Fermented Autumn Plum, its production method and use

Abstract

A fermented product of Prunus salicina is obtained by solvent extraction of Prunus salicina and then anaerobic fermentation. In addition, the fermented product of Prunus salicina is also suitable for preparing a composition for whitening, enhancing mitochondrial activity and reducing skin spots.

Description

Technical Field

[0001] The present invention relates to a fermented product of Autumn Plum, and in particular to a fermented product of Autumn Plum, a preparation method thereof, and use thereof in preparing whitening, enhancing mitochondrial activity and reducing skin spots. Background Art

[0002] Since the rise of the concept of organic and / or natural diets, biotech companies and food companies have actively invested in the research and development of natural plant-related products. In order to provide a scientific basis for the health benefits of plant-related products, the analysis of plant active ingredients and efficacy evaluation have become key projects in product development.

[0003] The Akishime plum (Prunus salicina), known as the king of Japanese plum varieties, is a new variety bred in Yamanashi Prefecture, Japan. The Akishime plum has large fruits, a bright red skin, thick and dense flesh, and a rich sweet taste and aroma. In addition, due to its translucent red appearance, it is also known as the "Japanese plum princess". Therefore, biotechnology companies and food companies are actively studying the active ingredient analysis and efficacy evaluation of Akishime plums and developing related products based on them. Summary of the invention

[0004] In some embodiments, a fermented product of Prunus salicina is obtained by solvent extraction of Prunus salicina followed by anaerobic fermentation.

[0005] In some embodiments, the anaerobic fermentation is performed using yeast and / or lactic acid bacteria.

[0006] In some embodiments, a fermented product of Prunus armeniaca is used for preparing a whitening composition.

[0007] In some embodiments, the fermented product of Prunus mume has the effect of reducing melanin and / or inhibiting the production of melanin.

[0008] In some embodiments, the fermented product of Prunus mume has the effect of inhibiting tyrosinase activity.

[0009] In some embodiments, a fermented product of Prunus armeniaca is used for preparing a composition for enhancing mitochondrial activity.

[0010] In some embodiments, a fermented product of Prunus armeniaca is used to prepare a composition for reducing skin pigmentation spots.

[0011] In some embodiments, the pigmented spot is a dark brown spot, a visible spot, or a combination thereof.

[0012] In some embodiments, the aforementioned fermented product of Prunus mume comprises 2,5-bis-(hydroxymethyl)furane, 5-hydroxyfuroic acid, trans-4-hydroxycyclohaxane carboxlic acid, tyrosol, 6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or a combination thereof.

[0013] In some embodiments, a method for preparing a fermented plum comprises extracting plums with a solvent to obtain a plum extract, and anaerobically fermenting the plum extract to obtain the plum fermented product.

[0014] In summary, the fermented product of Prunus mume in any embodiment can enhance mitochondrial activity or improve skin condition. In other words, the fermented product of Prunus mume in any embodiment is suitable for preparing a composition for enhancing mitochondrial activity or improving skin condition. In some embodiments, the fermented product of Prunus mume or a composition prepared therefrom also has one or more of the following functions: whitening, reducing melanin, inhibiting melanin production, inhibiting tyrosinase activity, reducing skin brown spots, and reducing skin spots. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention is a flow chart of a method for producing fermented Autumn Plum according to an embodiment of the present invention.

[0016] Figure 2 yes Figure 1 Flow chart of an embodiment of step S11 in FIG.

[0017] Figure 3 It is a bar graph showing the test results of total polyphenol content of Akieme plum extract, Akieme plum aerobic fermentation product, and Akieme plum fermentation product.

[0018] Figure 4 It is a bar graph of the results of cell experiments on relative tyrosinase activity.

[0019] Figure 5 A is a bar graph of the cell experiment results of relative melanin content.

[0020] Figure 6 It is a bar graph of the results of cell experiments on relative mitochondrial activity.

[0021] Figure 7 This is a fluorescent staining image of the cell experiment results of mitochondrial activity.

[0022] Figure 8 It is a bar graph of the human body test results of the relative skin spot levels at week 0 and week 8.

[0023] Fig. 9 It is a bar graph showing the results of a human experiment on the relative degree of skin brown spots at week 0 and week 8.

[0024] Fig.10 It is the HPLC fingerprint of the Akiko plum extract and Akiko plum fermentation product.

[0025] Fig.11 This is the hydrogen-NMR spectrum of TCI-GSF-01.

[0026] Fig.12 This is the hydrogen-NMR spectrum of TCI-GSF-03.

[0027] Fig.13 This is the hydrogen-NMR spectrum of TCI-GSF-04.

[0028] Fig.14 This is the hydrogen-NMR spectrum of TCI-GSF-05.

[0029] Fig.15 This is the carbon-NMR spectrum of TCI-GSF-05.

[0030] Fig.16 This is the hydrogen-NMR spectrum of TCI-GSF-07.

[0031] Fig.17 This is the hydrogen-NMR spectrum of TCI-GSF-09.

[0032] Fig.18 This is the carbon-NMR spectrum of TCI-GSF-09.

[0033] Fig.19 B is a bar graph of the cell experiment results of relative melanin content.

[0034] Wherein, the reference numerals are:

[0035] S10: Steps

[0036] S11: Steps

[0037] S111: Steps

[0038] S112: Steps DETAILED DESCRIPTION

[0039] The following describes some specific implementations of the present invention. Without departing from the spirit of the present invention, the present invention can be implemented in a variety of different forms, and the scope of protection should not be limited to the conditions specifically stated in the specification.

[0040] In this document, the unit "%" used when referring to the content generally refers to weight percentage.

[0041] See also Figure 1 In some embodiments, the fermented material of Prunus salicina is obtained by subjecting Prunus salicina to solvent extraction (step S10) and then anaerobic fermentation (step S11).

[0042] In some embodiments, in the extraction step (ie, step S10), the Autumn Plum subjected to extraction may be a plant, root, stem, leaf, or fruit of Autumn Plum. In some embodiments, the Autumn Plum subjected to extraction is a Autumn Plum fruit with skin after removing seeds.

[0043] In some embodiments, in the extraction step (i.e., step S10), the Autumn Plum to be extracted may be raw material (e.g., intact fruit with skin but without seeds), or may be physically pre-treated to be broken down into small-sized forms such as fragments, particles, or powder. The physical pre-treatment may include at least one of the following: coarse crushing, chopping, breaking, shearing, pounding, and grinding.

[0044] In some embodiments, in the extraction step (i.e., step S10), the Autumn Plums to be extracted may be freshly harvested and collected Autumn Plums, dried Autumn Plums, or frozen Autumn Plums. For example, in the extraction step (i.e., step S10), the dried Autumn Plums are extracted using a solvent.

[0045] In some embodiments, in the extraction step (ie, step S10), the solvent for extraction may be water or alcohol. In some embodiments, in the extraction step (ie, step S10), the solvent for extraction is water.

[0046] In some embodiments, the plums may be crushed into plum particles and then extracted with a solvent. For example, the plums with seeds and skin removed are crushed into plum particles with a particle size of less than 12 mm, and then the plum particles are extracted with water.

[0047] In some embodiments, the plums are extracted with a solvent at 80-100° C. for 0.5-1 hour to obtain a plum extract, and then bacteria are added for anaerobic fermentation to obtain a plum fermentation product. For example, plums (such as plum particles with a particle size of less than 12 mm) can be soaked in 95° C. water for 0.5 hour to dissolve the active ingredients in the plums into the water to obtain a plum extract.

[0048] In some embodiments, in the extraction step (ie, step S10), the weight ratio of the solvent to the plum is 10-30:1-3. In some embodiments, in the extraction step (ie, step S10), the weight ratio of the solvent to the plum is 10:1.

[0049] In some embodiments, after the extraction is completed and before the bacterial strain is added, the plum extract can be cooled to room temperature to obtain a cooled plum extract, and then the bacterial strain is added.

[0050] In some embodiments, the Autumn Plum extract is subjected to an anaerobic fermentation step (ie, step S11 ) without removing the extraction material (ie, including Autumn Plum).

[0051] In some embodiments, the anaerobic fermentation step (i.e., step S11) is performed in an environment without a continuous supply of oxygen. In some embodiments, in the anaerobic fermentation step (i.e., step S11), a plastic wrap is used to cover the outer body and cover hole of the fermentation tank to prevent external air from entering the fermentation tank.

[0052] In some embodiments, the anaerobic fermentation step (i.e., step S11) is to add multiple strains for anaerobic fermentation for 5-10 days to obtain the fermented product of autumn plum. In some embodiments, the anaerobic fermentation step (i.e., step S11) is to add multiple strains for anaerobic fermentation for 2-6 days to obtain the fermented product of autumn plum.

[0053] In some embodiments, the bacterial species is yeast and / or lactic acid bacteria. In some embodiments, the bacterial species is yeast and lactic acid bacteria.

[0054] In some embodiments, the anaerobic fermentation step (ie, step S11) is a two-stage fermentation. In some embodiments, the anaerobic fermentation step (ie, step S11) is to sequentially add yeast and lactic acid bacteria to the plum extract for fermentation.

[0055] See also Figure 2 In some embodiments, the anaerobic fermentation step (i.e., step S11) is to add yeast to the plum extract for a first fermentation to obtain a first primary fermentation liquid (step S111), and then add lactic acid bacteria to the first primary fermentation liquid for a second fermentation to obtain a second primary fermentation liquid (step S112).

[0056] In some embodiments, step S111 is to colonize 0.2% beer yeast (e.g., beer yeast with accession number BCRC20271) in the plum extract and ferment at 28°C-37°C for 1-3 days to obtain a first fermentation liquid. In some embodiments, the plum extract after colonization with beer yeast is fermented at 30°C for 3 days.

[0057] In some embodiments, in step S111, yeast is added to the plum extract to ferment the plum extract to generate alcohol, thereby facilitating the extraction of effective ingredients in the plum.

[0058] In some embodiments, the pH value of the first initial fermentation liquid is ≤4.0, and the sugar content is about 3.6°Bx (Degrees Brix). In some embodiments, the pH value of the first initial fermentation liquid is 3.95.

[0059] In some embodiments, step S112 is to colonize 0.06% Lactobacillus plantarum (e.g., Lactobacillus plantarum with accession number BCRC910760) in the first primary fermentation liquid and ferment at 28° C.-37° C. for 1 to 3 days to obtain a second primary fermentation liquid. In some embodiments, the first primary fermentation liquid after colonization with Lactobacillus plantarum is fermented at 30° C. for 3 days.

[0060] In some embodiments, in step S112, by adding lactic acid bacteria to the first primary fermentation liquid, the glucose in the first primary fermentation liquid is consumed to reduce the sugar content, and lactic acid is produced to reduce the pH value of the first primary fermentation liquid. Here, reducing the pH value of the first primary fermentation liquid is beneficial to further extracting other different effective components in the Autumn Plum.

[0061] In some embodiments, the pH value of the second primary fermentation broth is ≤3.5, and the sugar content is about 1.5° Bx. In some embodiments, the pH value of the second primary fermentation broth is 3.32.

[0062] In some embodiments, after step S112, the second initial fermentation liquid may be further filtered to obtain initial fermentation filtrate. In some embodiments, the second initial fermentation liquid is filtered through a 200-mesh filter to obtain initial fermentation filtrate.

[0063] In some embodiments, the second primary fermentation liquid or the primary fermentation filtrate can be concentrated under reduced pressure at 55° C. to 65° C. to obtain a fermentation concentrate. In some embodiments, the reduced pressure concentration can be performed at 60° C.

[0064] In some embodiments, the aforementioned primary fermentation filtrate or fermentation concentrate can be filtered to obtain a fermentation stock solution. In some embodiments, the filtration step can be performed using a 200 mesh filter.

[0065] In some embodiments, the pH value of the fermentation liquid is ≤3.5, and the sugar content is about 1.5° Bx. In some embodiments, the pH value of the fermentation liquid is 3.2.

[0066] In some embodiments, oligosaccharides can be added to the fermentation stock solution to adjust the sugar content of the fermentation stock solution to 24°Bx, thereby obtaining a fermentation stock solution after sugar content adjustment. In some embodiments, the oligosaccharide is an oligosaccharide polymerized from 3 to 10 monosaccharide molecules. In some embodiments, the oligosaccharide is, but is not limited to, fructooligosaccharide, galacto-oligosaccharide, xylooligosaccharide or isomaltooligosaccharide. In some embodiments, the oligosaccharide is an oligosaccharide solution containing 20% ​​to 50% isomaltooligosaccharide.

[0067] In some embodiments, the fermentation stock solution can be further subjected to liquid-liquid partition extraction with alcohol and water to obtain an alcohol-soluble portion. In some embodiments, the alcohol can be n-butanol, and the alcohol-soluble portion is an n-butanol-soluble portion.

[0068] In some embodiments, the n-butanol soluble portion can be further separated using alcohol as an eluent to obtain a variety of compounds.

[0069] It should be understood that the second primary fermentation liquid obtained by anaerobic fermentation, the primary fermentation filtrate, the fermentation concentrate, the fermentation liquid, the fermentation liquid after sugar content adjustment, the alcohol-soluble part, the separated compounds or any combination thereof can be used as the fermented product of Akikomi plum according to actual needs.

[0070] In some embodiments, the aforementioned fermented product of Prunus mume comprises 2,5-bis-(hydroxymethyl)furane, 5-hydroxyfuroic acid, trans-4-hydroxycyclohaxane carboxlic acid, tyrosol, 6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or a combination thereof. In some embodiments, the fermented product of Prunus mume can be composed of 2,5-furan dimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one and 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one.

[0071] In some embodiments, the aforementioned fermented product of Prunus azedarach has the ability to whiten the skin. In other words, the fermented product of Prunus azedarach can whiten the skin of an individual when administered to the individual. Therefore, the fermented product of Prunus azedarach is suitable for preparing a whitening composition.

[0072] In some embodiments, the fermented product of Prunus mume has the ability to reduce melanin, inhibit melanin production, inhibit tyrosinase activity, or any combination thereof. In other words, the fermented product of Prunus mume can reduce melanin, inhibit melanin production, inhibit tyrosinase activity, or any combination thereof when administered to an individual. Therefore, the fermented product of Prunus mume is suitable for preparing a composition that reduces melanin, inhibits melanin production, inhibits tyrosinase activity, or any combination thereof.

[0073] In some embodiments, the aforementioned fermented plum has the ability to enhance mitochondrial activity. In other words, when the fermented plum is administered to an individual, it can enhance the mitochondrial activity of the individual. Therefore, the fermented plum is suitable for preparing a composition for enhancing mitochondrial activity.

[0074] In some embodiments, the aforementioned fermented product of Prunus mume has the ability to reduce brown spots and / or spots on the skin. In other words, when the fermented product of Prunus mume is administered to an individual, the brown spots and / or spots on the skin of the individual can be reduced. Therefore, the fermented product of Prunus mume is suitable for preparing a composition for reducing brown spots and / or spots on the skin.

[0075] In some embodiments, the aforementioned individual may be a human.

[0076] In some embodiments, the prepared composition can be a pharmaceutical composition, a food composition, a cosmetic composition, or a skin care product composition.

[0077] In some embodiments, when the aforementioned composition is a pharmaceutical composition, the pharmaceutical composition comprises an effective amount of fermented plum. The pharmaceutical composition can be manufactured into a dosage form suitable for enteral, parenteral, oral, or topical administration using techniques well known to those skilled in the art.

[0078] In some embodiments, the enterally or orally administrable dosage form may be, but is not limited to, a tablet, a troche, a lozenge, a pill, a capsule, a dispersible powder or granules, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, or the like.

[0079] In some embodiments, the parenteral or topical dosage form may be, but is not limited to, an injection [e.g., a sterile aqueous solution or dispersion], a sterile powder, an external preparation, or the like.

[0080] In some embodiments, the administration of the injection may be, but is not limited to, intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection or intralesional injection.

[0081] In some embodiments, the pharmaceutical composition containing an effective amount of the fermented product of autumn plum may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. In some embodiments, the pharmaceutically 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 quantity of the selected carrier fall within the professional accomplishment and routine technical scope of ordinary technicians in the field. The solvent used as the pharmaceutically acceptable carrier may be water, normal saline, phosphate buffered saline (PBS) or an alcohol containing aqueous solution.

[0082] In some embodiments, the pharmaceutical composition containing an effective amount of the fermented product of Autumn Plum can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those of ordinary skill in the art. In some embodiments, the external preparation includes, but is not limited to: emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve, and bandage.

[0083] In some embodiments, when the aforementioned pharmaceutical composition is an external preparation, the pharmaceutical composition can be prepared by mixing an effective amount of the fermented product of Prunus armeniaca with a base well known to those skilled in the art.

[0084] In some embodiments, the substrate may include one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum, jelly and white petrolatum), waxes (such as paraffin and yellow wax), preservatives, antioxidants, surfactants, absorption enhancers, stabilizing agents, gelling agents (such as 974P( 974P), microcrystalline cellulose and carboxymethylcellulose], active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants, etc. The selection and amount of these additives fall within the professional qualities and routine technical scope of ordinary technicians in this field.

[0085] In some embodiments, when the aforementioned composition is a food composition, the food composition comprises a specific amount of fermented prunes, wherein the food composition may be in the form of powder, granules, solution, colloid or paste.

[0086] In some embodiments, the food composition containing the fermented product of Prunus armeniaca may be a food product or a food additive.

[0087] In some embodiments, the food product containing the fermented product of Autumn Plum can be beverages, fermented foods, bakery products, health foods or dietary supplements, etc. In some embodiments, the food product containing the fermented product of Autumn Plum can further include an adjuvant. For example, the adjuvant can be maltodextrin, malic acid, sucralose, citric acid, fruit flavors, honey flavors, steviol glycosides or a combination thereof. The type and quantity of the selected carrier fall within the professional accomplishment and routine technical scope of ordinary technicians in the field.

[0088] In some embodiments, the food additive containing fermented plum can be a seasoning, a sweetener, a flavoring, a pH adjuster, an emulsifier, a colorant or a stabilizer.

[0089] In some embodiments, when the aforementioned composition is a cosmetic composition or a skin care product composition, in other words, the cosmetic composition or the skin care product composition comprises an effective amount of fermented Autumn Plum.

[0090] In some embodiments, the cosmetic composition or skin care product composition containing the fermented product of Prunus akitaenas can be in any of the following forms: lotion, gel, frozen mask, mud mask, emulsion, cream, lipstick, foundation, pressed powder, loose powder, make-up remover oil, make-up remover milk, facial cleanser, shower gel, shampoo, hair conditioner, sunscreen, hand cream, nail polish, perfume, essence and facial mask.

[0091] In some embodiments, the cosmetic composition or skin care product composition containing the fermented product of Prunus mume may further include an acceptable component for external use as needed. In some embodiments, the acceptable component for external use may be, for example, an emulsifier, a penetration enhancer, a softener, a solvent, an excipient, an antioxidant, or a combination thereof.

[0092] Unless otherwise specified in the following examples, the experimental steps were performed at room temperature (about 25° C.) and normal pressure (1 atm).

[0093] Example 1

[0094] A. Raw materials:

[0095] 1. Dried fruits of Prunus salicina (Prunus salicina) with skin (without seeds), origin: China, hereinafter referred to as "Prunus salicina fruits".

[0096] 2. Secondary water, also known as RO water (Reverse Osmosis) or double distilled water, hereinafter referred to as "water".

[0097] 3. Brewer's yeast, which was purchased from the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Institute and has the deposit number BCRC20271.

[0098] 4. Lactobacillus germini, which was purchased from BCRC and has the deposit number BCRC910760.

[0099] 5. Oligosaccharide solution, which is a 20% to 50% isomaltooligosaccharide solution prepared by water and isomaltooligosaccharide.

[0100] B. Preparation process:

[0101] 1. Crush the plum fruits with a blender (model: 12Speed ​​Blender; brand: Osterizer) to form plum particles with a particle size of less than 12 mm.

[0102] 2. After heating the water to 95°C, add the Autumn Plum particles and soak the Autumn Plum particles in the 95°C water for 0.5 hours to form an Autumn Plum extract. Here, the weight ratio of the Autumn Plum particles to the water is 1:10.

[0103] 3. 0.2% brewer's yeast was added to the cooled Autumn Plum extract (containing Autumn Plum) and anaerobically fermented at 30°C for 3 days to obtain a first fermentation liquid. The pH value of the first fermentation liquid was 3.95, and its sugar content was about 3.6°Bx. Here, the anaerobic fermentation was carried out in the fermentation tank, and the outer body and cover hole of the fermentation tank were covered with plastic wrap to prevent external air from entering the fermentation tank.

[0104] 4. 0.06% Lactobacillus germinatus was colonized in the first fermentation liquid, and anaerobically fermented at 30°C for 3 days to obtain a second fermentation liquid. The pH value of the second fermentation liquid was 3.32, and its sugar content was about 1.5°Bx. Here, the anaerobic fermentation was carried out in the fermentation tank, and the outer body and cover hole of the fermentation tank were covered with plastic wrap to prevent external air from entering the fermentation tank.

[0105] 5. Filter the second primary fermentation liquid through a 200-mesh filter to obtain the primary fermentation filtrate.

[0106] 6. After the temperature of the concentrator (model: Rotavapor R-100; brand: BUCHI) was set to 60°C, the primary fermentation filtrate was concentrated under reduced pressure using the concentrator to obtain a fermentation concentrate.

[0107] 7. The fermentation concentrate was filtered through a 200-mesh filter to obtain a fermentation stock solution. The pH value of the fermentation stock solution was 3.2, and the sugar content was about 1.5° Bx.

[0108] 8. Add 20% to 50% isomalto-oligosaccharide solution to the fermentation solution to adjust the sugar content of the fermentation solution to 24° Bx to obtain Akiehime plum fermentation product A.

[0109] Example 2

[0110] A. Raw materials:

[0111] The raw materials used are the same as those in Example 1.

[0112] B. Preparation process:

[0113] 1. Crush the plum fruits with a crusher to form plum particles with a particle size of less than 12 mm.

[0114] 2. After heating the water to 95°C, add the Autumn Plum particles and soak the Autumn Plum particles in the 95°C water for 0.5 hours to form an Autumn Plum extract. Here, the weight ratio of the Autumn Plum particles to the water is 1:10.

[0115] 3. 0.2% brewer's yeast was added to the cooled Autumn Plum Extract (containing Autumn Plum) and fermented at 30°C for 3 days to obtain the first fermentation liquid. Here, the entire fermentation process was carried out in the fermentation tank, but the outer body of the fermentation tank and the lid hole were not covered with plastic wrap during the entire fermentation process.

[0116] 4. 0.06% Lactobacillus germinatus was colonized in the first primary fermentation liquid and fermented at 30°C for 3 days to obtain the second primary fermentation liquid. Here, the entire fermentation process was carried out in the fermentation tank, but the outer body of the fermentation tank and the lid hole were not covered with plastic wrap during the entire fermentation process.

[0117] 5. Filter the second primary fermentation liquid through a 200-mesh filter to obtain the primary fermentation filtrate.

[0118] 6. After the temperature of the concentrator (model: Rotavapor R-100; brand: BUCHI) was set to 60°C, the primary fermentation filtrate was concentrated under reduced pressure using the concentrator to obtain a fermentation concentrate.

[0119] 7. Filter the fermentation concentrate through a 200-mesh filter to obtain the fermentation stock solution.

[0120] 8. Add 20% to 50% isomalto-oligosaccharide solution to the fermentation solution to adjust the sugar content of the fermentation solution to 24° Bx to obtain Akiehime plum fermentation product B.

[0121] Example 3

[0122] Weigh 10.0 mg of gallic acid into a 10 mL volumetric flask and then add water (H 2 O) was quantified to 10 mL to obtain a stock solution of gallic acid (i.e., containing 1000 ppm of gallic acid). The stock solution of gallic acid was diluted 10 times, i.e., 100 μL of the stock solution of gallic acid was added with 900 μL of water to obtain an initial solution of 100 μg / mL gallic acid (i.e., containing 100 ppm of gallic acid). Then, 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL gallic acid standard solutions were prepared according to Table 1 below, and 100 μL of each concentration of the standard solution was taken into a glass test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck) to each glass test tube and mix well with the standard solution and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate and mix well and react for 30 minutes to obtain a standard reaction solution. Take 200 μL of the standard reaction solution to a 96-well plate and measure its absorbance at 750 nm to obtain a standard curve.

[0123] Table 1

[0124] Standard solution (μg / mL) 0 20 40 60 80 100 Initial solution (μL) 0 20 40 60 80 100 Water (μL) 100 80 60 40 20 0

[0125] Take the plum extract and plum fermentation product A prepared in Example 1, and plum fermentation product B prepared in Example 2 as samples. Dilute each sample 10 times with water and take 100 μL into a centrifuge tube. Then, add 500 μL of Folin phenol reagent to each glass test tube and mix it evenly with the sample and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate and mix it evenly and react for 30 minutes to obtain the reaction solution to be tested. After shaking the glass test tube containing the reaction solution to be tested to ensure that there are no bubbles, take 200 μL of the reaction solution to be tested into a 96-well plate, and measure the absorbance of the reaction solution to be tested at 750 nm.

[0126] Next, the absorbance value of the reaction solution to be tested is converted into the total polyphenol content using a standard curve and interpolation method, and then back-multiplied by the dilution factor to obtain the actual total polyphenol content.

[0127] Here, it can be obtained that the total polyphenol content of the autumn plum extract is 95.4 ppm (i.e., 95.4 μg / mL), the total polyphenol content of the autumn plum fermentation product B is 213.913 ppm (i.e., 213.913 μg / mL), and the total polyphenol content of the autumn plum fermentation product A is 283.47 ppm (i.e., 283.47 μg / mL). Figure 3As shown. It can be seen that compared with the content before fermentation (i.e., the extract of Plum Autumnii), the total polyphenol content of the Plum Autumnii fermented product after fermentation increased significantly. Among them, after anaerobic fermentation by microorganisms, the total polyphenol content can be increased by 3 times (297%). Moreover, the total polyphenol content of Plum Autumnii fermented product A obtained by anaerobic fermentation is significantly higher than that of Plum Autumnii fermented product B obtained by aerobic fermentation. Polyphenols are antioxidant substances rich in plants, and the literature has confirmed that they can effectively reduce the formation of free radicals in the body. It can be seen that the Plum Autumnii fermented product obtained by anaerobic fermentation can have a higher total polyphenol content, which can enhance the antioxidant activity of individuals.

[0128] Example 4

[0129] A. Materials and Instruments:

[0130] 1. Cell line: Mouse melanoma cells, purchased from ATCC (American type culture collection), cell number 6475, hereinafter referred to as B16F10 cells.

[0131] 2. Cell culture medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, product number 12800017), supplemented with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotics (purchased from Gibco, product number 15240-062).

[0132] 3. Kojic acid, purchased from Sigma, product number K3125.

[0133] 4. BSA stock solution: prepared with water and BSA (purchased from Sigma, product number A9647-100G).

[0134] 5. Cell lysis reagent: prepared with RIPA Lysis and Extraction Buffer (purchased from Thermo, product number 89900) and PMSF (phenylmethylsulfonyl fluorid, purchased from Thermo, product number 36978).

[0135] 6. Protein assay reagent: Prepared by diluting Bio-Rad Protein Assay Dye Reagent Concentrate (purchased from Bio-Rad, product number 5000006) with 4 times its volume of water.

[0136] 7.10 mM L-DOPA (3,4-dihydroxyphenylalanine): prepared with 9.8 mg L-DOPA (purchased from Sigma, product number D9628-5G) and 5 mL 0.1 mM phosphate buffer (phosphate buffer, pH 7.0, purchased from Gibco, product number 14200-075).

[0137] 8. ELISA reader, purchased from BioTek (USA).

[0138] B. Test process:

[0139] 1. Plate B16F10 cells at 1.5×10 per well. 5 The cells were inoculated into a 6-well culture dish containing cell culture medium at a density of 100 μg / mL and cultured at 37°C for 24 hours. Here, B16F10 cells were divided into five experimental groups, namely, blank group, kojic acid group, experimental group A, experimental group B and experimental group C. Each group was repeated three times (i.e., each group had three wells).

[0140] 2. After culturing for 24 hours, each group was replaced with an experimental culture medium and continued to be cultured at 37°C for 48 hours. The experimental culture medium of the blank group was a cell culture medium without a sample or kojic acid; the experimental culture medium of the kojic acid group was a cell culture medium containing 200 μg / mL of kojic acid, wherein kojic acid has been widely recognized as a substance with the effect of reducing melanin production; the experimental culture medium of experimental group A was a cell culture medium containing 0.0625% (v / v) of the Autumn Plum extract prepared in Example 1; the experimental culture medium of experimental group B was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product B prepared in Example 2; and the experimental culture medium of experimental group C was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product A prepared in Example 1.

[0141] 3. Remove the experimental culture medium of each group after culture and rinse twice with DPBS.

[0142] 4. After rinsing, add 200ul of cell lysis reagent to each well to lyse the cells to form a cell lysis solution.

[0143] 5. Centrifuge each set of test tubes to pellet the cells.

[0144] 6. Collect the supernatant in each group of centrifuge tubes for protein quantification.

[0145] 7. Standard curve establishment: Prepare 0 mg / mL, 0.15625 mg / mL, 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, and 2.5 mg / mL BSA standard solutions in a 96-well plate according to Table 2 below. Then, add 200 μL of protein assay reagent to each well, mix well with the standard solution and let stand for 5 minutes to obtain a standard reaction solution, and measure its absorbance at 595 nm to obtain a standard curve.

[0146] Table 2

[0147]

[0148] 8. Protein quantification of each group of supernatant: Add 1 μL of the supernatant collected in each group of centrifuge test tubes in step 6 to each well of the 96-well plate, and add 9 μL of water, mix well to obtain the supernatant protein quantification solution of each group. Then, add 200 μL of protein assay reagent to each well, mix well with the sample protein quantification solution of each group and let stand for 5 minutes to obtain the reaction solution to be tested, and measure its absorbance at 595 nm. Use the standard curve and interpolation method to convert the absorbance value of the reaction solution to be tested into the protein quantification value.

[0149] 9. Add the supernatant in each centrifuge tube containing 10 mg protein quantitative value to the corresponding wells of a new 96-well plate, and add cell lysis reagent to each well to make the final volume of each well 100 μL. (That is, make the protein concentration of each group the same, all 100 mg / mL)

[0150] 10. Add 10 μL of 10 mM L-DOPA to each well and incubate at 37°C in the dark for 20 minutes to perform oxidation reaction.

[0151] 11. After 20 minutes of reaction, the absorbance value (OD 405 value).

[0152] C. Test results:

[0153] The relative tyrosinase activity of all groups was calculated according to the following formula: Relative tyrosinase activity (%) = (OD 405 Value / Blank Group OD 405 value)×100%.

[0154] The statistically significant differences between the blank group and the other groups, and between experimental group B and experimental group C were obtained by student t-test statistical analysis. In the figures, "*" represents that the p-value is less than 0.05 when compared with the blank group, "**" represents that the p-value is less than 0.01 when compared with the blank group, and "***" represents that the p-value is less than 0.001 when compared with the blank group; and in the figures, "#" represents that the p-value is less than 0.05 when compared with experimental group B, "##" represents that the p-value is less than 0.01 when compared with experimental group B, and "###" represents that the p-value is less than 0.001 when compared with experimental group B.

[0155] See also Figure 4 . The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative tyrosinase activity of the blank group is set to 100%, the relative tyrosinase activity of the kojic acid group is 85.2%, the relative tyrosinase activity of the experimental group A is 97.7%, the relative tyrosinase activity of the experimental group B is 71.7%, and the relative tyrosinase activity of the experimental group C is 60%. In other words, relative to the blank group, the relative tyrosinase activity of the kojic acid group was significantly reduced by about 14.8%, the relative tyrosinase activity of the experimental group A was reduced by about 2.3%, the relative tyrosinase activity of the experimental group B was significantly reduced by about 28.3%, and the relative tyrosinase activity of the experimental group C was significantly reduced by about 40%. Moreover, relative to the experimental group B, the relative tyrosinase activity of the experimental group C was also significantly reduced by about 11.7%.

[0156] Tyrosinase is a key enzyme in the synthesis of melanin in the body. L-DOPA, under the action of tyrosinase, will produce dopaquinone and then form melanin. Therefore, through the detection of melanin, the activity of tyrosinase can be evaluated, and the ability of melanocytes to produce melanin can be evaluated.

[0157] The experimental results show that the plum extract, plum fermentation product A obtained by anaerobic fermentation, and plum fermentation product B obtained by aerobic fermentation can all reduce tyrosinase activity. The plum fermentation product A obtained by anaerobic fermentation has the ability to significantly reduce tyrosinase activity, and is superior to kojic acid, plum extract, and plum fermentation product B obtained by aerobic fermentation. In other words, the plum fermentation product obtained by anaerobic fermentation can significantly reduce tyrosinase activity, thereby inhibiting and / or reducing the synthesis of melanin, which is beneficial to reduce the individual's melanin and improve the transparency and whitening of the individual's skin.

[0158] Example 5

[0159] A. Materials and Instruments:

[0160] 1. Cell line: Mouse melanoma cells, purchased from ATCC, cell number 6475, hereinafter referred to as B16F10 cells.

[0161] 2. Cell culture medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, product number 12800017), supplemented with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotics (purchased from Gibco, product number 15240-062).

[0162] 3. Kojic acid, purchased from Sigma, product number K3125.

[0163] 4.1N NaOH: prepared with water and NaOH (purchased from Sigma, product number 221465).

[0164] 5. Trypsin: 10X trypsin (purchased from Thermo, product number 15400-054) was diluted with 9 times the volume of DPBS. That is, 10X trypsin was diluted ten times.

[0165] 6. ELISA reader, purchased from BioTek (USA).

[0166] B. Test process:

[0167] 1. Plate B16F10 cells at 1.5×10 per well. 5 The cells were inoculated into a 6-well culture dish with 2 mL of cell culture medium per well and cultured at 37°C for 24 hours. Here, B16F10 cells were divided into five experimental groups, namely, blank group, kojic acid group, experimental group A, experimental group B and experimental group C. Each group was repeated three times (i.e., each group had three wells).

[0168] 2. After culturing for 24 hours, each group was replaced with 2 mL of experimental culture medium and continued to be cultured at 37°C for 48 hours. The experimental culture medium of the blank group was a cell culture medium without sample or kojic acid; the experimental culture medium of the kojic acid group was a cell culture medium containing 200 μg / mL of kojic acid, wherein kojic acid has been widely recognized as a substance with the effect of reducing melanin production; the experimental culture medium of experimental group A was a cell culture medium containing 0.0625% (v / v) of the Autumn Plum extract prepared in Example 1; the experimental culture medium of experimental group B was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product B prepared in Example 2; and the experimental culture medium of experimental group C was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product A prepared in Example 1.

[0169] 3. Remove the experimental culture medium of each group after culture and rinse twice with DPBS.

[0170] 4. After rinsing, add 200 μL of trypsin to each well and react for 3 minutes. After the reaction, add cell culture medium to terminate the reaction. Then collect the suspended cells and cell culture medium in each well into the corresponding centrifuge tube.

[0171] 5. Centrifuge each centrifuge tube to precipitate the cells, remove the supernatant in each set of centrifuge tubes, and then wash and suspend the precipitated cells with DPBS. Repeat this process twice to obtain a cell suspension that is re-dissolved with 200 μL DPBS.

[0172] 6. Freeze the cell suspension with liquid nitrogen for about 10 seconds and place it at room temperature for about 30 minutes until it is completely thawed.

[0173] 7. After completely thawing, centrifuge the test tubes. Then remove the supernatant in the test tubes, add 120 μL 1NNaOH to each test tube, and heat in a 60°C dry bath for 1 hour to dissolve the melanin, so as to obtain the samples to be tested in each group.

[0174] 8. Take 100 μL of the sample to be tested from each group into a 96-well culture plate and use an enzyme immunoassay analyzer to measure the absorbance value (OD) at 405 nm in each well. 405 value).

[0175] C. Test results:

[0176] The relative melanin content of all groups was calculated according to the following formula: Relative melanin content (%) = (OD 405 Value / Blank Group OD 405 value)×100%.

[0177] The statistically significant differences between the blank group and the other groups, and between experimental group B and experimental group C were obtained by student t-test statistical analysis. In the figures, "*" represents that the p-value is less than 0.05 when compared with the blank group, "**" represents that the p-value is less than 0.01 when compared with the blank group, and "***" represents that the p-value is less than 0.001 when compared with the blank group; and in the figures, "#" represents that the p-value is less than 0.05 when compared with experimental group B, "##" represents that the p-value is less than 0.01 when compared with experimental group B, and "###" represents that the p-value is less than 0.001 when compared with experimental group B.

[0178] See also Figure 5 . The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative melanin content of the blank group is set to 100%, the relative melanin content of experimental group A is 107.3%, the relative melanin content of experimental group B is 104.7%, and the relative melanin content of experimental group C is 87.9%. In other words, relative to the blank group, the relative melanin content of experimental group A increased by about 7.3%, the relative melanin content of experimental group B increased by about 4.7%, and the relative melanin content of experimental group C decreased significantly by about 12.1%. Moreover, relative to experimental group B, the relative melanin content of experimental group C also decreased significantly by about 16.8%.

[0179] It can be seen that the Autumn Plum extract and the Autumn Plum fermentation product B obtained by aerobic fermentation cannot reduce the melanin content of B16F10 cells, but increase the melanin content of B16F10 cells. The Autumn Plum fermentation product A obtained by anaerobic fermentation has the ability to significantly reduce the melanin content, and is superior to the Autumn Plum extract and the Autumn Plum fermentation product B obtained by aerobic fermentation. In other words, the Autumn Plum fermentation product obtained by anaerobic fermentation can reduce melanin and / or inhibit melanin production, thereby reducing the melanin of an individual, thereby improving the transparency and whitening of the individual's skin.

[0180] Example 6

[0181] A. Materials and Instruments:

[0182] 1. Cell line: Human skin fibroblasts, purchased from BCRC (Bioresource Collection and Research Center), cell number 60153, hereinafter referred to as CCD-966SK cells.

[0183] 2. Cell culture medium: MEM (Minimum essential medium, purchased from Gibco, product number 11095080), supplemented with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028), 1% penicillin-streptomycin (purchased from Gibco, product number 15140122), 1 mM sodium pyruvate (purchased from Gibco, product number 11360-070), 1.5 g / L sodium bicarbonate (purchased from Sigma, product number S5761-500G) and 0.1 mM non-essential amino acids (purchased from Gibco, product number 11140050).

[0184] 3. Trypsin: 10X trypsin (purchased from Gibco, product number 15400-054) was diluted with 9 times the volume of DPBS.

[0185] 4. Assay buffer: Prepared by diluting 10X Assay buffer (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) and 9 times its volume of DPBS, and placed in a constant temperature water bath to maintain its temperature at 37°C.

[0186] 5. JC-1 reagent: Dissolve JC-1 dye (a reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) in 130 μL DMSO (purchased from ECHO, product number DA1101-000000-72EC) to form a JC-1 storage solution. Then, dilute the JC-1 storage solution with 99 times the volume of the Assay buffer.

[0187] 6. Flow cytometer, purchased from BD.

[0188] B. Test process:

[0189] 1. CCD-966SK cells were plated at 1×10 per well. 5The cells were inoculated into a 6-well culture dish with 2 mL of cell culture medium per well and cultured at 37°C for 24 hours. Here, CCD-966SK cells were divided into four experimental groups, namely, blank group, experimental group A, experimental group B and experimental group C. Each group was repeated three times (i.e., each group had three wells).

[0190] 2. After culturing for 24 hours, each group was replaced with an experimental culture medium and continued to be cultured at 37°C for 24 hours. The experimental culture medium of the blank group was a cell culture medium without a sample; the experimental culture medium of the experimental group A was a cell culture medium containing 0.0625% (v / v) of the Autumn Plum extract prepared in Example 1; the experimental culture medium of the experimental group B was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product B prepared in Example 2; and the experimental culture medium of the experimental group C was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product A prepared in Example 1.

[0191] 3. Remove the experimental culture medium of each group after culture and rinse twice with DPBS.

[0192] 4. After rinsing, add 200 μL of trypsin to each well and react for 5 minutes. After the reaction, add cell culture medium to terminate the reaction. Then collect the suspended cells and cell culture medium in each well into the corresponding centrifuge tube, and centrifuge each centrifuge tube to precipitate the cells.

[0193] 5. Remove the supernatant from each centrifuge tube, wash the precipitated cells twice with DPBS, and then resuspend the cells with 100 μL JC-1 reagent, mix well and incubate in the dark for 15 minutes.

[0194] 6. After the reaction, centrifuge each set of test tubes and remove the supernatant. Wash the precipitated cells twice with Assay buffer.

[0195] 7. After washing, add 500 μL DPBS to each set of centrifuge tubes to resuspend the cells to form a cell suspension.

[0196] 8. After setting the excitation light of the flow cytometer to 488 nm and the scattered light to 527 nm (FITC) and 590 nm (PE), the flow cytometer was used to detect the green (527 nm (FITC)) fluorescence signal and the red (590 nm (PE)) fluorescence signal of each group.

[0197] C. Test results:

[0198] The relative mitochondrial activity of all groups was calculated according to the following formula: relative mitochondrial activity (%) = (red fluorescence signal value of each group / red fluorescence signal value of the blank group) × 100%.

[0199] The statistically significant differences between the blank group and the test results of the other groups were obtained by Student's t-test. In the figure, "*" represents that the p value is less than 0.05 when compared with the blank group, "**" represents that the p value is less than 0.01 when compared with the blank group, and "***" represents that the p value is less than 0.001 when compared with the blank group.

[0200] See also Figure 6 . The blank group was not treated with samples, so the test results of the blank group represent the performance of CCD-966SK cells under normal physiological metabolism. Here, when the relative mitochondrial activity of the blank group is set to 100%, the relative mitochondrial activity of experimental group A is 126.9%, the relative mitochondrial activity of experimental group B is 120.95%, and the relative mitochondrial activity of experimental group C is 145.76%. In other words, compared with the blank group, the relative mitochondrial activity of experimental group A increased significantly by about 26.9%, the relative mitochondrial activity of experimental group B increased significantly by about 20.95%, and the relative mitochondrial activity of experimental group C increased significantly by about 45.76%.

[0201] It can be seen that the Autumn Plum extract, the Autumn Plum fermentation product A obtained by anaerobic fermentation, and the Autumn Plum fermentation product B obtained by aerobic fermentation can all significantly increase mitochondrial activity. The Autumn Plum fermentation product A obtained by anaerobic fermentation has the ability to significantly increase mitochondrial activity, and is superior to the Autumn Plum extract and the Autumn Plum fermentation product B obtained by aerobic fermentation. Mitochondria are the generators of cells and play an important role in energy transfer. The higher their activity, the more vigorous the cell physiological activity. In other words, the Autumn Plum fermentation product obtained by anaerobic fermentation can increase mitochondrial activity, thereby increasing cell physiological activity and regulating cell apoptosis and aging.

[0202] Example 7

[0203] A. Materials and Instruments:

[0204] 1. Cell line: Human skin fibroblasts, purchased from BCRC (Bioresource Collection and Research Center), cell number 60153, hereinafter referred to as CCD-966SK cells.

[0205] 2. Cell culture medium: MEM (Minimum essential medium, purchased from Thermo, product number 61100061), supplemented with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028), 1% penicillin-streptomycin (purchased from Gibco, product number 15140122), 1 mM sodium pyruvate (purchased from Gibco, product number 11360-070), 1.5 g / L sodium bicarbonate (purchased from Sigma, product number S5761-500G) and 0.1 mM non-essential amino acids (purchased from Gibco, product number 11140050).

[0206] 3. Assay buffer: Prepared by diluting 10X Assay buffer (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) and 9 times its volume of DPBS, and placed in a constant temperature water bath to maintain its temperature at 37°C.

[0207] 4. JC-1 reagent: Dissolve JC-1 dye (a reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) in 130 μL DMSO (purchased from ECHO, product number DA1101-000000-72EC) to form a JC-1 storage solution. Then, dilute the JC-1 storage solution with 99 times the volume of the Assay buffer.

[0208] 5. Hoechst stain, purchased from Thermo, product number D 33342.

[0209] 6. Microscope, purchased from ZEISS, product number Axio Vert A1.

[0210] B. Test process:

[0211] 1. CCD-966SK cells were plated at 1×10 per well. 4The cells were inoculated into a 24-well culture dish with a density of 0.5 mL of cell culture medium per well and cultured at 37°C for 24 hours. Here, CCD-966SK cells were divided into four experimental groups, namely: blank group, experimental group A, experimental group B and experimental group C. Each group was repeated three times (i.e., each group had three wells).

[0212] 2. After culturing for 24 hours, each group was replaced with an experimental culture medium and continued to be cultured at 37°C for 24 hours. The experimental culture medium of the blank group was a cell culture medium without a sample; the experimental culture medium of the experimental group A was a cell culture medium containing 0.0625% (v / v) of the Autumn Plum extract prepared in Example 1; the experimental culture medium of the experimental group B was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product B prepared in Example 2; and the experimental culture medium of the experimental group C was a cell culture medium containing 0.5% (v / v) of the Autumn Plum fermentation product A prepared in Example 1.

[0213] 3. Remove the experimental culture medium of each group after culture and rinse once with DPBS.

[0214] 4. After rinsing, add 300 μL of JC-1 reagent and shake gently to ensure that the bottom of the well plate is covered with JC-1 reagent, then let it stand for 15 minutes away from light.

[0215] 5. After standing in the dark, remove the JC-1 reagent from each group and rinse once with DPBS.

[0216] 6. After rinsing, add Hoechst dye (1:20000 dilution) and stain for 5 minutes in the dark.

[0217] 7. After staining in the dark, remove the Hoechst dye from each group and add 500 μL of cell culture medium to maintain the health of the cells during the imaging process.

[0218] 8. Use a microscope to photograph the staining results of each group. In the staining results, red fluorescence represents the signal of normal mitochondria; green fluorescence represents the signal of apoptotic mitochondria; and blue fluorescence represents the signal of cell nuclei.

[0219] C. Test results:

[0220] See also Figure 7. The green fluorescence ratio of the blank group was significantly higher than that of the red fluorescence. Compared with the blank group, the green fluorescence ratio of experimental group A decreased significantly, and the red fluorescence ratio increased significantly, indicating that the Qiuji plum extract can significantly increase mitochondrial activity, that is, significantly reduce cell apoptosis. Compared with the blank group, the green fluorescence and red fluorescence ratio of experimental group B was similar to that of the blank group, but its green fluorescence intensity decreased significantly, indicating that Qiuji plum fermentation product B obtained by aerobic fermentation can slightly increase mitochondrial activity, that is, slightly reduce cell apoptosis. Compared with the blank group, the green fluorescence ratio of experimental group C decreased significantly, and the red fluorescence ratio increased significantly, indicating that Qiuji plum fermentation product A obtained by anaerobic fermentation can significantly increase mitochondrial activity, that is, significantly reduce cell apoptosis.

[0221] It can be seen that the Autumn Plum extract, the Autumn Plum fermentation product A obtained by anaerobic fermentation, and the Autumn Plum fermentation product B obtained by aerobic fermentation can all increase mitochondrial activity. The Autumn Plum fermentation product A obtained by anaerobic fermentation has the ability to significantly increase mitochondrial activity, and is superior to the Autumn Plum extract and the Autumn Plum fermentation product B obtained by aerobic fermentation. Mitochondria are the generators of cells and play an important role in energy transfer. The higher their activity, the more vigorous the cell physiological activity. In other words, the Autumn Plum fermentation product obtained by anaerobic fermentation can increase mitochondrial activity, thereby increasing cell physiological activity and regulating cell apoptosis and aging.

[0222] Example 8

[0223] A. Test process:

[0224] Twelve subjects aged 20 to 55 who wanted to whiten or improve skin condition were asked to drink a 50 mL bottle of test drink (containing 6 mL of Akahime plum fermentation product A prepared in Example 1, and the rest was water and sweetener) every day for 8 consecutive weeks (i.e. 56 days).

[0225] The subjects underwent skin testing before starting to drink (face cleansed, week 0) and 56 days after drinking (face cleansed, week 8). Skin testing uses corresponding instruments and measurement methods based on different test items, records the values ​​of facial skin, and takes photos before and after use. Skin testing items include skin spots and skin brown spots. In addition, when testing before and after use, the temperature and humidity of the test area where the subjects are located are consistent to reduce the impact of external factors such as temperature and humidity on the skin.

[0226] The skin spots were tested using the VISIA Complexion Analysis System purchased from Canfield Scientific, USA, on the subjects' facial skin before drinking the test drink and 56 days after drinking it. This detector uses visible light (white light) to capture high-resolution skin images, and uses built-in software to analyze the number and area of ​​pigment spots visible to the naked eye to obtain a value that can represent the skin's spot condition (hereinafter referred to as the skin spot degree value). In addition, the higher the skin spot degree value obtained, the higher the skin spot degree. Then, the relative skin spot degree is calculated using the following formula: Relative skin spot degree (%) = (skin spot degree value of each group / skin spot degree value before use) × 100%.

[0227] The skin brown spots were tested using the VISIA Complexion Analysis System purchased from Canfield Scientific, USA, on the facial skin of the subjects before drinking the test drink and 56 days after drinking it. This detector uses RBX polarized light technology to take pictures of the facial skin and detect the melanin spots in the dermis that are invisible to the naked eye to obtain a value that can represent the skin's brown spot condition (hereinafter referred to as the skin brown spot degree value). In addition, the higher the skin brown spot degree value obtained, the higher the skin brown spot degree. Then, the relative skin brown spot degree is calculated using the following formula: Relative skin brown spot degree (%) = (skin brown spot degree value of each group / skin brown spot degree value before use) × 100%.

[0228] B. Test results:

[0229] 1. About the test results of "skin spots"

[0230] Reference Figure 8 . The skin spot level values ​​of the 12 subjects at week 0 were regarded as 100% relative skin spot level. At this time, at week 8 (i.e. after 8 weeks of continuous drinking of the test beverage), the average relative skin spot level was 96.4%. In other words, compared to before drinking the test beverage (week 0), after continuously drinking the test beverage containing Akahime Plum Fermentation A for 8 weeks, the relative skin spot level of these subjects was reduced by 3.6%, and the improvement rate reached 66.7% (8 people). It can be seen that the Akahime Plum Fermentation obtained by anaerobic fermentation can indeed reduce skin spots or fade visible spots, and improve the skin condition of the subjects, thereby enhancing the skin texture.

[0231] 2. Test results on "brown spots on skin"

[0232] Reference Fig. 9. The skin brown spot level values ​​of the 12 subjects at week 0 were regarded as 100% relative skin brown spot level. At this time, at week 8 (i.e. after 8 weeks of continuous drinking of the test beverage), the average relative skin brown spot level was 93.3%. In other words, compared to before drinking the test beverage (week 0), after continuously drinking the test beverage containing Akahime Plum Fermentation A for 8 weeks, the relative skin brown spot level of these subjects can be reduced by 6.7%, and the improvement rate is as high as 66.7% (8 people). It can be seen from this that the Akahime Plum Fermentation obtained by anaerobic fermentation can indeed reduce skin brown spots or deep spots, improve the skin condition of the subjects, and thus enhance the skin texture.

[0233] Example 9

[0234] A. Materials and Instruments:

[0235] 1. High-resolution liquid chromatography mass spectrometer: The measurement was carried out by connecting an ultra-high performance liquid chromatograph (Ultimate 3000 HPLC, ThermoFisher Scientific) and a high-resolution orbital ion trap mass spectrometer (Q-EXACTIVE System with Ion MaxSource, Thermo Fisher Scientific). The unit is m / z.

[0236] 2. High Performance Liquid Chromatography (HPLC): Hitachi chromaster 5260 series; elution solvent delivery system Hitachi chromaster 5110; column thermostat Hitachi chromaster 5310; diode array detector (DAD) Hitachi chromaster 5430, detection wavelength 210nm.

[0237] 3. HPLC analysis column: Mightysil RP-18GP 250 (Kanto, 250x4.6mm, 5μm).

[0238] 4. Column Chromatography Filling Materials:

[0239] (1) Macroporous resin: Diaion HP-20, purchased from Mitsubishi Chemical Corporation, Japan.

[0240] (2) Normal phase silica gel: Merck Kieselgel 60, 40-63 μm, purchased from Merck, Germany, product number Art.9385.

[0241] (3) Reverse phase silica gel: Merck RP-18, 40-63um, purchased from Merck, Germany, product number Art.0250.

[0242] 5. Solvent: methanol, purchased from Merck, Taiwan, China.

[0243] 6. Polyvinylidene fluoride membrane filters (PVDF), pore size 0.22 μm, purchased from Millipore, USA.

[0244] 7. Samples: (1) the Akihime plum extract prepared in Example 1, and (2) the Akihime plum fermentation product A prepared in Example 1.

[0245] B.HPLC analysis process:

[0246] 1. The extract of Prunus mume and the fermented product of Prunus mume A were filtered by polyvinylidene fluoride membrane filtration. In order to separate the fat-soluble components from the water-soluble components of the extract of Prunus mume and the fermented product of Prunus mume A, the extract of Prunus mume and the fermented product of Prunus mume obtained in Example 1 were subjected to liquid-liquid phase partition extraction using n-butanol as solvent to obtain the n-butanol-soluble part (fat-soluble component) of the extract of Prunus mume and the water-soluble part (water-soluble component) of the extract of Prunus mume, as well as the n-butanol-soluble part of the fermented product of Prunus mume A and the water-soluble part of the fermented product of Prunus mume A, respectively.

[0247] 2. Use HPLC to analyze the components of each sample to obtain the fingerprint of each sample. Here, methanol (A) and water (B) are used as solvents, 0.1% formic acid is added, and the flow rate is set to 1 mL / min. The extraction conditions are: at 0 minutes, methanol (A): water (B) = 2:98; at 10 minutes, A:B = 2:98; at 40 minutes, A:B = 70:30; at 50 minutes, A:B = 100:0; at 60 minutes, A:B = 100:0. Among them, the samples are autumn plum extract samples (including the n-butanol soluble part of the autumn plum extract and the water soluble part of the autumn plum extract) and autumn plum fermentation A samples (including the n-butanol soluble part of the autumn plum fermentation A and the water soluble part of the autumn plum fermentation A), and the sample concentrations are all 50 mg / mL, and the injection volume is 10 μL. Among them, during HPLC analysis, the column temperature is 40°C.

[0248] C.HPLC analysis results:

[0249] See also Fig.10. The HPLC fingerprint of the plum extract has no obvious peaks and a lower kurtosis. Compared with the plum extract, the HPLC fingerprint of plum fermentation product A has more obvious peaks (peaks 02-S, 01, 03, 05, 08, 04, 06, 07 and 09) and a higher kurtosis. In other words, compared with the plum extract, the plum fermentation product obtained by anaerobic fermentation of microorganisms can greatly increase the compounds it contains, and even has compounds that are not contained in the plum extract.

[0250] It can be seen that the fermented product of Akikomi Plum obtained by anaerobic fermentation has a richer and more diversified compound composition than the Akikomi Plum extract.

[0251] Example 10

[0252] A. Materials and Instruments:

[0253] 1. Nuclear Magnetic Resonance Spectrometer (NMR): 1D and 2D spectra were obtained using Ascend 400 MHz, Bruker Co., Germany. Chemical shift is expressed in δ and the unit is ppm.

[0254] 2. High-resolution liquid chromatography mass spectrometer: The measurement was carried out by connecting an ultra-high performance liquid chromatograph (Ultimate 3000 HPLC, ThermoFisher Scientific) and a high-resolution orbital ion trap mass spectrometer (Q-EXACTIVE System with Ion MaxSource, Thermo Fisher Scientific). The unit is m / z.

[0255] 3. Medium pressure liquid chromatography (MPLC): Rf+, Teledyne ISCO, Lincoln, NE.

[0256] 4. High Performance Liquid Chromatography (HPLC): Hitachi chromaster 5260 series; elution solvent delivery system Hitachi chromaster 5110; column thermostat Hitachi chromaster 5310; diode array detector (DAD) Hitachi chromaster 5430, detection wavelength 210 nm.

[0257] 5. HPLC analysis column: Mightysil RP-18GP 250 (Kanto, 250x4.6mm, 5μm).

[0258] 6.HPLC separation column: 5μm C18(2) (250x10mm), purchased from Phenomenex, USA.

[0259] 7. Column Chromatography Filling Materials:

[0260] (1) Macroporous resin: Diaion HP-20, purchased from Mitsubishi Chemical Corporation, Japan.

[0261] (2) Normal phase silica gel: Merck Kieselgel 60, 40-63 μm, purchased from Merck, Germany, product number Art.9385.

[0262] (3) Reverse phase silica gel: Merck RP-18, 40-63um, purchased from Merck, Germany, product number Art.0250.

[0263] 8. Thin-Layer Chromatography:

[0264] (1) TLC aluminum sheet: thin layer chromatography sheet, coated with silica gel 60F 254 (0.25 mm), purchased from Merck, Germany.

[0265] (2) TLC aluminum sheet: thin layer chromatography sheet, coated with RP-18F 254-S (0.25 mm), purchased from Merck, Germany.

[0266] 9. UV Lamp: UVP UVGL-25, wavelength is 254nm and 365nm.

[0267] 10. Solvent: n-hexane, ethyl acetate, acetone, methanol, ethanol, acetonitrile, chloroform-d1 (deuterated 99.5%), methanol-d4 (deuterated 99.5%), deuterium oxide (deuterated > 99.8%), and dimethyl sulfoxide-d6 (deuterated > 99.9%) were purchased from Merck, Taiwan, China.

[0268] 11. Sample: Fermented product A of Akiehime plum obtained in Example 1.

[0269] B. Compound separation and structure identification process:

[0270] 1. In order to separate the fat-soluble components and water-soluble components of the fermented product of Prunus akira, 5 L of Prunus akira fermented product A prepared in Example 1 was subjected to liquid-liquid partition extraction using n-butanol as solvent to obtain 12.6 g of n-butanol-soluble part (fat-soluble component) and 101.9 g of water-soluble part (water-soluble component).

[0271] 2. The separation and purification of the effective compounds is based on the bioassay guided fractionation method; and the efficacy of reducing the melanin of melanocytes is used as the basis for the selection of stratification and subsequent fine separation parts.

[0272] 3.12.6 g of the n-butanol soluble portion was further subjected to preliminary separation using Diaion HP-20 macroporous resin column chromatography, using pure water, pure water-methanol in a volume ratio of 1:1, and methanol as eluents, to obtain three separations (hereinafter referred to as BUF1 separation, BUF2 separation, and BUF3 separation).

[0273] 4. Take the BUF1 fraction and re-separate it using a reverse phase medium pressure liquid chromatography (RP-MPLC) to obtain a variety of eluents. Here, the eluent is linearly eluted from water to methanol, the eluent time is 60 minutes, and the flow rate is 10 ml per minute. Subsequently, the eluents with similar results are combined using thin layer chromatography (TLC aluminum sheet: thin layer chromatography sheet, coated with silica gel 60F254 (0.25 mm), purchased from Merck, Germany) to obtain three sub-separations (hereinafter referred to as BUF1-1 separation, BUF1-2 separation, and BUF1-3 separation).

[0274] 5. The BUF1-2 fraction was purified by reverse phase-high performance liquid chromatography (methanol / water = 1 / 9) to obtain two compounds TCI-GSF-01 and TCI-GSF-03. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR), it was confirmed by literature comparison that compound TCI-GSF-01 is 2,5-furan dimethanol (2,5-bis-(hydroxymethyl)furane), such as Fig.11 As shown; and, compound TCI-GSF-03 is 5-hydroxyfuroic acid (5-Hydroxymethyl-2-furonic acid), such as Fig.12 shown.

[0275] 6. Take another BUF-2 fraction and re-separate it using a reverse-medium pressure liquid chromatography (RP-MPLC) to obtain multiple eluents. Here, the eluent is linearly eluted from water to methanol, the eluent time is 100 minutes, and the flow rate is 10 ml per minute. Subsequently, the eluents with similar results are combined using thin layer chromatography (TLC aluminum sheet: thin layer chromatography sheet, coated with RP-18F254-S (0.25 mm), purchased from Merck, Germany) to obtain 6 sub-fractions (hereinafter referred to as BUF2-1 fraction, BUF2-2 fraction, BUF2-3 fraction, BUF2-4 fraction, BUF2-5 fraction and BUF2-6 fraction).

[0276] 7. The BU2-2 fraction was purified by reverse HPLC (methanol / water = 3 / 17) to obtain compound TCI-GSF-05. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and carbon-nuclear magnetic resonance spectroscopy (13C-NMR), it was confirmed by literature comparison that compound TCI-GSF-05 is 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, as Figure 14 to Figure 15 shown.

[0277] 8. The BU2-3 fraction was purified by reverse HPLC (methanol / water = 1 / 4) to obtain compound TCI-GSF-04. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR), it was confirmed by literature comparison that compound TCI-GSF-04 is trans-4-hydroxycyclohaxane carboxlic acid, such as Fig.13 shown.

[0278] 9. The BUF2-4 fraction was purified by reverse HPLC (methanol / water = 1 / 3) to obtain compound TCI-GSF-07. Its chemical structure was analyzed by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR). After comparison with literature, it was confirmed that compound TCI-GSF-07 was tyrosol. Fig.16 shown.

[0279] 10. The BUF2-5 fraction was purified by reverse HPLC (methanol / water = 3 / 7) to obtain compound TCI-GSF-09. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and carbon-nuclear magnetic resonance spectroscopy (13C-NMR), it was confirmed by literature comparison that compound TCI-GSF-09 is 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one, such as Figure 17 to Figure 18 shown.

[0280] The names and chemical structures of the aforementioned compounds are shown in Table 3 below.

[0281] Table 3

[0282]

[0283]

[0284] Example 11

[0285] A. Materials and Instruments:

[0286] 1. Cell line: Mouse melanoma cells, purchased from ATCC, cell number 6475, hereinafter referred to as B16F10 cells.

[0287] 2. Cell culture medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, product number 12800017), supplemented with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotics (purchased from Gibco, product number 15240-062).

[0288] 3. Kojic acid, purchased from Sigma, product number K3125.

[0289] 4.1N NaOH: prepared with water and NaOH (purchased from Sigma, product number 221465).

[0290] 5. Trypsin: 10X trypsin (purchased from Thermo, product number 15400-054) was diluted with 9 times the volume of DPBS. That is, 10X trypsin was diluted ten times.

[0291] 6. ELISA reader, purchased from BioTek (USA).

[0292] B. Test process:

[0293] 1. Plate B16F10 cells at 1.5×10 per well. 5 The cells were inoculated at a density of 100 μg / mL in a 6-well culture dish containing cell culture medium and cultured at 37°C for 24 hours. Here, B16F10 cells were divided into eight experimental groups, namely, blank group, kojic acid group, compound 01 group, compound 03 group, compound 04 group, compound 05 group, compound 07 group and compound 09 group. Each group was repeated three times (i.e., each group had three wells).

[0294] 2. After 24 hours of culture, each group was replaced with experimental medium and cultured at 37°C for another 48 hours. Among them, the experimental culture medium of the blank group is a cell culture medium without sample or kojic acid; the experimental culture medium of the kojic acid group is a cell culture medium containing 200 μg / mL kojic acid, wherein kojic acid has been widely recognized as a substance with the effect of reducing melanin production; the experimental culture medium of the compound 01 group is a cell culture medium containing 100 μM of the compound TCI-GSF-01 isolated from Example 9; the experimental culture medium of the compound 03 group is a cell culture medium containing 100 μM of the compound TCI-GSF-03 isolated from Example 9; the experimental culture medium of the compound 04 group is a cell culture medium containing 100 μM of the compound TCI-GSF-04 isolated from Example 9; the experimental culture medium of the compound 05 group is a cell culture medium containing 100 μM of the compound TCI-GSF-05 isolated from Example 9; the experimental culture medium of the compound 07 group is a cell culture medium containing 100 μM of the compound TCI-GSF-07 isolated from Example 9; and the experimental culture medium of the compound 09 group is a cell culture medium containing 100 μM of the compound TCI-GSF-09 isolated from Example 9.

[0295] 3. Remove the experimental culture medium of each group after culture and rinse twice with DPBS.

[0296] 4. After rinsing, add 200 μL of trypsin to each well and react for 3 minutes. After the reaction, add cell culture medium to terminate the reaction. Then collect the suspended cells and cell culture medium in each well into the corresponding centrifuge tube.

[0297] 5. Centrifuge each centrifuge tube to precipitate the cells, remove the supernatant in each set of centrifuge tubes, and then wash and suspend the precipitated cells with DPBS. Repeat this process twice to obtain a cell suspension that is re-dissolved with 200 μL DPBS.

[0298] 6. Freeze the cell suspension with liquid nitrogen for about 10 seconds and place it at room temperature for about 30 minutes until it is completely thawed.

[0299] 7. After completely thawing, centrifuge the test tubes. Then remove the supernatant in the test tubes, add 120 μL 1NNaOH to each test tube, and heat in a 60°C dry bath for 1 hour to dissolve the melanin, so as to obtain the samples to be tested in each group.

[0300] 8. Take 100 μL of the sample to be tested from each group into a 96-well culture plate and use an enzyme immunoassay analyzer to measure the absorbance value (OD) at 405 nm in each well. 405 value).

[0301] C. Test results:

[0302] The relative melanin content of all groups was calculated according to the following formula: Relative melanin content (%) = (OD 405 Value / Blank Group OD 405 value)×100%.

[0303] The statistically significant differences between the blank group and the test results of the other groups were obtained by Student's t-test statistical analysis. In the figure, "*" represents that the p value is less than 0.05 when compared with the blank group, "**" represents that the p value is less than 0.01 when compared with the blank group, and "***" represents that the p value is less than 0.001 when compared with the blank group.

[0304] See also Fig.19 . The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative melanin content of the blank group is set to 100%, the relative melanin content of the kojic acid group is 82.5%, the relative melanin content of the compound 01 group is 53.5%, the relative melanin content of the compound 03 group is 85.7%, the relative melanin content of the compound 04 group is 80.6%, the relative melanin content of the compound 05 group is 61.6%, the relative melanin content of the compound 07 group is 83.3%, and the relative melanin content of the compound 09 group is 86.1%. That is, compared with the blank group, the relative melanin content of the kojic acid group was significantly reduced by about 17.5%, the relative melanin content of the compound 01 group was significantly reduced by about 46.5%, the relative melanin content of the compound 03 group was significantly reduced by about 14.3%, the relative melanin content of the compound 04 group was significantly reduced by about 19.4%, the relative melanin content of the compound 05 group was significantly reduced by about 38.4%, the relative melanin content of the compound 07 group was significantly reduced by about 16.7%, and the relative melanin content of the compound 09 group was significantly reduced by about 13.9%.

[0305] It can be seen that compounds TCI-GSF-01, TCI-GSF-03, TCI-GSF-04, TCI-GSF-05, TCI-GSF-07 and TCI-GSF-09 can significantly reduce melanin. Among them, compound TCI-GSF-01 has a better ability to reduce melanin content than other compounds. In other words, the 2,5-bis-(hydroxymethyl)furane, 5-hydroxyfuranic acid, trans-4-hydroxycyclohaxane carboxlic acid, tyrosol, 6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one and 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one contained in the fermented product of Akiehime plum all have the ability to reduce melanin and / or inhibit the production of melanin, thereby helping to reduce the individual's melanin and thereby improving the transparency and whitening of the individual's skin.

[0306] In summary, the fermented product of Prunus mume in any embodiment can enhance mitochondrial activity or improve skin condition. In other words, the fermented product of Prunus mume in any embodiment is suitable for preparing a composition for enhancing mitochondrial activity or improving skin condition. In some embodiments, the fermented product of Prunus mume or a composition prepared therefrom also has one or more of the following functions: whitening, reducing melanin, inhibiting melanin production, inhibiting tyrosinase activity, reducing skin brown spots, and reducing skin spots.

[0307] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, technicians familiar with the field may make various corresponding changes and deformations based on the present invention, but these corresponding changes and deformations should all fall within the scope of protection of the claims of the present invention.

Claims

1. A fermented product of akihime plum for reducing skin pigmentation spots, characterized in that: The fermented Autumn Plum is obtained by extracting Autumn Plum with water or alcohol and then undergoing anaerobic fermentation. The anaerobic fermentation is performed by performing a first-stage anaerobic fermentation with 0.2% brewer's yeast and then performing a second-stage anaerobic fermentation with 0.06% germ lactobacillus.

2. The fermented product of Akiehime plum according to claim 1, characterized in that The fermented product of autumn plum contains 2,5-furan dimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or a combination thereof.

3. The fermented product of Akiehime plum according to claim 1, characterized in that The fermented product of Akiehime plum has the effect of reducing melanin and / or inhibiting the production of melanin.

4. The fermented product of Akiehime plum according to claim 1, characterized in that The fermented product of Akie Plum has the effect of inhibiting the activity of tyrosinase.

5. Use of the fermented product of Prunus mume as claimed in claim 1 for preparing a composition for enhancing mitochondrial activity.

6. The fermented product of Akiehime plum according to claim 1, characterized in that The pigmented spots are dark brown spots, visible spots or a combination thereof.

7. The fermented product of Akiehime plum according to any one of claims 3, 4 or 6, characterized in that The fermented product of autumn plum contains 2,5-furan dimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or a combination thereof.

8. A method for preparing a fermented product of Autumn Plum for reducing skin pigmentation spots, comprising: Extracting Autumn Plum with a solvent to obtain an Autumn Plum extract, wherein the solvent is water or alcohol; as well as The Autumn Plum extract is anaerobically fermented to obtain the Autumn Plum fermented product. The anaerobic fermentation is performed by using 0.2% brewer's yeast for the first stage of anaerobic fermentation and then using 0.06% germ lactobacillus for the second stage of anaerobic fermentation.

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