TWI774168B - Morus alba ferment, manufacturing method thereof and use thereof - Google Patents

Abstract

The present invention provides aMorus alba ferment, which is obtained from aMorus alba water-extract pre-fermented by yeast and lactic acid bacteria, and post-fermented by acetic acid bacteria. The present invention further provides use of theMorus alba ferment for manufacturing of a composition for improving skin condition and/or inhibiting fat accumulation. Said improving skin condition comprising reducing the production of melanin, enhancing skin fibroblast mitochondrial activity, increasing the secretion of hyaluronic acid, reducing the production of inflammatory response factors, inhibiting the activity of tyrosinase, reducing UV spots, increasing skin moisture content, improving skin elasticity, and/or promoting collagen proliferation. The present invention further provides a manufacturing method of theMorus alba ferment.

Description

White mulberry fermented product and its preparation method and use

The present invention relates to a white mulberry fermented product and its preparation method and use, in particular to the use of the white mulberry fermented product for preparing a composition for improving skin condition and/or inhibiting fat accumulation.

It is human nature to love beauty. Skin beauty and slimming and fat reduction have become a major topic that modern people pay attention to. Not only women strive for skin whitening and slimming, but in recent years, boys focus on skin whitening and health care. The reason why the skin turns dark is that the skin is directly or indirectly exposed to sunlight every day. In order to protect the skin from ultraviolet rays, the human body will synthesize melanin to defend against ultraviolet rays in the sun. Can cause dull, sallow, blotchy, or acne-prone skin.

In addition, with age, the skin will develop wrinkles or sagging, which is known to be caused by the reduction of the collagen production capacity of dermal fibroblasts; and aging will reduce the skin's moisture content, causing the skin to lose Firming and softening, it is related to the loss of hyaluronic acid, which can be used to increase the moisture level of the skin and strengthen the protection of the skin's surface layer.

In today's society, the concept of natural organic and natural ingredients skin care products is gradually emerging, and biotechnology companies and skin care products related industries are actively investing in the research and development of natural plant-related products. Component analysis and efficacy evaluation have become key items in product development.

White mulberry ( Morus alba , also known as white mulberry) is a fast-growing small and medium-sized mulberry tree, which can grow to 10-20 meters high, and it is usually regarded as a short-lived tree species, native to northern China and India is currently widely planted around the world, including: the United States, Turkey, Mexico, Australia, Argentina, and Iran. It is mainly grown by silk producers to feed the silkworms used in production. The leaves of white mulberries can grow as long as 30 cm in the shape of a serrated circle, while the fruit is about 1-1.5 cm long and is edible when ripe.

The well-known mulberry fruit is red or black-purple, but the white mulberry fruit is white, so it is also called "white jade king", its taste is sweeter than the ordinary black mulberry, and the mature white mulberry has a little purple, After it is fully cooked, the sweetness can reach 19 degrees; the white mulberry contains low calories and is suitable for obese people, and it contains trace elements such as protein, fat, vitamins, carbohydrates and dietary fiber, and is rich in nutritional value. However, whether white mulberry has other effects and uses has not been widely understood and applied.

In view of this, the object of the present invention is to provide a kind of white mulberry fermented product, wherein this white mulberry fermented product is a white mulberry water extract obtained by a white mulberry fruit through water extraction, and then through a yeast and a lactic acid bacteria to carry out a It is obtained by pre-fermentation and post-fermentation with an acetic acid bacteria.

In some embodiments of the present invention, the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria is 1-20%(v/v).

In some embodiments of the present invention, the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.

In some embodiments of the present invention, the white mulberry fermented product is further filtered with a mesh screen of 200-400 mesh, and concentrated under reduced pressure at ^{55-65 ℃} , and then 40-70% isomalt is added. The oligosaccharides are sized and sterilized by heating at 95-105 ^o C for 100-120 minutes.

In some embodiments of the present invention, the total polyphenol content of the white mulberry fermentation product is 2-5 times the total polyphenol content of the white mulberry water extract.

Another object of the present invention is to provide a use of the aforementioned white mulberry fermented product for preparing a composition for improving skin condition and/or inhibiting fat accumulation.

In some embodiments of the present invention, the improving skin condition is reducing melanin production, increasing skin fibroblast mitochondrial activity, increasing hyaluronic acid secretion, reducing UV pigmentation, increasing skin moisture content, increasing skin elasticity, and/or Promote collagen growth.

In some embodiments of the present invention, the composition achieves the effect of reducing melanin production by reducing the production of an inflammatory response factor and inhibiting the activity of tyrosinase, wherein the inflammatory response factor is nitric oxide.

In some embodiments of the present invention, the concentration of the white mulberry fermentation product is at least 0.5% (v/v).

In some embodiments of the present invention, the composition is a pharmaceutical product, a food product or a skin care product.

Another object of the present invention is to provide a method for preparing a white mulberry fermented product. Acetobacter is subjected to a post-fermentation, filtered with a 200-400 mesh sieve, concentrated under reduced pressure at 55-65 ^o C, and adjusted by adding 40-70% isomalt oligosaccharide, then adjusted at 95-105 ^o C Heating for 100-120 minutes for sterilization; wherein the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria 1-20% (v/v); wherein the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.

Some specific implementations of this case will be further described below in conjunction with the drawings. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. The scope of protection should not be limited to the conditions specifically stated in the description, while some changes and modifications may be made within the spirit and scope.

The case used Excel software for statistical analysis. Data are presented as mean ± standard deviation (SD), and differences between groups were analyzed by Student's t-test. In the graph, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference. The "###" symbol represents a p-value less than 0.001 compared to the control group.

The numerical values used herein are approximate, and all experimental data are expressed within the range of plus or minus 20%, preferably within the range of plus or minus 10%, and most preferably within the range of plus or minus 5%.

Herein, "wt%" refers to weight percent, and "vol%" refers to volume percent. As used herein, "white mulberry" generally refers to the white mulberry fruit.

The "effective concentration" or "effective amount" described herein means that it can effectively reduce the production of melanin, increase the mitochondrial activity of skin fibroblasts, increase the secretion of hyaluronic acid, inhibit the accumulation of fat, reduce the production of inflammatory response factors, and inhibit the production of tyrosinase The concentration of the white mulberry fermented product of the present invention required for activity, reducing UV spots, increasing skin moisture content, improving skin elasticity, and/or promoting collagen proliferation. The effective concentration may vary depending on the target, but the effective concentration can be determined experimentally by, for example, dose escalation.

In some embodiments, the white mulberry fermented product is obtained by performing a pre-fermentation process with the water extract of white mulberry, yeast and lactic acid bacteria, and then performing a post-fermentation process with an acetic acid bacteria.

In some embodiments, the white mulberry water extract comprises white mulberry and water, the ratio of the white mulberry and water is 1:5-20, and 5% glucose is added according to the total weight, so that its Brix (Degrees Brix)>8.5 , and heated at 95 ^o C for 60 minutes.

In the pre-fermentation process, first add 0.01%-0.5% yeast to the white mulberry water extract, and ferment the white mulberry water extract and yeast for 1 to 2 days, and then add 0.01%-0.25% lactic acid bacteria. Fermentation 1 to 2 days to complete the previous fermentation process. In other words, the addition amount of the yeast is 0.01-0.5% (v/v), and the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), wherein the fermentation time of the pre-fermentation is 1-3 days, and It is carried out at 28°C-37°C.

In the post-fermentation procedure, add 1-20% acetic acid bacteria for 3-10 days, and stop the fermentation when the Brix value is less than 3.9 and the pH value is equal to 3.7±1.

In some embodiments, the yeast may be Saccharomyces cerevisiae , and in some embodiments, the lactic acid bacteria may be Streptococcus thermophilus , Lactobacillus helveticus , or Lactobacillus plantarum , in some embodiments, the acetic acid bacteria can be acetic acid bacteria ( Acetobacter aceti ).

In some embodiments, the white mulberry fermented product can be further subjected to mesh filtration, reduced pressure concentration, specification adjustment and heat sterilization.

In some embodiments, the mesh filtration is performed with a mesh filter with a mesh size of 200 to 400 mesh, and concentrated under reduced pressure at 55-65 ^° C.

In some embodiments, the specification can be adjusted by adding 40-70% excipients to adjust the Brix of the fermentation stock. In some embodiments, the excipient used to adjust the sugar content may be isomalt-oligosaccharide.

In some embodiments, the heat sterilization procedure may be to sterilize the raw material mixture at 95°C to 105°C for 100-120 minutes, and cool the sterilized mixture to room temperature (ie, 25°C to 30°C) to form white Mulberry fermented product. In some embodiments, the sterilized raw material mixture can be cooled to room temperature by natural cooling.

In some embodiments, the pH of the white mulberry fermentation is 4.0±1.0, and the Brix of the white mulberry fermentation is 39±2.

In some embodiments, the preparation method of the white mulberry fermented product includes extracting a white mulberry water extract obtained from a white mulberry through water, performing a pre-fermentation through a yeast and a lactic acid bacteria, and then using an acetic acid bacteria Carry out a post-fermentation, and filter with a 200-400 mesh sieve, then concentrate under reduced pressure at 55-65 ^o C, add 40-70% isomalt oligosaccharide for adjustment, and heat at 95-105 ^o C Sterilize for 100-120 minutes; wherein the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria is 1 -20% (v/v); wherein the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.

In some embodiments, the aforementioned white mulberry fermented product is used to prepare a composition for improving skin condition and/or inhibiting fat accumulation. In some embodiments, the improving skin condition is reducing melanin production, increasing skin fibroblast mitochondrial activity, increasing hyaluronic acid secretion, reducing UV pigmentation, increasing skin moisture content, increasing skin elasticity, and/or promoting collagen hyperplasia. In some embodiments, the composition achieves the reduction in melanin production by reducing inflammatory response factor production and inhibiting tyrosinase activity. In some embodiments, the inflammatory response factor is nitric oxide. Wherein, the receptor can be human.

In some embodiments, the concentration of the white mulberry fermentation is at least 0.5% (v/v).

In some embodiments, the composition is a pharmaceutical product, a food product, or a skin care product. In other words, the medicine, food or skin care product contains an effective content of white mulberry fermented product.

In some embodiments, the aforementioned pharmaceutical products can be manufactured for parenterally, parenterally, orally, or topically using techniques well known to those skilled in the art Dosage form.

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

In some embodiments, the aforementioned pharmaceutical products may include pharmaceutically acceptable carriers that are widely used in pharmaceutical manufacturing techniques. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent, disintegrant ( decomposer), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, glue gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. Regarding the type and quantity of the carrier selected, it falls within the scope of the professional quality and routine skills of those who are familiar with the technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier can be water, normal saline, phosphate buffered saline (PBS), or an alcohol containing aqueous solution ).

In some embodiments, any of the foregoing compositions may be edible compositions. In other words, the edible composition contains a specific amount of white mulberry fermented product. In some embodiments, the aforementioned edible composition may be a food product or a food additive. In some embodiments, the food products can be, but are not limited to, beverages, fermented foods, bakery products, health foods, or dietary supplements.

In some embodiments, the aforementioned edible compositions can be administered to a recipient orally. Wherein, the form of the edible composition can be powder, granule, solution, colloid or paste.

In some embodiments, any of the foregoing compositions can be a cosmetic or skin care product. In other words, cosmetics or skin care products contain a specific content of white mulberry fermented product.

In some embodiments, the aforementioned cosmetics or skin care products can be in any of the following forms: lotion, gel, jelly film, mud film, lotion, cream, lipstick, foundation, pressed powder, powder, makeup remover, makeup remover Milk, facial cleanser, body wash, shampoo, conditioner, sunscreen, hand cream, nail polish, perfume, serum and mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, emollients, solvents, excipients, antioxidants, or combinations thereof.

Embodiment 1. Fermented white mulberry preparation

First, mix the white mulberry ( Morus alba , the origin of white mulberry is Turkey) and water at a ratio of 1:10 (that is, the white mulberry is one time by weight, and the water is ten times the weight), and then add 5% glucose solution. The sugar content was adjusted to 8.5, and heated at 95° C. for 60 minutes to obtain a white mulberry water extract.

Then, 0.1% brewer's yeast ( Saccharomyces cerevisiae ) (purchased from the Biological Resource Conservation and Research Center (BCRC), Institute of Food Industry Development, accession number BCRC20271) was propagated into the white mulberry water extract and fermented at about 30°C for 1 day , to obtain the first primary fermentation juice. Next, 0.05% Streptococcus thermophilus (purchased from BCRC, accession number BCRC910636) was propagated into the first primary fermentation juice and fermented at about 30° C. for 1 day to obtain a second primary fermentation juice. Finally, 5% acetic acid bacteria ( Acetobacter aceti ) (purchased from BCRC, accession number BCRC11688) was propagated in the second primary fermentation juice and fermented at about 30°C for 5 days to obtain the third primary fermentation juice.

Then, the third primary fermentation juice is filtered through a mesh screen with a mesh size of 200, and then concentrated under reduced pressure at about 60° C. to obtain a fermentation stock solution. Finally, after adding 60% isomalt oligosaccharide to the fermentation stock solution, it was sterilized at about 100° C. for 2 hours to obtain a white mulberry fermented product.

Here, the sugar content of the white mulberry water extract and the white mulberry fermented product were detected respectively. Among them, the sugar content of white mulberry water extract is about 8.5, and the sugar content of white mulberry fermented product is about 40.

Example 2. Total polyphenol content test

10.0 mg of gallic acid (Gallic acid, purchased from Sigma, material number: G7384) was weighed into a 10 mL volumetric flask, and then quantified to 10 mL with water (H ₂ O) to obtain a stock solution of gallic acid (stock solution). The stock solution of gallic acid was diluted 10-fold, ie, 100 μL of the stock solution of gallic acid plus 900 μL of water, to obtain an initial solution of 100 μg/mL gallic acid (ie, containing 1000 ppm of gallic acid). Then, prepare standard solutions of gallic acid of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL according to Table 1, and take 100 μL of the standard solutions of each concentration. into a glass test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck, part number: 1.09001.0100) to each glass test tube and mix it with the standard solution and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate After mixing uniformly, the reaction was carried out for 30 minutes to obtain a standard reaction solution. Take 200 μL of the standard reaction solution into a 96-well plate, and measure its absorbance at 750 nm to obtain a standard curve.

Table I Standard concentration (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

The test sample of the experimental group (ie the white mulberry fermented product of Example 1) and the test sample of the control group (ie the white mulberry water extract of Example 1) were diluted 20 times with water respectively. Take 100 μL of each sample into a glass test tube. Next, add 500 μL of Folin phenol reagent to the glass test tube and mix with the sample evenly and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well, 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 no air bubbles, take 200 μL of the reaction solution to be tested into a 96-well plate, and measure the absorbance value of the reaction solution to be tested at 750 nm. The absorbance value of the reaction solution to be tested was first divided by the sugar content of the sample, and then converted into the total polyphenol content by interpolation of the standard curve.

The results of total polyphenol content are shown in Figure 1. The total polyphenol content of the control group is 90 ppm and the total polyphenol content of the experimental group is 250 ppm. Compared with the control group, the total polyphenol content of the sample group has been significantly improved. . Here, it can be inferred that the actual total polyphenol content of the white mulberry water extract and the white mulberry fermented product (that is, the white mulberry water extract and the white mulberry fermented product obtained by the first preparation method of this case) before the dilution are respectively 1800ppm and 5000ppm. It can be seen that the total polyphenol content of white mulberry can be increased by about 2.77 times after microbial fermentation. That is, compared with the white mulberry water extract, the white mulberry fermented product will release a large amount of total polyphenols, and can enhance the antioxidant activity, which can effectively reduce the accumulation of free radicals.

Example 3. Detection of Nitric Oxide, One of Anti-inflammatory Ability

In order to investigate whether the fermentation product of white mulberry has the effect of reducing inflammatory response, in this example, macrophage RAW264.7 was cultured as a cell platform, and then lipopolysaccharide (LPS) treatment was used to induce inflammatory response, and inflammatory response factors were detected. The amount of nitric oxide (NO) generated was used to evaluate whether the sample achieved the effect of reducing the inflammatory response.

Materials and Instruments 1. Cell line: mouse macrophage RAW264.7, obtained from American Type Culture Collection (American Type Culture Collection, ^ATCC® , Cat. TIB-71). 2. Medium: DMEM modified medium (Dulbecco's modified Eagle's medium, DMEM, purchased from Gibco, Cat. 11965-092) was added with additional components to contain 10 vol% fetal bovine serum (Fetal Bovine Serum, FBS, purchased from Gibco, Cat. .10437-028) and 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number Cat. 14200-075. 4. Lipopolysaccharide (LPS): purchased from Sigma, product code is SI-L2880-25MG. 5. Griess reagent kit: purchased from Life technologies, product number 1445263. 6. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.

Experimental steps: 1. The experiment will be divided into experimental group (experimental group A and experimental group B), control group, and control group; wherein the experimental group A is the white mulberry water extract group, and the experimental group B is the white mulberry fermentation RAW264.7 cells were seeded into 96-well cell culture plates at a density of 1 x 10 ⁴ cells/well, the volume of culture medium was 200 μl/well, and the cells were incubated at 37°C, 5 24 hours in a %CO ₂ constant temperature incubator. 3. After removing the medium in the well plate to be tested, LPS (concentration of 200 ng/ml) was added to the experimental group and the control group; at the same time, the culture medium of experimental group A was additionally added with white mulberry water extract to make it. The concentration was 0.5 mg/mL; the culture medium of experimental group B was additionally added with white mulberry fermented product to make the concentration of 0.5 mg/mL, and it was treated for 24 hours. (Use a medium without FBS) 4. Take a 96-well plate, add 130 μl of secondary water to each well, and add 150 μl of the culture medium from each well of step 3 after the reaction to the above-mentioned 130 μl secondary water. water in a 96-well dish. 5. Prepare Griess reagent (reagent A: B=1:1), add 20 μl of the culture medium to each well of a 96-well plate, and react in the dark for 30 minutes. 6. Using an ELISA reader (purchased from BioTek), measure the absorbance at OD548 nm (the larger the OD reading, the higher the NO concentration). 7. Statistical analysis was performed using student t-test (* table p < 0.05, ** table p < 0.01, *** table p < 0.001).

Experimental results

Referring to FIG. 2 , it is a graph showing the change in the relative generation amount of nitric oxide after the white mulberry water extract and the white mulberry fermented product of the present invention are treated. Nitric oxide is an important inflammatory response factor. The amount of production was used as an indicator of inflammatory response capacity. As shown in the figure, the production of nitric oxide in the control group was 122.86% relative to the control group, the production of nitric oxide in the experimental group A (white mulberry water extract) relative to the control group was 116.33%, while the experimental group B (white mulberry water extract) produced 116.33% of nitric oxide. Fermentation) was 106% nitric oxide production relative to the control group. It can be seen from the experimental results that compared with the control group, the white mulberry water extract and the white mulberry fermented product reduced the production of nitric oxide by about 6.53% and 16.86%, respectively, and the RAW264.7 macrophages treated with the white mulberry fermented product were significantly reduced. The production of nitric oxide was significantly lower than that of the control group, indicating that RAW264.7 macrophages could reduce the production of inflammatory response factors after treatment with white mulberry fermentation, indicating that white mulberry fermentation had the effect of resisting inflammatory response and secreting anti-inflammatory factors. And its effect is better than that of white mulberry water extract, and the secretion of anti-inflammatory factors can reduce the effect of melanin precipitation.

Example 4. Detection of tyrosinase activity

Tyrosinase is the key to melanin synthesis in living organisms. Tyrosinase catalyzes the conversion of tyrosine into L-Dopa, and then produces Dopaquinone to form melanin. Here, the tyrosinase activity was detected to understand the effect of white mulberry fermentation on the ability of melanocytes to produce melanin. When the tyrosinase activity was low, the ability of melanocytes to produce melanin was relatively reduced.

Materials and instruments 1. Cell line: mouse melanoma cell B16F10, purchased from American Type Culture Collection (American Type Culture Collection, ^ATCC® , No. 6475), hereinafter referred to as B16F10 cell. 2. Medium: DMEM modified medium (Dulbecco's modified Eagle's medium, DMEM, purchased from Gibco, Cat. 12100-038) was added with additional components to contain 10 vol% FBS (fetal bovine Serum, purchased from Gibco, product number 10438-026) ) and 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. Kojic acid: purchased from Sigma-Aldrich, product number K3125-5G. 5. Full name Radioimmunoprecipitation Buffer Protein Lysis Buffer (RIPA Lysis Buffer (PMSF)): purchased from Sigma-Aldrich, product number 10837091001. 6. Protein quantification kit (Bio-rad Protein Assay, purchased from Biotek, product number Epoch). 7. 10 mM L-Dopa (L-Dopa): 9.8 mg of L-Dopa (purchased from Sigma-Aldrich, product number D9628-5G) was dissolved in 5 mL of 0.1 mM PBS, pH 7.0. 8. Trypsin: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times in 1X PBS solution. 9. White mulberry water extract and white mulberry fermented product: obtained by the preparation method of Example 1 of this case.

Experimental steps:

2 mL of the above medium was added to each well, and B16F10 cells were seeded in a 6-well dish at a density of 1.5 x 10 ⁵ per well and incubated at 37°C for 24 hours.

The experiment was divided into control group, control group, experimental group A and experimental group B. Among them, 2 mL of fresh DMEM medium was added to each group. The culture medium of the control group was additionally added with Kojic acid at a concentration of 0.5 mg/mL, of which kojic acid has been widely recognized as a substance that inhibits cellular tyrosinase activity; the culture medium of experimental group A was additionally White mulberry water extract was added to make its concentration 0.5 mg/mL; the culture medium of experimental group B was additionally added white mulberry fermented product to make its concentration 0.5 mg/mL. Three replicates were performed for each group, and the reaction was incubated for 48 hours.

After incubation, the medium was removed and washed twice with 1x PBS(). Then trypsin-EDTA was added to act on the cells for 3 minutes, the suspended cells were recovered in a 15 mL centrifuge tube, and the cells were pelleted by centrifugation at 400 xg for 5 minutes.

After the pelleted cells were washed twice with 1x PBS, the cells were suspended with 200 μL of cell lysate, mixed by shaking, and centrifuged at 12,000 x g for 20 minutes.

Remove the supernatant into a 1.5 mL centrifuge tube for protein concentration detection:

First, mix Bio-rad stain with deionized water (1:4 by volume), and dispense 500 μL into microcentrifuge tubes. Then, 10, 8, 6, 4, 2, 1, and 0 μL of 2 mg/mL BSA were added to the aforementioned microcentrifuge tubes to prepare standard protein concentration. Add another 2 μL of test sample for detection. 200 μL of the test sample after the aforementioned reaction was taken out in a 96-well plate, and the absorbance at 595 nm was measured with an ELISA reader (purchased from BioTek).

Next, 400 μg of protein was added to each well, followed by 90 μL of cell lysate, including control group and control group. In the dark, add 10 μL of 10 M L-Dopa at 37 °C and observe every 10 min until the suspension turns black. The absorbance at 405 nm was then measured.

The analysis formula of tyrosinase content is: tyrosinase inhibition (%)=(sample absorbance/control group absorbance)×100%. The obtained values were then statistically analyzed with Microsoft EXCEL software and Student's t test, and the results are shown in Figure 3.

It can be seen from the results in FIG. 3 that the effect of inhibiting the activity of tyrosinase can be achieved under the treatment of the white mulberry sample of the present invention. Specifically, the tyrosinase activity of the control group relative to the control group was 69.87%, the tyrosinase activity of the experimental group A (white mulberry water extract) relative to the control group was 86.44%, and the experimental group B (white mulberry fermentation The tyrosinase activity relative to the control group was 74.13%. Compared with the control group, the tyrosinase activity of the experimental group A decreased by about 13.56%, and the tyrosinase activity of the experimental group B was decreased by about 25.87% compared with the control group. The tyrosinase inhibitory ability was higher than that of experimental group A (white mulberry water extract), and the efficacy of the white mulberry fermented product in this case was similar to that of kojic acid in inhibiting tyrosinase activity. Therefore, the white mulberry fermented product prepared by the embodiment of the present invention can indeed reduce the activity of tyrosinase, thereby reducing the generation of melanin, so it can be applied to reduce skin dark spots, improve skin luster, whiteness and skin whitening. components of the composition.

Embodiment 5. Detection of melanin production

Here, an enzyme-linked immunosorbent assay reader was used to measure the change of the melanin content of the melanoma cell line B16F10 after being treated with the white mulberry fermentation product.

Materials and Instruments 1. Cell line: mouse melanoma cell B16F10, purchased from American Type Culture Collection (American Type CμLture Collection, ATCC®, No. 6475), hereinafter referred to as B16F10 cell. 2. Medium: DMEM (Dulbecco's modified minimal essential medium, available from Gibco, Cat. 12100-038) was added with additional components to contain 1 vol% of an antibiotic solution (Antibiotic Antimycotic Solution, available from Gibco, 15240-062) and 10 vol% of FBS (fetal bovine Serum, available from Gibco, 10437-028). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. A solution of 1N NaOH (purchased from Sigma, product number 221465) was prepared in double distilled water. 5. Enzyme immunoassay analyzer (ELISA reader, purchased from BioTek, product number FLx 800). 6. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.

Experimental Procedures The experiment will be divided into four groups: experimental group A, experimental group B, control group and control group. Each group will carry out three repeated experiments: 1. B16F10 cells are inoculated in 1.5×10 ⁵ cells per well. in 6-well plates containing 3 ml of medium per well. 2. Place the culture plate in an environment of 5% CO ₂ and 37° C. for 24 hours. 3. Then, without disturbing the attached cells, remove the medium from each well. 4. This experiment is divided into control group, control group, experimental group A and experimental group B. Among them, 2 mL of fresh DMEM medium was added to each group. The culture medium of the control group was additionally added with kojic acid to a concentration of 0.5 mg/mL, of which kojic acid has been widely recognized as a substance that has the effect of reducing the production of melanin; the culture medium of the experimental group A was additionally added with white mulberry The water extract was added to make the concentration of 0.5 mg/mL; the culture medium of experimental group B was additionally added with white mulberry fermentation product to make the concentration of 0.5 mg/mL. Groups were replicated in triplicate and incubated at 37°C for 24 hours. 5. The experimental group A, the experimental group B and the control group after the reaction were moved to a blue light box, and subjected to blue light irradiation at room temperature (25±5°C) for 3 hours. Additionally, the control group was moved to a dark room and allowed to stand at room temperature for 3 hours. 6. After the reaction, the cells were incubated at 37°C for 48 hours. 7. Afterwards, the medium was removed and the cells were washed twice with PBS solution. 8. Add 200 μl of trypsin (Trypsin-EDTA (10X), purchased from Gibco; product number 15400-054) to each well for 3 minutes of reaction. After the reaction, 6 mL of medium solution was added to stop the reaction. Then, the suspended cells and medium in each well were collected into corresponding 15ml centrifuge tubes, and each centrifuge tube was centrifuged at 400 xg for 5 minutes to pellet the cells. 9. After washing the pelleted cells twice with PBS solution, resuspend the cells with 200 μL PBS solution. 10. Freeze the cell suspension in liquid nitrogen for 10 minutes and allow to thaw completely at room temperature for about 30 minutes. 11. After complete thawing, centrifuge the tube at 12,000 g for 3 minutes. 12. Remove the supernatant, resuspend the cell pellet with 120 μL of 1N sodium hydroxide solution, and dry the test tube at 60° C. for 1 hour to obtain the sample to be tested. 13. Transfer 100 μL of the sample to be tested into a 96-well plate, and use an ELISA plate reader to measure the absorbance of the cell solution at 450 nm.

Experimental results

The relative expression of melanin in the experimental group and the control group was calculated according to the following formula: relative expression of melanin (%)=(OD ₄₅₀ value of each group/OD ₄₅₀ value of control group)×100%. Since the experiment was performed in triplicate, the results of the triplicate experiments were averaged and shown in Figure 4 .

As shown in FIG. 4 , under the treatment of the white mulberry sample of the present invention, the effect of reducing the production of melanin can be achieved. Specifically, the melanin production of the control group relative to the control group was 83.9%, the melanin production of experimental group A (white mulberry water extract) relative to the control group was 93.54%, and the experimental group B (white mulberry fermented product) was relatively control The melanin production of the group was 86.05%. Compared with the control group, the production of melanin in the experimental group A was reduced by about 6.46%, and the production of melanin in the experimental group B was reduced by about 13.95% compared with the control group. The effect of acid to reduce melanin production. Therefore, the white mulberry fermented product prepared in the embodiment of the present invention can indeed effectively reduce the melanin produced by melanocytes, so it can be used as an ingredient in a related composition for reducing skin dark spots, enhancing skin luster, and whitening skin.

Example 6. Skin cell mitochondrial activity experiment

In order to explore the effect of white mulberry fermentation product on the mitochondrial function of skin cells, flow cytometry was used to evaluate the changes in mitochondrial activity of human skin fibroblasts CCD-966sk treated with white mulberry fermentation product. In this experiment, the aggregation ability of JC-1 was used as an indicator of mitochondrial activity. In healthy mitochondria with normal membrane potential, JC-1 exists in the form of a polymer, and when the mitochondrial membrane potential decreases, JC-1 exists in the form of a polymer. JC-1 becomes a monomer, which can be used as an indicator for the assessment of mitochondrial activity.

Materials and Instruments 1. Cell Line: Human skin fibroblasts CCD-966sk (BCRC No. 60153). 2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). Among them, the basal medium is made of Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319) with additional components to make it contain 1 mM sodium pyruvate (sodium pyruvate, purchased from Gibco), 1.5 g/L sodium bicarbonate (purchased from Sigma) and 0.1 mM non-essential amino acid solution (purchased from Gibco) were prepared. 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco. 4. Mitochondrial membrane potential detection kit (BD ^TM MitoScreen(JC-1) kit, model 551302). Among them, the mitochondrial membrane potential detection kit has JC-1 dye (lyophilized) and 10X analysis buffer. 10X Assay Buffer was diluted 10-fold in IX PBS prior to use to form IX Assay Buffer. 130 μL of DMSO was added to JC-1 stain (lyophilized) to form a JC-1 stock solution. The JC-1 stock solution was then diluted with 1X assay buffer to form the JC-1 working solution (ie, JC-1 mitochondrial specific stain). The dilution ratio was 1:100 for JC-1 stock solution to 1X assay buffer. 5. Trypsin: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times in 1X PBS solution. 6. Flow cytometer: purchased from BD Pharmingen Company, model BDTM Accuri C6 Plus. 7. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.

Experimental steps 1. CCD-966sk cells were seeded in a 6-well culture dish containing 2 mL of cell culture medium per well at 1×10 ⁵ cells per well. 2. Replace the medium in each well of the plate with 2 mL of experimental medium. Wherein, the experimental medium of experimental group A was the white mulberry water extract obtained in Example 1 containing 0.5 mg/mL. The experimental medium of experimental group B was a cell culture medium containing 0.5 mg/mL of the white mulberry fermented product obtained in Example 1. The experimental medium of the control group was pure cell culture medium (ie, no white mulberry sample). 3. Place the culture plate in 5% CO ₂ at 37°C for 24 hours. 4. Remove the experimental medium from the plate and rinse twice with 1 mL of 1X PBS solution. 5. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, cell culture medium was added to terminate the reaction. Collect the cells and cell culture medium in each well into a corresponding 1.5 mL centrifuge tube, and centrifuge the centrifuge tube containing the cells and cell culture medium at 400 x g for 10 minutes. 6. After centrifugation, remove the supernatant and re-dissolve the cell pellet with 1 mL of 1X PBS or transfer the cell pellet to a 15 mL centrifuge tube to form a cell suspension. 7. Centrifuge the tube containing the cell suspension at 400 x g for 5 minutes. 8. After centrifugation, remove the supernatant from each centrifuge tube and add 100 μL of JC-1 working reagent to each centrifuge tube. 9. Vortex the cell pellet in each centrifuge tube with JC-1 working reagent and incubate in the dark for 15 minutes. 10. After 15 minutes, centrifuge each tube at 400 x g for 5 minutes. 11. After centrifugation, remove the supernatant from each centrifuge tube, re-dissolve the cell pellet in each tube with 1 mL of 1X PBS solution and centrifuge at 400 x g for 5 min. Repeat this step twice. 12. After centrifugation, remove the supernatant from each centrifuge tube and resuspend the cells with 500 μL of 1X PBS to obtain the cell solution to be tested. 13. Measure the mitochondrial membrane potential of the cells to be tested in each well by flow cytometry to analyze mitochondrial activity. Since the experiment was carried out in three repetitions, the results of the three repeated experiments in each group were averaged to obtain the average value, and then the average value of the control group was taken as 100% of the relative accumulation of JC-1, and the average value of the experimental group was converted into a relative value. The aggregated amount of JC-1 is shown in Figure 5.

Experimental results

Referring to FIG. 5 , it is a bar graph comparing the relative amount of JC-1 aggregation in the experimental group treated with the white mulberry water extract and the white mulberry fermented product of the present invention and the untreated control group. It can be seen from the figure that the relative aggregate amount of JC-1 in experimental group A is about 97.5%, while the relative amount of JC-1 aggregate in experimental group B is about 117.19%. In other words, compared with the control group, the mitochondrial activity of human skin fibroblasts in experimental group B increased by about 17.19%. It is shown that the white mulberry fermented product can enhance the mitochondrial activity of skin cells and achieve the effect of enhancing the activity of skin cells.

Embodiment 7. Detection of the ability to secrete hyaluronic acid

Here, the human hyaluronic acid detection kit was used in conjunction with an ELISA reader to measure the changes in the secretion of hyaluronic acid from human primary skin keratinocytes HPEK-50 treated with white mulberry fermentation.

Materials and Instruments 1. Cell line: Human primary epidermal keratinocytes (Human primary epidermal keratinocytes) HPEKp (CELLnTEC Company (Switzerland), HPEK-50). 2. Culture medium: serum-free keratinocyte-SFM (Gibco Company, USA, No. #10724-011). 3. Human Hyaluronic acid (HA ELISA Kit): purchased from Cusabio Biotech. 4. Enzyme immunoassay analyzer (ELISA reader), purchased from BioTek Company. 5. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.

Experimental procedure

The experiment will be divided into three groups: experimental group A, experimental group B, and control group (without the addition of white mulberry samples): 1. Inoculate human primary skin keratinocytes at a density of 1×10 ⁴ per well into each well of culture In a 96-well culture plate containing 2 ml of medium in each well, the water extract of white mulberry was additionally added to the culture medium of experimental group A to make the concentration of 0.5 mg/mL; the culture medium of experimental group B was additionally added white mulberry fermentation substance to a concentration of 0.5 mg/mL. Each group was replicated in triplicate and incubated at 37°C for 24 hours. 2. Take 100 μL of medium per well, add it to a pre-coated ELISA plate, and incubate at 37°C for 2 hours. 3. Remove medium from each well without washing. 4. Add 100 μL of Biotin-antibody to each well and incubate at 37°C for 1 hour. 5. After the reaction is completed, remove the medium from each well and wash with 200 μL of Wash Buffer. Each wash is allowed to stand for 2 minutes, and this step is repeated three times. After the last wash, remove all remaining wash buffer by aspiration. (Confirm complete removal of liquid) 6. Add 100 μL of HRP-avidin to each well and incubate at 37°C for 1 hour. 7. After the reaction is completed, repeat the cleaning method in step 6. 8. Add 90 μL of TMB Substrate to each well and incubate at 37°C for 15-30 minutes in the dark. 9. Add 50 μL of Stop Solution to each well and tap the plate lightly to ensure adequate mixing. 10. Use an ELISA reader to measure the absorbance at 450 nm in each well within 5 minutes. 11. Statistical analysis of the obtained results was performed with Excel software.

Experimental results:

FIG. 6 is a graph showing the results of keratinocytes secreting hyaluronic acid after being treated with the white mulberry water extract or the white mulberry fermented product of the present invention. Specifically, the relative hyaluronic acid secretion of experimental group A was about 130%, while the relative hyaluronic acid secretion of experimental group B was about 170%. In other words, compared with the control group, the relative hyaluronic acid secretion rates of both the experimental group A and the experimental group B increased. Among them, the relative hyaluronic acid secretion of the experimental group A (white mulberry water extract) increased by 30% compared with the control group Compared with the control group, the relative hyaluronic acid secretion of experimental group B (white mulberry fermented product) increased by 70%. It can be seen that the ability of white mulberry fermented product to increase hyaluronic acid secretion is better than that of white mulberry water extract. The results of this example show that the white mulberry fermented product of the present invention has the effect of promoting the secretion of hyaluronic acid, thereby helping to retain skin collagen, increase skin water content, and provide skin elasticity and flexibility.

Hyaluronic acid prevents natural aging of the skin and protects the skin from the sun's UV rays, tobacco smoke and air pollutants. Hyaluronic acid also helps increase skin moisture and tightens the skin structure to reduce the appearance of fine lines and wrinkles. In addition, hyaluronic acid also plays a key role in wound healing. Its concentration increases when skin cells need to be repaired or damaged, and its application to skin wounds has been shown to reduce wound size and pain, and can also help reduce Risk of wound cell infection.

In addition, hyaluronic acid is also helpful for osteoarthritis. Experiments have shown that consuming 80-200 mg of hyaluronic acid every day for at least two months can significantly reduce knee pain in patients with osteoarthritis, and also help reduce gastric acid reflux symptoms. With excellent moisturizing properties, it is also commonly used in the treatment of dry eyes, slowing osteoporosis and reducing bladder pain syndrome.

Embodiment 8. Test on the efficacy of inhibiting fat accumulation

Fat is stored in fat cells in the form of lipid droplets. Based on this, the dyed oil droplets were analyzed in this experiment to observe the number of oil droplets in the cells, thereby confirming the state of fat accumulation. Subsequently, the dye was dissolved and analyzed as a quantitative numerical indicator.

Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used in this experiment, and OP9 cells were purchased from the OP9 cell line (ATCC CRL-2749) of the American Type Culture Collection (ATCC ^® ).

First, take a 24-well culture plate and inoculate each well with 8×10 ⁴ OP9 cells and 500 μL of medium (Medium), which is MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, product number Cat. 12000-022) cell culture Add 20% Fetal Bovine Serum (Fetal Bovine Serum, available from Gibco, Product No. Cat. 10437-028) and 0.1% Penicillin-streptomycin (available from Gibco, Product No. Cat. 15240-062) ), cultured at 37°C for 7 days. The medium was changed every 3 days during this 7-day cell culture period. After 7 days, intracellular oil droplet formation was observed under a microscope (ZEISS; magnification 400x) to confirm that the cells had fully differentiated into adipocytes.

Then, the differentiated adipocytes were divided into two groups: experimental group A, experimental group B and control group.

Experimental group: 500 μL medium per well contains 125 μg of white mulberry water extract (experimental group A) or white mulberry fermented product (experimental group B) prepared by the method of Example 1 of this case, and its concentration is 0.25 mg/ mL and incubated at 37°C for 7 days. The medium was changed every 3 days during the 7 days of cell treatment.

Control group: without any treatment, that is, without additionally adding other compounds to the differentiation medium containing differentiated adipocytes, and cultured at 37° C. for 7 days. The medium was changed every 3 days during this 7-day cell treatment period.

Next, oil red O staining was performed according to the following procedure. After 7 days of cell treatment, the medium was removed, the adipocytes were washed twice with 1 mL of Phosphate buffered saline (PBS), and then 1 mL of 10% formaldehyde was added and reacted at room temperature for 30 minutes to fix the fat. cell. After removing the formaldehyde, the adipocytes were gently washed twice with 1 mL of PBS, and then 1 mL of 60% isopropanol was added to each well of cells. After 1 minute of reaction, the isopropanol was removed and 1 mL of Oil Red O was added. The action solution reacted with adipocytes, reacted at room temperature for 1 hour, then removed the oil red O action solution that acted on adipocytes and quickly decolorized the adipocytes with 1 mL of 60% isopropanol for 5 seconds. Use a microscope to observe and photograph the cells.

Subsequently, the dyed groups were then quantified for Oil Red O according to the following steps. Add 100% isopropanol to the stained cells, and place on a shaker to react for 10 minutes to dissolve the oil droplets, then take 100 μL into a 96-well culture dish to measure the OD of each group with an ELISA reader (BioTek). _510nm reading. Among them, use Excel software to perform student t-test to determine whether there is a statistically significant difference between the two sample groups, as shown in Figure 7 ("*" in the diagram represents p value less than 0.05, "**" represents p Values less than 0.01, and "***" represent p-values less than 0.001. The more "*", the more statistically significant the difference).

Referring to Figure 7, when the fatty oil droplet content of the control group is regarded as 1 (i.e. 100%), the fatty oil droplet content of the experimental group B is 82.6%, which means that it can be significantly reduced by about 17.4% after being treated with the white mulberry fermented product. of fat accumulation. The results show that the white mulberry fermented product can effectively block the enlargement of adipocytes, so as to reduce the content of oil droplets in adipocytes, and achieve the effect of inhibiting fat accumulation.

Embodiment 9. Skin improvement efficacy test of white mulberry enzyme drink

Make 10 subjects with dull skin, loose and not firm, drink a bottle of 50 mL white mulberry enzyme drink (which contains 12 vol% of the white mulberry fermented product obtained in Example 1 and 88 vol% of water) every day, Drink continuously for 4 weeks. And, before drinking (that is, the 0th week) and after 4 weeks of drinking (that is, the 4th week), use a VISIA skin detector (VISIA Complexion Analysis System, purchased from Canfield, USA) and a skin surface humidity tester (Corneometer CM825, Purchased from C+K Company, Germany) to detect skin condition. Here, the VISIA skin tester was used to measure the changes of skin texture, skin UV spots, skin wrinkles, skin elasticity and collagen, and the skin moisture content was measured by the skin surface humidity tester.

See Figure 8. The graph shows the average improvement percentage of ultraviolet pigmentation after subjects drank the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average content of UV spots before drinking was 100%, the average content of UV spots after 4 weeks of drinking was 93.3%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average UV-stained content was reduced by 6.7%. Thus, it can be seen that the white mulberry fermented product of the present invention has the effect of reducing the generation of ultraviolet stains, so as to achieve the effect of lightening.

See Figure 9. The graph is the average percentage improvement of skin moisture content after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin moisture content of the subjects before drinking was 100%, the average skin moisture content after drinking for 4 weeks was 120.2%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin water content increased by 20.2%, which was statistically significant (the "***" symbol represents that the p value is less than that before drinking 0.001). It can be seen that the white mulberry fermented product of the present invention has the functions of increasing the moisture content of the skin, locking the moisture in the skin, and enhancing the moisturizing effect of the skin.

See Figure 10. The graph is the average percentage improvement of skin elasticity after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin elasticity of the subjects before drinking was 100%, the average skin elasticity after drinking for 4 weeks was 104%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin elasticity increased by 4%, which was statistically significant. It can be seen that the white mulberry fermented product of the present invention has the effect of enhancing skin elasticity, making the skin more elastic.

See Figure 11. The graph is the average percentage change of skin collagen after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin collagen content of the subjects before drinking was 100%, the average skin collagen content after drinking for 4 weeks was 115.5%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin collagen content increased by 15.5%, which was statistically significant. It can be seen that the white mulberry fermented product of the present invention has the effect of increasing the content of collagen in the skin. Collagen is related to the elasticity of the skin, and can delay the aging of the skin, reduce wrinkles, improve cellulite, and make the skin more elastic.

Referring to Figure 8 to Figure 11, compared to before drinking (week 0), drinking white mulberry fermented drink for 4 weeks can reduce the content of UV spots on the skin by about 6.7%, significantly increase the water content of the skin by about 20.2%, and make the skin elastic Increases by about 4%, and significantly increases skin collagen content by 15.5%. It can be seen that long-term use of white mulberry fermented drink can improve skin condition, that is, white mulberry fermented product has the effect of improving skin condition.

To sum up, according to any embodiment of the white mulberry fermented product of the present invention, it can be used to prepare a composition for improving skin condition and/or inhibiting fat accumulation. In other words, the aforementioned composition has one or more of the following functions: reducing melanin production, enhancing mitochondrial activity of dermal fibroblasts, enhancing hyaluronic acid secretion, reducing inflammatory response factor production, inhibiting tyrosinase activity, reducing ultraviolet pigmentation, enhancing Skin moisture content, improve skin elasticity, promote collagen proliferation and inhibit fat accumulation.

none

FIG. 1 is a bar graph showing the total polyphenol content of the white mulberry water extract and the white mulberry fermented product according to Example 2 of the present invention. FIG. 2 is a graph showing the relative nitric oxide generation ratio between the experimental group and the control group according to the third embodiment of the present invention. FIG. 3 is a graph showing the relative tyrosinase activity of the experimental group and the control group in Example 4 of the present invention. FIG. 4 is a graph showing the relative melanin production ratio between the experimental group and the control group in Example 5 of the present invention. FIG. 5 is a graph showing the relative mitochondrial activity of skin cells in the experimental group and the control group in Example 6 of the present invention. 6 is a graph showing the relative production ratio of hyaluronic acid between the experimental group and the control group in Example 7 of the present invention. FIG. 7 is a graph showing the relative proportion of fatty oil droplets in the experimental group and the control group in Example 8 of the present invention. FIG. 8 is a graph showing the change of relative ultraviolet light spots after the experimenter in Example 9 of the present invention used the white mulberry fermented drink for 4 weeks. FIG. 9 is a graph showing the change of relative skin water content after the experimenter in Example 9 of the present invention consumed the white mulberry fermented drink for 4 weeks. Fig. 10 is a graph showing the relative skin elasticity change of the experimenter in Example 9 of the present invention after using the white mulberry fermented drink for 4 weeks. Fig. 11 is a graph showing the relative changes in collagen after the experimenter in Example 9 of the present invention consumed the white mulberry fermented drink for 4 weeks.

Claims (10)

A white mulberry fermented product, wherein the white mulberry fermented product is a white mulberry water extract obtained from a white mulberry fruit through water extraction, and then sequentially subjected to a pre-fermentation through a yeast and a lactic acid bacteria, and then an acetic acid bacteria Obtained by post-fermentation. The white mulberry fermented product as described in item 1 of the claimed scope, wherein the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and The addition amount of the acetic acid bacteria is 1-20% (v/v); the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days. The white mulberry fermented product as described in item 1 of the patent application scope, wherein the white mulberry fermented product is further filtered with a mesh screen of 200-400 meshes, and concentrated under reduced pressure at 55-65° C., and then added 40- 70% isomalt oligosaccharide is adjusted for specification, and then heated at 95-105°C for 100-120 minutes for sterilization. The white mulberry fermented product as described in item 1 of the claimed scope, wherein the total polyphenol content of the white mulberry fermented product is 2-5 times the total polyphenol content of the white mulberry water extract. A use of the white mulberry fermented product according to any one of the claims 1 to 4 of the application scope for preparing a composition for improving skin condition and/or inhibiting fat accumulation. The use as described in item 5 of the scope of the patent application, wherein the improvement of skin condition is to reduce the production of melanin, enhance the mitochondrial activity of skin fibroblasts, enhance the secretion of hyaluronic acid, reduce UV spots, enhance skin moisture content, and enhance skin elasticity , and/or promote collagen proliferation. The use according to claim 6, wherein the composition reduces the production of melanin by reducing the production of inflammatory response factors and inhibiting the activity of tyrosinase. The use according to item 7 of the claimed scope, wherein the inflammatory response factor is nitric oxide. The use as described in claim 5, wherein the concentration of the white mulberry fermented product is at least 0.5% (v/v). The use as described in item 5 of the scope of the application, wherein the composition is a medicine, a food or a skin care product.

Images