluble in DMSO, and 2M potassium hydroxide (commercial product).

[0098] Commercially available NMN (manufacturer: Wuxi Xima Biotechnology Co., Ltd.) was serially diluted to prepare NMN samples of different concentrations, such as 0.04 mM to 0.000625 mM. To prepare a reference line of NMN concentrations, 250 μL of each was taken and placed in a 96-well plate, and the concentrations were converted to ppm.

[0099] Next, the test solutions of the experimental and control groups, as well as the above-mentioned NMN samples of different concentrations, were measured for UV radiation at 445 nm with an excitation wavelength of 382 nm using a microplate detector (Microplate Reader; manufacturer: Thermo Fisher Scientific) to obtain corresponding readings, and the NMN contents of the experimental and control groups were converted by interpolation using the NMN concentration reference line created by the NMN samples of different concentrations, as shown in Figure 3.

[0100] Please refer to Figure 3. The nicotinamide mononucleotide content of the experimental group is 5264.15 ppm, and the nicotinamide mononucleotide content of the control group is 2526.38 ppm. In other words, the nicotinamide mononucleotide content of the yeast powder produced by Saccharomyces cerevisiae in the experimental group is 2.1 times that of the control group.

[0101] As can be seen from this, fermentation using a fermentation medium containing nicotinamide (NAM), tryptophan and nicotinic acid (Niacin) can improve the nicotinamide mononucleotide content in yeast powder.

[0102] Example 4 Fermentation Process - Fermentation Media Testing (Plant Extracts and Compounds) Here, the nicotinamide mononucleotide (NMN) contained in the yeast powder is used as the judgment indicator, and the nicotinamide mononucleotide content ratio (hereinafter referred to as the NMN relative content ratio) of each group is determined based on the index of the nicotinamide mononucleotide content contained in the yeast of Example 1.

[0103] The control group was the yeast powder produced in Example 1, and the composition of the fermentation medium used therein was composed of 99.6631% basal medium, 0.1% nicotinamide, 0.2% tryptophan, and 0.0369% nicotinic acid. The composition of the basal medium was as described in Examples 1 and 2, so a detailed description is omitted.

[0104] The experimental groups are the banana peel extract group, the chocolate berry extract group and the black tomato extract group. The yeast powder of the experimental group is the yeast powder produced according to the production flow of Example 1 except that the fermentation medium used in Example 1 is replaced. That is, the difference between the production flow of the experimental group and the production flow of Example 1 is that the fermentation medium used contains 0.1% nicotinamide, 0.2% tryptophan, 0.0369% nicotinic acid and the corresponding percentage of plant extract composition. And the plant extract used in the banana peel extract group is 0.05% banana peel extract, the plant extract used in the chocolate berry extract group is 0.085% chocolate berry extract, and the plant extract used in the black tomato extract group is 0.05% black tomato extract, and the remaining amount is made up to 100% with the basal medium.

[0105] Here, for the banana peel extract used in the banana peel extract group, the peel and pulp of banana (Musa spp.) are separated, and the banana peel is treated as follows to obtain a banana peel extract: 1. The banana peel is mixed with an extraction solvent (the volume ratio of banana peel to extraction solvent is 1:6, and the extraction solvent system is provided in a volume ratio of citric acid:water=1:100), and extracted at 85°C for 0.5 hours to obtain a crude extract. 2. The crude extract of step 1 is centrifuged at a rotation speed of 5000 rpm. After taking the supernatant for 10 minutes, it is filtered through a 400 mesh filter to obtain a filtrate. 3. The filtrate of step 2 is concentrated under reduced pressure at 55±5°C to obtain a concentrated extract. 4. The concentrated extract of step 3 is freeze-dried to obtain a dry product (i.e., a banana peel extract). The chocolate berry extract used in the chocolate berry extract group is prepared by cutting black chokeberry (Aronia melanocarpa) fruits into blocks and grinding them homogeneously to form a black chokeberry homogenate, and then extracting the black chokeberry homogenate with water at 40°C to 60°C for 0.5 to 2 hours, with a liquid-solid ratio of water to black chokeberry homogenate of 5-20:1-5. The black tomato extract used in the black tomato extract group is prepared by cutting black tomatoes (Solanum lycopersicum 'Kumato') into blocks and grinding them homogeneously to form a black tomato homogenate, and then extracting the black tomato homogenate with water at 40°C to 60°C for 0.5 to 2 hours, with a liquid-solid ratio of water to black tomato homogenate of 5-20:1-5.

[0106] The control groups are the tannic acid group, the licorice extract group, and the passionflower extract group, respectively. The yeast powder of the control group is yeast powder produced in vitro according to the production flow of Example 1 by replacing the fermentation medium used in Example 1. That is, the difference between the production flow of the control group and the production flow of Example 1 is that the fermentation medium used contains 0.1% nicotinamide, 0.2% tryptophan, 0.0369% nicotinic acid, and the corresponding percentages of plant extracts or compound compositions. And the compound used in the tannic acid group is 5 μM tannic acid (manufacturer: Sigma-Aldrich), the plant extract used in the licorice extract group is 0.5% licorice extract, and the plant extract used in the passionflower extract group is 4% passionflower extract, and the remaining amount is made up to 100% with a basic medium.

[0107] The licorice extract used in the licorice extract group is prepared by finely chopping licorice, mixing the chopped licorice with a solid:liquid ratio of 1:2 to 1:10, and uniformly mixing the mixed solution with an aqueous solution containing 0.5% to 2% citric acid at 50°C to 100°C for 30 to 120 minutes. The mixed solution is then cooled to room temperature and filtered through a 200 micron filter to obtain the licorice extract. The passionflower extract used in the passionflower extract group is prepared by washing and drying the seeds of Passiflora edulis (Passiflora spp.) and crushing them in a homogenizer to form a passionflower seed homogenate, mixing the passionflower seed homogenate with water in a material:water ratio (weight) of 1:5, and then extracting at 55°C for 1 hour and then extracting at 85°C for 1 hour.

[0108] Weigh out 1 gram of yeast powder for the control group, experimental group, and control group, respectively, and dissolve them in 9 grams of water to form yeast solutions, and shatter the yeast cells on ice with a cell disrupter (manufacturer: Shangwei Co., Ltd.). Next, place the yeast solutions and reaction solutions that have been subjected to cell disruption treatment for the control group, experimental group, and control group into 96-well plates, react for 2 minutes on ice, add 88% formic acid (commercial product used), and further react for 10 minutes at 37°C to obtain test solutions. The reaction solution contains 20% acetophenone (commercial product used) soluble in DMSO, and 2M potassium hydroxide (commercial product used).

[0109] Commercially available NMN (manufacturer: Wuxi Xima Biotechnology Co., Ltd.) is serially diluted to prepare NMN samples of different concentrations, such as 0.04 mM to 0.000625 mM, and 250 μL of each is taken and placed in a 96-well plate for the creation of the subsequent NMN concentration reference line.

[0110] Next, the test solutions of the control group, experimental group, and control group, as well as the above-mentioned multiple different concentration NMN samples, are measured for UV radiation at 445 nm with an excitation wavelength of 382 nm using a microplate detector (Microplate Reader; manufacturer: Thermo Fisher Scientific) to obtain corresponding readings, and the NMN content of the control group, experimental group, and control group is converted by interpolation using the NMN concentration reference line created by the multiple different concentration NMN samples. Here, as shown in Figure 4, the reading value obtained in the control group is set to 1 to convert the relative NMN content ratio of the experimental group and the control group.

[0111] Please refer to Figure 4. The NMN relative content ratio of the control group is 1.0. Compared to the control group, the NMN relative content ratio of the tannic acid group in the control group is 0.9, the NMN relative content ratio of the licorice extract group is 0.62, and the NMN relative content ratio of the passionflower extract group is 0.74. Compared to the control group, the NMN relative content ratio of the banana peel extract group in the experimental group is 1.1, the NMN relative content ratio of the chocolate berry extract group is 1.47, and the NMN relative content ratio of the black tomato extract group is 1.19. In other words, compared to the control group and the control group, the yeast powders of each group produced by adding 0.05% banana peel extract, 0.085% chocolate berry extract, and 0.05% black tomato extract to the fermentation medium respectively have high nicotinamide mononucleotide content. And when 0.085% chocolate berry extract is added to the fermentation medium, the produced yeast powder can have nearly 1.5 times the nicotinamide mononucleotide content. Also, as can be seen from the experimental results of the control group, not all plant extracts can improve the nicotinamide mononucleotide content of yeast powder.

[0112] As can be seen from this, when banana peel extract, chocolate berry extract and black tomato extract are added to the fermentation medium, the nicotinamide mononucleotide content in the yeast powder produced therefrom can be improved.

[0113] Example 5 Cell experiment - Suppression of ROS generation (hydrogen peroxide treatment) Here, we combined the fluorescent probe DCFH-DA with flow cytometry to measure the change in reactive oxygen species (ROS) content in mouse brain neuroblastoma cells (Neuro2a) after treating them with yeast powder containing nicotinamide mononucleotide (NMN).

[0114] The medium used is Dulbecco's Modified Eagle Medium (DMEM; brand: Gibco) supplemented with 10% by volume of fetal bovine serum (FBS; brand: Gibco) and 1% by volume of penicillin / streptomycin (brand: Gibco), hereinafter referred to as cell medium. The DCFH-DA solution used is a reaction solution prepared by dissolving 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl sulfoxide (DMSO; purchased from Sigma, product number SI-D6883-50MG).

[0115] First, 2×10 5 Mouse neuroblastoma cells (ATCC CCL-131; hereafter referred to as Neuro2a cells) are placed into a 6-well cell culture plate containing 2 mL of cell medium in each well and cultured at 37 °C for 24 h.

[0116] After the Neuro2a cells in each well are attached to the bottom of a 6-well cell culture plate, the Neuro2a cells are divided into a blank group, a control group, a control group, and an experimental group. Then, the cell culture medium of each group is replaced with an experimental medium, followed by continuous culture at 37°C for 24 hours. The experimental medium of the blank group and the control group is a simple cell culture medium. The experimental medium of the control group is a cell culture medium to which chemically synthesized nicotinamide mononucleotide (manufacturer: Sigma-Aldrich) with a volume of 10 μL and a concentration of 200 mM has been added. The experimental medium of the experimental group is a cell culture medium containing 0.25% by volume of the NMN-rich yeast powder produced in Example 1.

[0117] Next, 2 μL of 5 μg / mL DCFH-DA solution was added to the experimental medium of each well in each group, and Neuro2a cells were treated for 15 minutes. After the DCFH-DA solution reacted, 10 μL of 200 mM hydrogen peroxide (Sigma-Aldrich) was added to the experimental medium of each group, and the mixture was allowed to react at 37°C for 1 hour.

[0118] Next, after removing the experimental medium of each group, wash the Neuro2a cells of each group twice with 1 mL of 1X PBS solution. Then, add 200 μL of trypsin to each well and react for 5 minutes. After the reaction, add 1 mL of cell culture medium to each well to terminate the reaction. Collect the Neuro2a cells and cell culture medium in each well individually in a corresponding 1.5 mL microcentrifuge tube, and centrifuge the microcentrifuge tube containing the Neuro2a cells and cell culture medium at 400 x g for 5 minutes. After centrifugation, remove the supernatant in the microcentrifuge tube of each group, and further re-dissolve the precipitate of Neuro2a cells with 1X PBS solution and centrifuge at 400 x g for 5 minutes. After centrifugation again, remove the supernatant in the microcentrifuge tube of each group, and resuspend the Neuro2a cells in 200 μL of 1X PBS solution per centrifuge tube in the dark to obtain the test cell solution of each group.

[0119] Flow cytometry (manufacturer: Beckman; Catalog No. 660519) is used to detect the fluorescent signal of DCFH-DA in the test cell solution of each group. The excitation wavelength of the fluorescence detection used is 450-490 nm, and the emission wavelength is 510-550 nm. When DCFH-DA enters Neuro2a, it is first hydrolyzed to DCFH (dichlorodihydrofluorescein), and then oxidized to DCF (dichlorofluorescein) that can emit green fluorescence by reactive oxygen species (ROS). Therefore, the fluorescence intensity of the DCFH-DA-treated Neuro2a cells can indicate the reactive oxygen species (ROS) content in the Neuro2a cells, and the proportion of the number of cells in which reactive oxygen species (ROS) are highly expressed in the Neuro2a cells to the number of original cells can be known. The experiment was repeated twice, and the measurement results of the two repeated experiments for each group were averaged to obtain the average value. The average value of the blank group was then converted to the relative ROS generation amount, with the average value of the control group, control group, and experimental group being taken as 100% relative ROS generation amount, as shown in Figure 5. Then, the expression of Neuro2a cells in each group was observed under a fluorescence microscope (Beckman).

[0120] Please refer to Figure 5. The blank group cells were not treated with hydrogen peroxide water, and the relative ROS generation was taken as the reference standard of 100%. Under the fluorescent microscope, the Neuro2a cells were blue, indicating that no cellular oxidative stress was induced. After the control group was treated with hydrogen peroxide water, the relative ROS generation (high fluorescence expression) was significantly increased to 487.6% compared to the blank group, and under the fluorescent microscope, the Neuro2a cells were fluorescent green, indicating that hydrogen peroxide water treatment certainly generated ROS in the cells, which further caused subsequent damage to the Neuro2a cells. After the control group was treated with hydrogen peroxide water, the relative ROS generation was increased to 452.5% compared to the blank group, but decreased by 35.1% compared to the control group. Under the fluorescent microscope, the Neuro2a cells in the control group showed blue-green interspersed but more obvious green, indicating that the chemically synthesized nicotinamide mononucleotide in the cell culture medium slightly cooperated with the Neuro2a cells in resisting the cellular oxidative stress caused by hydrogen peroxide. After the experimental group was treated with hydrogen peroxide, the relative ROS generation increased by 411.2% compared to the blank group, but decreased by 76.4% compared to the control group. Under the fluorescent microscope, the Neuro2a cells showed mainly blue but still had a little green, indicating that the natural nicotinamide mononucleotide in the cell culture medium can effectively reduce the generation or accumulation of reactive oxygen species (ROS) in the cells and further cooperate well with the Neuro2a cells in resisting the cellular oxidative stress caused by hydrogen peroxide. Furthermore, compared to the control group, the relative ROS production in Neuro2a cells in the experimental group was significantly reduced (about 2.17-fold), indicating that natural nicotinamide mononucleotide in yeast powder can more effectively reduce the generation or accumulation of reactive oxygen species (ROS) in cells and help cells resist ROS damage than chemically synthesized nicotinamide mononucleotide, and has antioxidant and anti-aging capabilities.

[0121] In other words, yeast powder rich in nicotinamide mononucleotide can be used as an active oxygen substance scavenger, i.e., yeast powder rich in nicotinamide mononucleotide can reduce the reactive oxygen substance (ROS) content in cells, thereby reducing oxidative damage to cells caused by active oxygen substances, etc.

[0122] Based on these, when subjects take yeast powder rich in nicotinamide mononucleotide, they can effectively resist ROS damage and further achieve the effects of antioxidant and anti-aging.

[0123] Example 6 Cell experiment - mitochondrial activity Here, we use flow cytometry to evaluate changes in mitochondrial activity in mouse brain neuroblastoma cells (Neuro2a) after they are treated with yeast powder containing nicotinamide mononucleotide (NMN).

[0124] The medium used is Dulbecco's Modified Eagle Medium (DMEM; brand: Gibco) supplemented with 10% by volume of fetal bovine serum (FBS; brand: Gibco) and 1% by volume of penicillin / streptomycin (brand: Gibco), hereinafter referred to as cell medium. The mitochondrial activity test method used is to measure the mitochondrial membrane potential using a mitochondrial membrane potential detection set (BDTM MitoScreen (JC-1) kit, model number 551302) and to perform mitochondrial activity analysis. The mitochondrial membrane potential detection set contains JC-1 dye (lyophilized) and 10x analysis buffer. Before use, the 10x analysis buffer is diluted 10 times with 1x PBS to form 1x analysis buffer. 130 μL of DMSO is added to the JC-1 dye (lyophilized) to form a JC-1 stock solution. The JC-1 stock solution was then further diluted with 1× assay buffer to form the JC-1 working solution (i.e., JC-1 mitochondrial-specific stain). The dilution ratio of the JC-1 stock solution to 1× assay buffer was 1:100.

[0125] First, 1×10 5 Mouse neuroblastoma cells (ATCC CCL-131; hereafter referred to as Neuro2a cells) are placed into a 6-well cell culture plate containing 2 mL of cell medium in each well and cultured at 37 °C for 24 h.

[0126] After the Neuro2a cells in each well are attached to the bottom of a 6-well cell culture plate, the Neuro2a cells are divided into an experimental group, a control group, and a control group. Then, the cell culture medium of each group is replaced with an experimental medium, followed by continuous culture at 37°C for 24 hours. The experimental medium of the experimental group is a cell culture medium of 0.25% by volume of yeast powder produced in Example 1. The experimental medium of the control group is a cell culture medium supplemented with 0.25% by volume of chemically synthesized nicotinamide mononucleotide (manufacturer: Sigma-Aldrich). The experimental medium of the control group is a simple cell culture medium.

[0127] Next, after removing the experimental medium of each group, wash the Neuro2a cells of each group twice with 1 mL of 1 × DPBS solution. Then, add 200 μL of trypsin to each well and react for 5 minutes. After the reaction, add 1 mL of cell culture medium to each well to terminate the reaction. Collect the Neuro2a cells and cell culture medium in each well individually in a corresponding 1.5 mL microcentrifuge tube, and centrifuge the microcentrifuge tube containing the Neuro2a cells and cell culture medium at 400 x g for 5 minutes. After centrifugation, remove the supernatant in the microcentrifuge tube of each group, and further re-dissolve the precipitate of Neuro2a cells with 1 × PBS solution, and centrifuge at 400 x g for 5 minutes. After centrifugation again, remove the supernatant in the microcentrifuge tube of each group, and add 100 μL of JC-1 working reagent to each microcentrifuge tube, and mix the precipitate of Neuro2a cells with JC-1 working reagent uniformly, and then leave it in the dark for 15 minutes. After standing for 15 minutes, the microcentrifuge tubes containing Neuro2a cells and JC-1 working reagent are centrifuged at 400xg for 5 minutes. Then, the supernatant in each group of microcentrifuge tubes is removed, 1x assay buffer is added to redissolve the Neuro2a cell pellets, and centrifuged at 400xg for 5 minutes, and repeated twice. The supernatant in each group of microcentrifuge tubes is removed, and the Neuro2a cell pellets are resuspended in 1x DPBS solution containing 2% by volume of bovine serum albumin to obtain the test cell solution.

[0128] The membrane potential of the cell mitochondria in the test cell solution of each group is measured by flow cytometry (Flow cytometry; Manufacturer: Beckman; Catalog No. 660519) to perform mitochondrial activity analysis. The wavelength of the excitation light set in the flow cytometer is 488 nm, and the wavelength of the scattered light is 527 nm and 590 nm. Since the experiment is repeated three times, the results of the three repeated experiments of each group are averaged to obtain an average value, and then, as shown in Figure 6, the average value of the control group is set as 100% of the relative JC-1 accumulation amount, and the average relative JC-1 accumulation amount of the experimental group and the control group is converted. In Figure 6, "**" indicates that the p value is smaller than 0.01 compared to the control group. Then, the mitochondrial expression of Neuro2a cells of each group is observed with a fluorescence microscope (Beckman), and green fluorescence represents non-activated cell mitochondria, and red fluorescence represents activated cell mitochondria.

[0129] Please refer to FIG. 6. The relative JC-1 accumulation amount of the control group is 100%. Under a fluorescent microscope, the mitochondria in the Neuro2a cells of the control group exhibit red-green fluorescence intermingled with green fluorescence, which indicates that the mitochondria in the Neuro2a cells of the control group are partially activated but mostly inactivated. The relative JC-1 accumulation amount of the control group is 42.93%, and under a fluorescent microscope, the mitochondria in the Neuro2a cells of the control group exhibit green fluorescence mainly, which indicates that the mitochondria in the Neuro2a cells of the control group are mostly inactivated. The relative JC-1 accumulation amount of the experimental group is 108.89%, and under a fluorescent microscope, the mitochondria in the Neuro2a cells of the experimental group exhibit red fluorescence mainly, which indicates that the mitochondria in the Neuro2a cells of the control group are partially activated but mostly inactivated. experimentThis indicates that most of the mitochondria in the Neuro2a cells of the experimental group are in an activated state. In other words, the mitochondrial activity of the Neuro2a cells of the experimental group is significantly improved (about 1.09 times) compared with the control group. It has been shown that yeast powder rich in nicotinamide mononucleotide can improve the mitochondrial activity of nerve cells and achieve the effect of improving the activity of nerve cells. Also, as can be seen from Figure 6, chemically synthesized nicotinamide mononucleotide cannot activate the mitochondrial activity of nerve cells.

[0130] Based on these findings, when a subject takes yeast powder rich in nicotinamide mononucleotide, it can more effectively improve the mitochondrial activity of nerve cells, thereby achieving improved nerve cell activity and anti-aging effects.

[0131] Example 7 Cell experiment - Anti-aging related genes Here, the detected anti-aging-related genes are anti-aging genes, including the CCT5 gene (Gene ID: 22948), the CCT6A gene (Gene ID: 908), the CCT7 gene (Gene ID: 10574), the CCT8 gene (Gene ID: 10694), the PARP2 gene (Gene ID: 10038), the Parkin gene (Gene ID: 5071), the Atg1 gene (Gene ID: 8408), the Atg8 gene (Gene ID: 11345), the FOXO gene (Gene ID: 2308), the SIRT1 gene (Gene ID: 23411), the NADSYN gene (Gene ID: 55191), and the MRPS5 gene (Gene ID: 64969).

[0132] The medium used was X-VIVO™ 15 Serum-free Hematopoietic Cell Medium (Manufacturer: Lonza; Product No.: 04-418Q) supplemented with 10% by volume of fetal bovine serum (FBS; Brand: Gibco) and 1% by volume of penicillin / streptomycin (Brand: Gibco), hereinafter referred to as the cell medium.

[0133] First, 1×10 6 Human peripheral blood mononuclear cells (PBMCs, isolated from human tissue; purchased from ATCC, product number PCS-200-010 TM ; hereafter referred to as PBMC cells) are placed in a 6-well cell culture plate containing 2 mL of cell medium in each well and cultured at 37°C for 24 hours.

[0134] PBMC cells are divided into an experimental group, a control group and a control group. The cell culture medium of each group is removed and replaced with 2 mL of experimental medium in each well, followed by continuous cultivation at 37°C for 24 hours, respectively. The experimental medium of the experimental group is a cell culture medium of 0.25% by volume of yeast powder produced in Example 1. The experimental medium of the control group is a cell culture medium supplemented with 0.25% by volume of chemically synthesized nicotinamide mononucleotide (manufacturer: Sigma-Aldrich). The experimental medium of the control group is a simple cell culture medium.

[0135] PBMC cells of each group are collected, and RNA of each group is extracted with RNA extraction reagent kit (purchased from Geneaid, Taiwan, China, Lot No. FC24015-G). Then, for each group, 1000ng of RNA is taken as template, and RNA is reverse transcribed into corresponding cDNA by SuperScript® III reverse transcriptase (Invitrogene, USA, No. 18080-051). Furthermore, quantitative real-time reverse transcription polymerase chain reaction is performed on the cDNA of each group by ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000) and primers in Table 1 (SEQ ID NO: 1 to SEQ ID NO: 24) to observe the expression level of anti-aging related genes in PBMC cells. The quantitative real-time reverse transcription polymerase chain reaction instrument setting conditions are 95℃ for 20 seconds, then 95℃ for 3 seconds, 60℃ for 30 seconds, and repeat 40 cycles, and gene quantification is performed using the 2-ΔCt method, as shown in Figures 7 to 10. By performing quantitative real-time reverse transcription polymerase chain reaction with cDNA, the mRNA expression level of the gene can be indirectly quantified, and the expression level of the protein encoded by the gene can be further estimated.

[0136] It should be noted that the gene expression in Figures 7 to 10 is expressed as relative expression fold, the standard deviation is calculated using the STDEV formula in Excel software, and the statistically significant difference between each group is statistically analyzed by Student's t-test. In Figures 7 to 10, "*" represents that the p value is smaller than 0.05 compared to the control group, "**" represents that the p value is smaller than 0.01 compared to the control group, and "***" represents that the p value is smaller than 0.001 compared to the control group.

[0137] [Table 1]

[0138] In Table 1, F is a forward primer and R is a reverse primer.

[0139] Please refer to Figure 7. The relative expression fold of PARP2 gene of each group in the control group is 1.00 (i.e., the expression of gene of each group in the control group is 100%). Compared with the control group, the relative expression fold of PARP2 gene of the experimental group is 0.47, and the relative expression fold of PARP2 gene of the control group is 0.67. And, compared with the control group, the expression of PARP2 gene of the experimental group is significantly decreased, which indicates that the naturally synthesized nicotinamide mononucleotide-rich yeast powder can more effectively suppress the expression of PARP2 gene than chemically synthesized nicotinamide mononucleotide. In other words, when the subject takes the naturally synthesized nicotinamide mononucleotide-rich yeast powder, it can effectively suppress the expression of PARP2 gene, and further promote cell rejuvenation and telomerase activity, thereby achieving anti-aging function.

[0140] Please refer to Figure 8. The relative expression fold of the CCT gene in each group of the control group is 1.00 (i.e., the expression amount of the CCT gene in each group of the control group is 100%). Compared with the control group, the relative expression fold of the CCT5 gene in the experimental group is 1.77, the relative expression fold of the CCT6A gene is 1.68, the relative expression fold of the CCT7 gene is 1.0736 (represented as 1.07 in Figure 8), and the relative expression fold of the CCT8 gene is 1.85. The relative expression fold of the CCT5 gene in the control group is 1 . At 55, the relative expression fold of the CCT6A gene was 1.62, the relative expression fold of the CCT7 gene was 1.0672 (shown as 1.07 in FIG. 8), and the relative expression fold of the CCT8 gene was 1.72. As can be seen, the expression level of the CCT gene in the experimental group was significantly higher than that of the control group, which indicates that the naturally synthesized nicotinamide mononucleotide-rich yeast powder can more effectively improve the expression of the CCT gene than the chemically synthesized nicotinamide mononucleotide, and can even restore mature cells to young cells and achieve anti-aging effects. In other words, when the subject takes the naturally synthesized nicotinamide mononucleotide-rich yeast powder, it can effectively improve the expression level of the CCT gene, and can even promote cell rejuvenation and achieve anti-aging functions.

[0141] Please refer to Figure 9. The relative expression fold of anti-aging related genes (here, Parkin gene, Atg1 gene, Atg8 gene, FOXO gene, SIRT1 gene) of each group in the control group is 1.00 (i.e., the expression amount of each gene in the control group is 100%). Compared with the control group, the relative expression fold of Parkin gene in the experimental group is 1.68, the relative expression fold of Atg1 gene is 1.28, the relative expression fold of Atg8 gene is 1.33, the relative expression fold of FOXO gene is 1.31, and the relative expression fold of SIRT1 gene is 5.52, while the relative expression fold of Parkin gene in the control group is 1.61, the relative expression fold of Atg1 gene is 1.25, the relative expression fold of Atg8 gene is 1.02, the relative expression fold of FOXO gene is 0.79, and the relative expression fold of SIRT1 gene is 4.30. And, the expression of anti-aging related genes in the experimental group was significantly improved compared with the control group, which indicates that the yeast powder rich in naturally synthesized nicotinamide mononucleotide can more effectively promote the expression of Parkin gene, Atg1 gene, Atg8 gene, FOXO gene, and SIRT1 gene compared with chemically synthesized nicotinamide mononucleotide. In other words, when the subject takes the yeast powder rich in naturally synthesized nicotinamide mononucleotide, it can effectively improve the expression of Parkin gene, Atg1 gene, Atg8 gene, FOXO gene, and SIRT1 gene, and further promote cell rejuvenation and achieve anti-aging function.

[0142] Please refer to Figure 10. The relative expression fold of NADSYN gene and MRPS5 gene of each group in the control group is 1.00 (i.e., the expression fold of NADSYN gene and MRPS5 gene of each group in the control group is 100%). Compared with the control group, the relative expression fold of NADSYN gene of the experimental group is 7.31, and the relative expression fold of MRPS5 gene is 1.10, while the relative expression fold of NADSYN gene of the control group is 5.84, and the relative expression fold of MRPS5 gene is 0.80. As can be seen, the expression fold of NADSYN gene and MRPS5 gene of the experimental group is significantly higher than that of NADSYN gene and MRPS5 gene of the control group, which indicates that the naturally synthesized nicotinamide mononucleotide-rich yeast powder can more effectively improve the expression of NADSYN gene and MRPS5 gene than chemically synthesized nicotinamide mononucleotide, and further improve the anti-aging and anti-oxidation ability of cells. In other words, when subjects take yeast powder rich in naturally synthesized nicotinamide mononucleotide, it can effectively improve the expression levels of NADSYN gene and MRPS5 gene, and further achieve the functions of antioxidant and anti-aging, and promote cell rejuvenation.

[0143] Also refer to Figure 9 and Figure 10. The expression levels of FOXO gene and MRPS5 gene in the control group are both lower than those in the control group, which indicates that chemically synthesized nicotinamide mononucleotide suppresses the expression levels of FOXO gene and MRPS5 gene. In other words, nicotinamide mononucleotide cannot promote the expression of all anti-aging genes.

[0144] Example 8 Human testing In order to further confirm the effect of yeast powder rich in nicotinamide mononucleotide (NMN) on the human body, capsules each containing 100 mg of the yeast powder produced in Example 1 were provided to eight subjects, who were to take one capsule per day for eight consecutive weeks. The eight subjects were healthy men and women aged 50 to 75 years.

[0145] Before taking capsules containing yeast powder (considered as week 0) and 8 weeks after taking capsules containing yeast powder (considered as week 8), eight subjects were asked to take blood samples and analysis, skin type detection, brain age detection, and a questionnaire on physical sensation. Blood test items included the expression of anti-aging-related genes in the blood, inflammation indicators in the blood (C-reactive protein (CRP) content), low-density cholesterol content in the blood, and the low-density cholesterol / high-density cholesterol ratio. Skin type test items included skin wrinkle analysis and skin texture (roughness) analysis. The questionnaire on physical sensation included skin type, hair loss status, and fatigue.

[0146] Of particular note is that statistically significant differences between measurements at weeks 0 and 8 were analyzed statistically by Student's t-test.

[0147] Example 8-1. Human Testing - Blood Analysis For eight subjects, 6 mL of venous blood was collected using purple-capped blood collection tubes containing EDTA anticoagulant before (i.e., week 0) and after (i.e., week 8) taking the yeast powder, and anti-aging-related gene expression analysis, blood inflammation index analysis, blood low-density cholesterol content, and low-density cholesterol / high-density cholesterol ratio analysis were performed.

[0148] Example 8-1-1. Analysis of anti-aging related gene expression in blood The anti-aging-related genes detected here were the SIRT1 gene (Gene ID: 23411) and the SOD3 gene (Gene ID: 6649).

[0149] First, the collected venous blood is centrifuged at a rotation speed of 300xg for 15 minutes. 2mL of the buffy coat white blood cell layer is taken from the centrifuged venous blood, and the white blood cell layer is diluted to 4mL with 2mL of phosphate buffer (1xPBS; hereafter referred to as 1xPBS buffer). Next, the diluted white blood cell layer is slowly added to a centrifuge tube containing 3mL of cell separation solution (Ficoll-Paque Plus cell separation solution). During the addition, the diluted white blood cell layer and the cell separation solution should not be mixed and should be in a stratified state. Next, the centrifuge tube containing the layered diluted white blood cell layer and cell separation solution is centrifuged at 400xg for 40 minutes, the supernatant is removed after centrifugation, and 2mL to 3mL of peripheral blood mononuclear cells (PBMCs; hereafter referred to as PBMCs) are taken from the intermediate layer in the centrifuge tube after the above centrifugation. PBMCs are washed with 3 volumes of 1x PBS buffer, and then mixed uniformly with the above-mentioned 1x PBS buffer to form a PBMC mixture. The PBMC mixture is then centrifuged at a rotation speed of 300xg for 10 minutes to form a PBMC supernatant and a PBMC precipitate, and the PBMC precipitate is decomposed with 600μL of RNA decomposition buffer, after which RNA is extracted. Next, the extracted RNA is reverse transcribed into cDNA as in the step of Example 7, and then quantitative real-time reverse transcription polymerase chain reaction is performed on the cDNA using two groups of primers (SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:25 and SEQ ID NO:26) in Table 2, as shown in Figure 11, to observe the expression levels of SIRT1 gene and SOD3 gene in blood at 0 weeks and 8 weeks.

[0150] [Table 2]

[0151] In Table 2, F is a forward primer and R is a reverse primer.

[0152] It is important to note that the gene expression in Figure 11 is expressed as a relative expression percentage, the standard deviation was calculated using the STDEV formula in Excel software, and the statistically significant differences between groups were statistically analyzed by Student's t-test. In Figure 11, "*" indicates that the p-value is less than 0.05 compared to the control group.

[0153] Please refer to Figure 11. If the relative gene expression percentages of SIRT1 gene and SOD3 gene of 8 subjects at week 0 are 100%, the relative gene expression percentages of SIRT1 gene at week 8 are 162.6%, and the relative gene expression percentages of SOD3 gene are 259.0%. As can be seen, when a subject takes 100mg of yeast powder every day for 8 consecutive weeks, the expression levels of SIRT1 gene and SOD3 gene in the subject's blood are improved, which indicates that the expression levels of proteins encoded by SIRT1 gene and SOD3 gene are improved. And SIRT1 gene and SOD3 gene act as NAD + As a downstream gene of SIRT1, it is related to mitochondrial activity, and when the SIRT1 gene and SOD3 gene in the blood are elevated, the NAD + It has been shown that the content of yeast powder increases, which can activate mitochondria and achieve anti-aging function. In other words, taking yeast powder can improve the expression levels of SIRT1 gene and SOD3 gene in blood, which indicates that yeast powder can activate mitochondria and has the potential for anti-aging.

[0154] Example 8-1-2. Analysis of the degree of inflammation in blood In this experiment, blood samples were taken from eight subjects at week 0 and week 8 to detect changes in the degree of inflammation before and after taking capsules containing 100 mg of yeast powder prepared in Example 1. C-Reactive Protein (CRP) is a special protein produced by the liver, an acute phase reactant protein, and an indicator of tissue destruction during acute inflammatory reactions. The higher the C-Reactive Protein content in the blood measured using enzyme-linked immunosorbent assay (ELISA), the more severe the anti-inflammatory reaction in the body. In this example, the C-Reactive Protein content in the blood of the subjects was entrusted to Li Ren Laboratory (Taiwan, China) for measurement.

[0155] The standard deviation of C-reactive protein content was calculated using the STDEV formula in Excel software, and the statistically significant differences between groups were statistically analyzed by Student's t-test, and "*" represents a p value less than 0.05 compared with the control group.

[0156] See Figure 12. The average blood C-reactive protein (CRP) content of the eight subjects at week 0 was 0.091 mg / dL, and the average blood C-reactive protein (CRP) content at week 8 was 0.070 mg / dL, a decrease of 0.021 mg / dL (a decrease of 23.1%). In other words, taking yeast powder reduced the blood C-reactive protein (CRP) content, which can indicate that yeast powder has anti-inflammatory potential.

[0157] Based on these findings, daily administration of 100 mg of yeast powder in the study subjects significantly reduced inflammatory indicators (C-reactive protein (CRP) content) and further reduced chronic and recurrent unpleasant symptoms.

[0158] Example 8-1- 3 . Analysis of blood lipids in blood Low-density lipid cholesterol can invade endothelial cells, oxidize cells, and increase the risk of cardiovascular disease, so it is generally desirable to reduce the content of low-density lipid cholesterol.On the contrary, high-density lipid cholesterol can suppress the occurrence of cardiovascular disease, and the cholesterol in high-density lipid protein particles is considered as a factor that protects the health of the body's cardiovascular system, so it is generally desirable to improve the content of high-density lipid cholesterol.

[0159] Blood lipid analysis, including detection of low-density cholesterol content and low-density cholesterol / high-density cholesterol ratio, was outsourced to Riren Laboratory (Taiwan, China) for measurement.

[0160] For blood samples taken from eight subjects at week 0 and week 8, the low-density cholesterol content and the low-density cholesterol / high-density cholesterol ratio were detected, as shown in Figures 13 and 14, respectively. The low-density cholesterol content in Figure 13 and the low-density cholesterol / high-density cholesterol ratio in Figure 14 were used to calculate the standard deviation using the STDEV formula in Excel software, and the statistically significant differences between the groups were statistically analyzed by Student's t-test, and "*" indicates that the p-value is less than 0.05 compared to the control group.

[0161] Please refer to Figure 13. The average blood low-density cholesterol content of the eight subjects at week 0 is 131.3 mg / dL, while the average blood low-density cholesterol content at week 8 is 116.2 mg / dL. As can be seen, after the subjects take the product for 8 weeks, the blood low-density cholesterol content is reduced by 15.1 mg / dL, which represents that the average blood low-density cholesterol content of the eight subjects at week 8 is reduced by 11.5% compared to the average blood low-density cholesterol content of the eight subjects at week 0. In other words, by taking yeast powder, the blood low-density cholesterol content is reduced, and arteriosclerosis caused by the oxidation of low-density lipid protein is prevented, which indicates that yeast powder has the potential for regulating blood lipids and cardiovascular health care.

[0162] Please refer to Figure 14. The average blood low-density cholesterol / high-density cholesterol ratio of the eight subjects at week 0 is 2.22, while the average blood low-density cholesterol / high-density cholesterol ratio at week 8 is 1.85, a decrease of 0.37 (16.7%). Since a higher low-density cholesterol / high-density cholesterol ratio indicates a higher risk of coronary artery sclerosis, a decrease in the low-density cholesterol / high-density cholesterol ratio indicates a lower risk of coronary artery sclerosis. In other words, by taking yeast powder, the low-density cholesterol / high-density cholesterol ratio in the blood is regulated and decreased, which can indicate that yeast powder has the potential for blood lipid regulation, risk reduction of coronary artery sclerosis, and cardiovascular health care.

[0163] Based on these, if a subject takes 100 mg of yeast powder every day, it can significantly reduce inflammation indicators (C-reactive protein (CRP) content), reduce the risk of coronary atherosclerosis, and further reduce the risk of suffering from cardiovascular disease, and achieve cardiovascular health care functions.

[0164] Example 8-2. Human testing - skin type analysis Here, the skin condition is analyzed using a VISIA high-level digital skin quality detector (VISIA Complexion Analysis System (Canfield scientific, USA)). For skin wrinkles, the facial skin of the same subject before and after taking yeast powder is photographed with a high-resolution camera lens, and standard white light is irradiated to detect changes in the shadow of the skin, thereby detecting the position of the texture and obtaining values ​​representing the smoothness of the skin and the skin texture. For skin texture, the facial skin of the same subject before and after taking yeast powder is detected, and the principle is to take high-resolution skin images with visible light and perform roughness analysis based on the unevenness of the skin using built-in software. The higher the measured value, the rougher the skin.

[0165] Please refer to Figure 15. The average skin wrinkle percentage detected by the VISIA advanced digital skin quality detector before the 8 subjects took yeast powder (week 0) is set as 100%. After the subjects took the yeast powder for 8 consecutive weeks, the average skin wrinkle percentage was reduced to 89.7%. In other words, compared to before taking yeast powder (week 0), after taking capsules containing 100 mg of yeast powder for 8 consecutive weeks, the skin wrinkle percentage of these subjects can be reduced by 10.3%. As can be seen from this, taking yeast powder rich in nicotinamide mononucleotide (NMN) for a long period of time can reduce the skin wrinkles of the subjects and improve the skin tone of the subjects, that is, yeast powder has the effect of smoothing fine wrinkles.

[0166] And, one of the subjects had many and dense wrinkles (WK-0) photographed by the VISIA high-level digital skin quality detector before taking yeast powder (week 0), but after taking yeast powder, rear The wrinkles in the WK-8 photograph taken at week 8 were reduced in number and sparse. Based on this, the long-term administration of yeast powder rich in nicotinamide mononucleotide can reduce the number of wrinkles on the face of the subject.

[0167] Please refer to Figure 16. The average skin texture percentage detected by VISIA advanced digital skin quality detector before taking yeast powder (week 0) of 8 subjects is 100%. After taking yeast powder for 8 consecutive weeks, the average skin texture percentage of the subjects was reduced to 92.0%. In other words, compared to before taking yeast powder (week 0), after taking capsules containing 100 mg of yeast powder for 8 consecutive weeks, the skin texture percentage of these subjects can be reduced by 8.0%. As can be seen from this, taking yeast powder rich in nicotinamide mononucleotide for a long period of time can reduce the skin texture of the subjects, reduce the roughness of the subjects' skin, and improve the skin condition of the subjects, that is, yeast powder has the effect of making the skin fine.

[0168] And, one of the subjects had a large number of bumps and dents in the uneven area T-0 photographed by the VISIA high-level digital skin quality detector before taking yeast powder (week 0), but the number of bumps and dents in the uneven area T-8 photographed after taking yeast powder (week 8) was reduced and sparse. Based on this, taking yeast powder rich in nicotinamide mononucleotide for a long period of time can reduce the bumps and dents on the subject's face, further reduce rough skin, and make the subject's skin finer.

[0169] Example 8-3. Human Testing - Brain Age Analysis Here, the brain age of eight subjects is tested using the "Test Your Brain Age" software before (week 0) and after (week 8) taking yeast powder. Specifically, the "Test Your Brain Age" software calculates the brain age of subjects based on the overall correct answer rate and answer speed of the subjects, and the test items include clicking the numbers displayed on the screen in ascending order before the numbers disappear.

[0170] "Test Your Brain Age" software site: https: / / apps.apple.com / tw / app / test-your-brain-age / id969455998.

[0171] Please refer to Figure 17. The average brain age of the eight subjects is 42 years old before taking yeast powder (week 0). After the subjects take it for 8 consecutive weeks, their average brain age is reduced to 30 years. In other words, compared to before taking yeast powder (week 0), after taking capsules containing 100 mg of yeast powder for 8 consecutive weeks, the average brain age of these subjects can be reduced by 12 years, which indicates that after the subjects take yeast powder for 8 consecutive weeks, their mental clarity and answering speed are improved.

[0172] As can be seen from this, long-term administration of yeast powder rich in nicotinamide mononucleotide (NMN) can improve mental clarity, reduce the brain detection age of the subject, and even achieve anti-aging effects, that is, yeast powder has the effects of improving mental clarity and anti-aging.

[0173] Example 8-4. Human Testing - Physical Experience Questionnaire Here, the subjects answered a questionnaire including an overall physical condition evaluation at week 0 and week 8. As shown in Table 3, the subjects rated the severity of their physical condition (e.g., overall poor skin condition, hair loss, thinning hair, physical fatigue, etc.).

[0174] [Table 3]

[0175] In Table 3, "none" represents 1 point, "minor" represents 2 points, "obvious" represents 3 points, "severe" represents 4 points, and "very serious" represents 5 points. The scores are summed up to show the severity of each test item.

[0176] Please refer to Figure 18. The average severity of "general poor skin condition" of the eight subjects before taking yeast powder (week 0) is set as 100%. After taking it continuously for eight weeks (i.e., week 8), the average severity of "general poor skin condition" is reduced to 83.3%. In other words, compared to before taking it (week 0), after taking capsules containing yeast powder for eight consecutive weeks, the eight subjects' sense of "general poor skin condition" can be reduced by 16.7%, which indicates that yeast powder rich in nicotinamide mononucleotide can improve the skin condition of the subjects.

[0177] The average severity of the "hair loss situation" of the eight subjects before taking yeast powder (week 0) is set as 100%. After taking it for eight consecutive weeks (i.e., week 8), the average severity of the "hair loss situation" was reduced to 75.0%. In other words, compared to before taking it (week 0), after taking capsules containing yeast powder for eight consecutive weeks, the "hair loss" feeling of the eight subjects can be reduced by 25.0%, which indicates that the yeast powder rich in nicotinamide mononucleotide can reduce hair loss in the subjects.

[0178] The average severity of the "thinning hair condition" of the eight subjects before taking yeast powder (week 0) is set as 100%. After taking it for eight consecutive weeks (i.e., week 8), the average severity of the "thinning hair condition" is reduced to 73.3%. In other words, compared to before taking it (week 0), after taking capsules containing yeast powder for eight consecutive weeks, the "thinning hair" feeling of the eight subjects can be reduced by 26.7%, which indicates that the yeast powder rich in nicotinamide mononucleotide can reduce the thinning hair of the subjects and improve the hair density of the subjects.

[0179] The average severity of "physical fatigue" of the eight subjects before taking yeast powder (week 0) is set as 100%. After taking it continuously for eight weeks (i.e., week 8), the average severity of "physical fatigue" is reduced to 50.0%. In other words, compared to before taking it (week 0), after taking capsules containing yeast powder for eight consecutive weeks, the sensation of "physical fatigue" of the eight subjects can be reduced by 50.0%, which indicates that yeast powder rich in nicotinamide mononucleotide can reduce the subject's physical fatigue, improve the subject's mental state, and make the subject more energetic.

[0180] As can be seen from these, long-term use of yeast powder rich in nicotinamide mononucleotide can improve the overall skin condition, hair loss status, and fatigue of the subjects.

[0181] Example 9. Sequencing analysis of peptides in nicotinamide mononucleotide-rich yeast powder After appropriately diluting the nicotinamide mononucleotide-rich yeast powder produced in Example 1, 200 μl of the supernatant was aspirated by centrifugation (13,000 rpm, 2 min), desalted and concentrated with a C18-ZipTip (Millpore), and the sample was redissolved, and 1 / 2 of the volume was taken and subjected to LC-MS / MS analysis under the following conditions. The MS / MS spectrum was subjected to repository search analysis using the Mascot analysis program to obtain the analysis results, and further obtain the protein sequence and identity identification information of the peptides contained in the yeast powder described above.

[0182] The reaction conditions are as follows:

[0183] Mass spectrometer: LTQ XL (Thermo Scientific)

[0184] LC System: Agilent 1200 Series

[0185] Buffer solution A: ddH2O / 0.1%formic acid

[0186] Buffer solution B: 100%ACN / 0.1%formic acid

[0187] Analytical column: C18 reverse phase column

[0188] Gradient: 0.00 min B 5%, 10.02 min B 5%, 10.05 min B 5%, 45.00 min B 40%, 50.00 min B 85%, 60.00 min B 85%, 63.00 min B 5%, 75.02 min B 5%, 75.05 min B 5%, 90.00 min B 5%.

[0189] Analysis results: Protein identity information of peptides contained in the aforementioned nicotinamide mononucleotide-rich yeast powder was Q 10It is a biosynthetic protein, and the protein sequence of the analyzed peptide is MVAKAHSKK (SEQ ID NO: 27). This shows that yeast powder rich in nicotinamide mononucleotide contains Q 10 Peptide fragments of biosynthetic proteins are included.

[0190] As described above, according to the method for producing yeast powder rich in nicotinamide mononucleotide of any one of the embodiments of the present invention, yeast powder containing at least 5000 ppm of nicotinamide mononucleotide can be produced, which solves the problem that conventional nicotinamide mononucleotide can only be obtained by chemical synthesis and harmful by-products are generated in the process, and also solves the technical bottleneck that conventional nicotinamide mononucleotide cannot be eaten and can only be used externally. The composition components of the fermentation medium used in the production method of any one of the embodiments include nicotinamide, tryptophan and nicotinic acid. The composition components of the fermentation medium used in the production method of any one of the embodiments further include one or more extracts of Solanum genus plant extract, Aronia genus plant extract, and Musa genus plant extract. The yeast powder produced by the production method of any one of the embodiments can be used to produce a composition for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue. In other words, the above-mentioned composition has one or more of the functions of resisting cellular oxidative stress, improving cellular mitochondrial activity, regulating anti-aging related genes (i.e., suppressing PARP2 gene, enhancing CCT gene, Parkin gene, Atg1 gene, Atg8 gene, FOXO gene, SIRT1 gene, NADSYN gene, MRPS5 gene and SOD3 gene), reducing skin wrinkles and skin texture, making the brain younger (reducing brain age), reducing hair loss, reducing thinning hair, and reducing body fatigue. And, the yeast powder of any one of the embodiments is Q 10 Further included are biosynthetic proteins.

[0191] The technical content of the present invention has been disclosed above through preferred embodiments, but this is not intended to limit the present invention. Any slight changes and modifications made by a person skilled in the art without departing from the spirit of the present invention are all considered to be included within the scope of the present invention. The scope of protection of the present invention shall be based on the scope of the patent application attached later.

[0192] Of course, the present invention may have various other embodiments, and those skilled in the art may make various corresponding modifications and variations according to the present invention without departing from the spirit and essence of the present invention, and all of these corresponding modifications and variations shall fall within the scope of protection of the patent application of the present invention.

[0193] Food Industry Development Research Institute Foundation (Taiwan, China); October 24, 2019; Deposit number: BCRC 920118.

[0194] German Microorganism Collection (Germany); March 27, 2020; Deposit number: DSM33480. [Industrial Applicability]

[0195] The present invention discloses a yeast powder rich in nicotinamide mononucleotide, its preparation method and use. The preparation method includes the steps of preparing a first medium, a second medium and a third medium, inoculating yeast into the first medium for fermentation to obtain a first fermentation broth, inoculating the first fermentation broth into a second medium for fermentation to obtain a second fermentation broth, inoculating the second fermentation broth into a third medium for fermentation to obtain a third fermentation broth, centrifuging the third fermentation broth to obtain a fermented product, and drying the fermented product to obtain a yeast powder. The composition components of the first medium, the second medium and the third medium include nicotinamide, tryptophan and nicotinic acid. The content of nicotinamide mononucleotide contained in the yeast powder is at least greater than 5000 ppm.

Claims

1. preparing a first medium, a second medium and a third medium, the components of which include nicotinamide, tryptophan and nicotinic acid; A step of inoculating yeast into the first medium and fermenting the yeast to obtain a first fermentation liquid; Inoculating the first fermentation broth into the second medium and fermenting it to obtain a second fermentation broth; Inoculating the second fermentation broth into the third medium and fermenting it to obtain a third fermentation broth; and a step of centrifuging the third fermentation liquor to obtain a fermentation product, and drying the fermentation product to obtain a yeast powder, wherein the content of nicotinamide mononucleotide contained in the yeast powder is at least greater than 5000 ppm.

2. 2. The method according to claim 1, wherein the yeast is inoculated into the first medium at a seeding amount of 5% by volume, the first fermentation broth is inoculated into the second medium at a seeding amount of 6% by volume, and the second fermentation broth is inoculated into the third medium at a seeding amount of 10% by volume.

3. The method according to claim 1, characterized in that the concentration of the nicotinamide is 0.01% by weight to 0.3% by weight, the concentration of the tryptophan is 0.1% by weight to 0.5% by weight, and the concentration of the nicotinic acid is 0.001% by weight to 0.06% by weight.

4. 2. The method according to claim 1, wherein the volume ratio of the first culture medium, the second culture medium and the third culture medium is 3:50:

500.

5. The method of claim 1 , wherein the yeast powder further comprises Q10 biosynthetic protein.

6. The method according to claim 1, wherein the compositional components of the first medium, the second medium and the third medium further contain one or more of an extract of a plant of the genus Solanum, an extract of a plant of the genus Aronia, and an extract of a plant of the genus Musa.

7. 1. Use of yeast powder for the manufacture of a composition for improving skin condition, hair care, anti-inflammation, cardiovascular health care, antioxidant, anti-aging and / or improving physical fatigue, comprising:

2. Use, characterized in that the yeast powder is obtainable by the method of claim 1 and the content of nicotinamide mononucleotide contained in the yeast powder is at least 5000 ppm.

8. The use according to claim 7, characterized in that the yeast powder further comprises Q10 biosynthetic protein.

9. 8. The use according to claim 7, wherein the improvement in skin condition is increased skin firmness, reduced wrinkles, reduced roughness, or a combination thereof.

10. 8. The use according to claim 7, characterized in that the hair care consists in reducing hair loss or in reducing the extent of thinning hair.

11. 8. The use according to claim 7, characterized in that the yeast powder is for regulating blood lipids.

12. The use according to claim 11, characterized in that the yeast powder is for reducing the expression level of C-reactive protein in a specimen.

13. The use according to claim 7, characterized in that the yeast powder achieves anti-aging by regulating the expression level of anti-aging related genes, activating mitochondria or reducing brain age.

14. The use according to claim 13, characterized in that the anti-aging related genes include CCT gene, PARP2 gene, Parkin gene, Atg gene, FOXO gene, SIRT1 gene, NADSYN gene, MRPS5 gene, SOD3 gene or combinations thereof.

15. The use according to claim 7, characterized in that the yeast powder achieves an antioxidant function by reducing damage caused by active oxygen substances.

16. 8. Use according to claim 7, characterized in that the daily dose of yeast powder is 100 mg.

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