CN112438922B - Application of mangosteen fermentation liquor in preparation of composition for beautifying skin and/or reducing fat - Google Patents

Abstract

The invention discloses the use of a mangosteen fermented liquid for preparing skin beautification and/or fat-reducing composition, and the composition has one or more of the following functions: enhancing antioxidant activity, inhibiting the formation of end-products of glycation, and inhibiting tyrosinase Formation, inhibition of fat accumulation, reduction of body fat in recipients and improvement of skin condition. In addition, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of bacterial strains through a fermentation process.

Description

Use of mangosteen fermented liquid for preparing skin beautifying and/or fat reducing composition

technical field

The invention relates to a mangosteen-related product, in particular to the use of mangosteen fermented liquid for preparing skin-enhancing and/or fat-reducing compositions.

Background technique

Since the rise of the concept of organic and natural diets, biotechnology companies and food companies have actively invested in the research and development of related products related to natural plants. In order to provide a basis for scientific verification of plant-related products to help health, the analysis and efficacy evaluation of plant active ingredients has become a key item in product development. The mangosteen (Garcinia mangostana) native to Southeast Asia has also become one of the objects of research and development.

Contents of the invention

In one embodiment, the use of a mangosteen fermented liquid is used to prepare a composition for enhancing antioxidant activity. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, a mangosteen fermented liquid is used to prepare a composition for inhibiting the formation of end-saccharification products. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, the use of a mangosteen fermented liquid is used to prepare a composition for inhibiting the formation of tyrosinase. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, a mangosteen fermented liquid is used for preparing a composition for inhibiting fat accumulation. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, a mangosteen fermented liquid is used for preparing a composition for reducing body fat of a subject. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In one embodiment, a mangosteen fermented liquid is used for preparing a composition for improving skin condition. Wherein, the mangosteen fermented liquid is obtained from a mangosteen culture liquid and a plurality of strains through a fermentation procedure. The mangosteen culture solution includes mangosteen and water, and the ratio of mangosteen to water is 1:2-5. Compared with the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In summary, according to any embodiment of the mangosteen fermented liquid of the present invention, it can prepare a composition for beautifying skin and/or reducing fat. In other words, the aforementioned composition has one or more of the following functions: enhancing antioxidant activity, inhibiting the formation of glycation end products, inhibiting the formation of tyrosinase, inhibiting fat accumulation, reducing body fat of recipients, and improving skin condition.

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but not as a limitation of the present invention.

Description of drawings

FIG. 1 is a histogram showing the changes in the total polyphenol content of mangosteen before and after fermentation.

Fig. 2 is a histogram showing the relative formation of glycation end products of each group.

Fig. 3 is a histogram showing the relative melanin content of each group.

Fig. 4 is a histogram showing the relative fat accumulation of each group.

FIG. 5 is a histogram showing changes in whole body fat percentage before and after use of an exemplary mangosteen fermented liquid.

FIG. 6 is a histogram showing changes in waist circumference of an exemplary mangosteen fermented liquid before and after use.

Fig. 7 is a histogram showing the changes of the relative deep spots of the skin before and after the use of an exemplary mangosteen fermented liquid.

Fig. 8 is a histogram of the change of relative skin wrinkles before and after use of an exemplary mangosteen fermented liquid.

Fig. 9 is a histogram of the relative changes in skin texture of an exemplary mangosteen fermented liquid before and after use.

Fig. 10 is a histogram of changes in the relative skin moisture content of an exemplary mangosteen fermented liquid before and after use.

Detailed ways

As used herein, the concentration symbol "%" generally refers to weight percent concentration, and the concentration symbol "vol%" generally refers to volume percent concentration. As used herein, "mangosteen" generally refers to the mangosteen fruit.

In some embodiments, the mangosteen fermented liquid is obtained by performing a fermentation process on the mangosteen culture liquid and a plurality of strains. Wherein, the mangosteen culture solution includes mangosteen and water. In the mangosteen culture solution, the ratio of mangosteen to water is 1:2-5. In the fermentation procedure, relative to the mangosteen culture broth, these strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria.

In some embodiments, the yeast may be Saccharomyces cerevisiae. In some embodiments, the lactic acid bacteria may be Lactobacillus plantarum or Lactobacillus plantarum. In some embodiments, the acetic acid bacteria may be Acetobacter aceti.

In some embodiments, in the fermentation process, 0.01%-0.5% of yeast is firstly added to the mangosteen culture broth, and the mangosteen culture broth and the yeast are fermented for 1 to 3 days to form a first primary fermentation broth. In other words, the first mixed liquid formed by mixing 0.01%-0.5% yeast and the mangosteen culture liquid is fermented for 1 to 3 days to form the first primary fermentation liquid. In some embodiments, the first mixture is fermented at 28°C-37°C.

After the first primary fermentation liquid is formed, 0.01%-0.25% lactic acid bacteria are added to the first primary fermentation liquid, and the first primary fermentation liquid and the lactic acid bacteria are fermented for 1 to 5 days to form the second primary fermentation liquid. In other words, the second mixed liquor formed by mixing 0.01%-0.25% lactic acid bacteria and the first primary fermentation liquid is fermented for 1 to 5 days to form the second primary fermentation liquid. In some embodiments, the second mixture is fermented at 28°C-37°C.

After the formation of the second primary fermentation liquid, add 1%-15% acetic acid bacteria to the second primary fermentation liquid, and ferment the second primary fermentation liquid with acetic acid bacteria for 3 to 10 days to form the third primary fermentation liquid. In other words, 1%-15% of the acetic acid bacteria and the second primary fermentation liquid are mixed to form a third mixed liquid and fermented for 3 to 10 days to form the third primary fermentation liquid. In some embodiments, the third mixture is fermented at 28°C-37°C.

After the third primary fermentation liquid is formed, the third primary fermentation liquid is filtered to obtain a mangosteen fermentation liquid. In the first example, the step of filtering the third primary fermentation liquid includes filtering the third primary fermentation liquid through a filter screen with meshes of 200 mesh to 400 mesh. In the second example, the step of filtering the third primary fermentation liquid includes concentrating under reduced pressure at 40° C. to 70° C. and filtering the third primary fermentation liquid through a filter with a mesh size of 200 mesh to 400 mesh. In the third example, the step of filtering the third primary fermentation liquid includes concentrating under reduced pressure at 40° C. to 70° C. and filtering the third primary fermentation liquid through a filter screen with a mesh size of 200 mesh to 400 mesh to obtain a fermenting the raw liquid, and adjusting the sugar content of the fermenting raw liquid to form a mangosteen fermented liquid.

In some embodiments, the sugar content of the fermentation stock solution can be adjusted by adding 55%-70% of excipients. In some embodiments, the excipient used to adjust the sugar content may be isomaltooligosaccharide.

In some embodiments, the pH of the mangosteen fermentation liquid is 3.7±1.0, and the Brix of the mangosteen fermentation liquid is 40±2.

In some embodiments, the polyphenol content of the mangosteen fermentation broth is 691 ppm.

In some embodiments, the preparation method of mangosteen culture solution is as follows. First, crush the mangosteen to form mangosteen granules, and then mix the mangosteen granules with water at a ratio of 1:2-5 to obtain a raw material mixture. Next, the obtained raw material mixture is sterilized to obtain mangosteen culture fluid (also called mangosteen juice).

In some embodiments, the mangosteen selected here can be whole fruit with shells. In other words, the whole mangosteen fruit and shell are broken into mangosteen particles. In some embodiments, the mangosteen selected here may be ripe and edible mangosteen fruit. In some embodiments, the particle size of the mangosteen particles may be less than 12 mm (millimeter).

In some embodiments, the sterilization procedure of the raw material mixture can be to sterilize the raw material mixture at 80° C. to 100° C. for 0.2 hour to 1 hour, and cool the sterilized raw material mixture to room temperature (ie, 25° C. to 30°C) to form a mangosteen culture solution. In some embodiments, the sterilized raw material mixture can be cooled to room temperature by natural cooling.

In some embodiments, the mangosteen fermented liquid has the effects of beautifying the skin and/or reducing fat. In some embodiments, the mangosteen fermented liquid can achieve the effect of reducing body fat by reducing the body fat of a subject. In some embodiments, the mangosteen fermented liquid can achieve the effect of beautifying the skin by improving the skin condition of a recipient. In some examples, improving the recipient's skin condition can be increasing the recipient's skin moisture content, reducing the recipient's deep spots, reducing the recipient's wrinkles, reducing the recipient's skin texture, or a combination thereof. In some embodiments, the mangosteen fermented liquid can achieve skin beautification and/or fat reduction through one or more of the following effects at the cell level: enhancing antioxidant activity, inhibiting the formation of end-products of glycation, inhibiting the formation of tyrosinase, and inhibit fat accumulation. Wherein, the receptor can be human.

In some embodiments, the mangosteen fermented liquid can be used to prepare a composition for enhancing antioxidant activity.

In some embodiments, mangosteen fermentation broth can be used to prepare a composition for inhibiting the formation of end-saccharification products.

In some embodiments, mangosteen fermentation broth can be used to prepare a composition for inhibiting the formation of tyrosinase.

In some embodiments, the mangosteen fermented liquid can be used to prepare a composition for inhibiting fat accumulation.

In some embodiments, mangosteen fermentation broth can be used to prepare a composition for reducing body fat in a subject.

In some embodiments, mangosteen fermented liquid can be used to prepare a composition for improving skin condition. Wherein, improving skin condition can be to increase skin moisture content, reduce deep spots, reduce wrinkles, reduce skin texture or a combination thereof.

In some embodiments, any of the aforementioned compositions can be a pharmaceutical. In other words, the medicine contains an effective amount of mangosteen fermented liquid.

In some embodiments, the aforementioned pharmaceuticals can be manufactured into dosage forms suitable for enteral, parenterally, oral, or topically administered using techniques well known to those skilled in the art .

In some embodiments, the dosage form for enteral or oral administration can be, but not limited to, 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 may be, but not limited to, 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 pharmaceuticals may contain pharmaceutically acceptable carriers (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, decomposing 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 amount of carrier to be used is within the professionalism and routine skill of those skilled in the art. In some embodiments, the solvent as the pharmaceutically acceptable carrier can be water, normal saline (normal saline), phosphate buffered saline (phosphate buffered saline, PBS), or an aqueous solution containing alcohol (alcohol containing aqueous solution) .

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

In some embodiments, the aforementioned edible composition can be administered orally to a recipient. In some embodiments, the edible composition can be a powder, granule, solution, gel or paste.

In some embodiments, any of the aforementioned compositions can be a cosmetic or skin care product. In other words, cosmetics or skin care products contain a specific amount of mangosteen fermented liquid.

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, cleansing oil, Makeup remover, facial cleanser, body wash, shampoo, hair conditioner, sunscreen, hand cream, nail polish, perfume, essence and mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients. In some embodiments, the topically acceptable ingredient can be, for example, an emulsifier, penetration enhancer, emollient, solvent, excipient, antioxidant, or a combination thereof.

Example 1: Preparation of mangosteen fermented liquid

First, the whole mangosteen fruit (including the shell) is crushed into mangosteen particles with a particle diameter of less than 12 mm. Mix mangosteen granules and water evenly at a ratio of 1:3 to obtain a raw material mixture. Then, the raw material mixture was sterilized at about 100° C. for 0.5 hour, and the sterilized raw material mixture was cooled to about 30° C. to obtain a mangosteen culture solution.

0.1% brewer's yeast (purchased from the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Institute (BCRC), registration number BCRC20271) was colonized in the mangosteen culture medium and fermented at about 30°C for 1 day to obtain the first primary fermentation liquid . Next, 0.05% Lactobacillus plantarum (purchased from BCRC, deposit number BCRC910760) was colonized in the first primary fermentation liquid and fermented at about 30° C. for 1 day to obtain a second primary fermentation liquid. Finally, 10% acetic acid bacteria (purchased from BCRC, deposit number BCRC11688) were colonized in the second primary fermentation broth and fermented at about 30° C. for 5 days to obtain a third primary fermentation broth.

Then the third primary fermentation liquid was concentrated under reduced pressure at about 60° C. and filtered through a mesh filter to obtain a fermentation stock solution. Finally, 60% isomaltooligosaccharide is added to the fermentation stock solution and then sterilized to obtain a mangosteen fermentation solution.

Here, the mangosteen culture solution, the fermentation stock solution and the mangosteen fermentation solution were tested for quality. Among them, the sugar content of the mangosteen culture liquid is about 10.6, the sugar content of the fermentation stock solution is about 3.9, and the sugar content of the mangosteen fermentation liquid is about 40.

Example 2: Total polyphenol content test

Weigh 10.0 mg of gallic acid (Gallic acid) into a 10 mL volumetric flask, and then quantify it to 10 mL with water (H ₂ O) to obtain a stock solution of gallic acid. The stock solution of gallic acid was diluted 10 times, that is, 100 μL of the stock solution of gallic acid was added to 900 μL of water to obtain an initial solution of 100 μg/mL of 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 the following table 1, and take 100 μL of standard solutions of each concentration to the glass in a test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (Folin-Ciocalteu's phenol reagent, purchased from Merck) to each glass test tube, mix well with the standard solution and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well and react for 30 minutes. A standard reaction solution was obtained. 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 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

The mangosteen culture broth and fermentation stock solution obtained in Example 1 were used as samples respectively. Take 100 mL of each sample into a glass test tube. Next, add 500 μL of Folin’s phenol reagent to the glass test tube, mix it with the sample and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix it evenly, and react for 30 minutes to obtain the reaction solution to be tested. Shake the glass test tube containing the reaction solution to be tested to ensure that there are no bubbles, then 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.

Next, the absorbance value of the reaction solution to be tested corresponding to each sample is first divided by the sugar content of the sample, and then converted into the total polyphenol content by interpolation using the standard curve. Here, the total polyphenol content of the mangosteen culture broth is 334 ppm and the total polyphenol content of the mangosteen fermentation broth is 691 ppm, as shown in FIG. 1 . It can be seen that the total polyphenol content can be increased by 2.1 times after the mangosteen is fermented by microorganisms. That is, compared with the mangosteen culture solution, the mangosteen enzyme solution can enhance the antioxidant activity.

Example 3: Anti-glycation test

Prepare 60 mg of 0.06% NaN ₃ with 200 mM sodium phosphate buffer (Sodium phosphate buffer, pH7.4), sodium azide (Sodiumazide, NaN ₃ ) and bovine serum albumin (Bovine serum albumin, BSA, brand: Gibco) /mL BSA solution.

A 1.5M D-fructose solution was prepared with 200 mM sodium phosphate buffer and D-(-)-Fructose (C ₆ H ₁₂ O ₆ ).

A 3 mM AG solution was prepared with 200 mM sodium phosphate buffer and aminoguanidine hydrochloride (AG, CH ₆ N ₄ ·HCl).

Get 0.25mL of the mangosteen fermented liquid obtained in Example 1, add 0.25mL of BSA solution and D-fructose solution and mix evenly to obtain the test solution of the experimental group.

Take 0.25mL of 3mM AG solution, add 0.25mL of BSA solution and D-fructose solution and mix evenly to obtain the test solution of the control group.

Take 0.1mL of the solution to be tested in each group as the zero point solution of each group. Use a fluorescence spectrophotometer (ThermoFisher Scientific) to measure the fluorescence value of 0.1mL zero-point solution of each group with 360nm excitation light and 460nm emission light to obtain the zero-point fluorescence value before the reaction.

Take 0.45mL of the test solution of each group and incubate at 50°C for 24 hours to obtain the end point solution of each group. Take 0.1 mL of the end point solution of each group and use a fluorescence spectrophotometer to measure the fluorescence value of the 0.1 mL end point solution of each group with 360nm excitation light and 460nm radiation light to obtain the fluorescence value of the end point of the reaction.

Then, according to the following formula (1), calculate the formation amount (%) of the relative glycation end products (Advanced Glycation Endproducts, AGEs) of each group to know its antiglycation activity (Antiglycative activity). In other words, here, the relative AGEs formation (%) of each group is calculated by considering the AGEs formation of the control group as 1 (that is, the relative AGEs formation of the control group is 100%).

Among them, Fluorescence sample _24hr represents the fluorescence value of the end point of the experimental group, Fluorescencesample _0hr represents the fluorescence value of the zero point of the experimental group, Fluorescence control _24hr represents the fluorescence value of the end point of the control group, and Fluorescence control _0hr represents the fluorescence value of the zero point of the control group.

Referring to FIG. 2 , compared with the control group, the experimental group significantly reduced the formation of 70% of the end products of glycation. It can be known that the mangosteen fermented liquid can effectively inhibit the formation of saccharification end products, that is, it has the effect of anti-saccharification.

Example 4: Detection of melanin content

Here, the cell culture medium used was Dubacco's with 1vol% penicillin/streptomycin (penicillin/streptomycin, brand: Gibco) and 10vol% fetal bovine serum (fetal bovine serum, FBS, brand: Gibco). Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM, brand: Gibco).

First, the mouse melanoma cell line B16F10 (purchased from the American Type Culture Collection (ATCC), number CRL-6475) was inoculated in 6 cells containing 3 mL of cell culture medium at a cell number of 1.5×10 ⁵ cells per well. Wells in each well of the culture plate and incubated at 37°C for 24 hours.

After culturing for 24 hours, the B16F10 cells were divided into 4 groups: two experimental groups (ie, experimental group A and experimental group B), a control group and a control group. The cell culture medium of each group was removed and replaced with 3mL experimental culture medium per well, and then cultured at 37°C for 48 hours. Wherein, the experimental culture medium of the experimental group A is a cell culture medium containing 0.125 vol% of the mangosteen fermented liquid obtained in Example 1. The experimental culture medium of the experimental group B is a cell culture medium containing 0.125 vol% of the mangosteen culture solution obtained in Example 1. The experimental medium of the control group was a cell culture medium containing 0.125 mg/mL kojic acid (kojic acid). The experimental culture medium of the control group is a simple cell culture medium (that is, no mangosteen fermentation broth, no mangosteen culture fluid, and no kojic acid).

After culturing for 48 hours, the experimental medium in each well was removed and rinsed twice with 1 times (1x) phosphate buffered saline (PBS, brand: Gibco). After washing, trypsin was added to each well to treat the cells for 3 minutes. After 3 minutes of treatment, the suspended cells in each well were individually collected in a 15 mL centrifuge tube, and then centrifuged at 400×g for 5 minutes to separate the cell pellet and supernatant. After resuspending the cell pellet in 1xPBS and centrifugation were repeated twice, the cell pellet was resuspended in 200 μL of 1xPBS to obtain a cell solution. Next, the cell solution was placed in liquid nitrogen for 10 minutes, and then allowed to stand at room temperature for 30 minutes to thaw. After thawing was complete, each centrifuge tube was centrifuged at 12,000 xg for 30 minutes. After centrifugation for 30 minutes, the supernatant in each centrifuge tube was removed and 120 μL of 1N NaOH (in ddH ₂ O) was added to mix with the precipitate in each centrifuge tube. After uniform mixing, each centrifuge tube containing the mixed solution was left to stand in a 60° C. dry bath for 1 hour. Afterwards, take 100 μ L of the mixed solution from each centrifuge tube to a 96-well culture plate and use an ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay) reader (brand: BioTek) at a wavelength of 450 nm to read 96 The absorbance value (OD450) of each well in the well plate.

After the measurement, the relative melanin content (%) was calculated by substituting the measured absorbance value into the following formula (2). In other words, here, the relative melanin content (%) of each group is calculated by considering the melanin content of the control group as 1 (ie, the relative melanin content of the control group is 100%). And, the statistically significant difference between the groups was statistically analyzed by student t-test (student t-test), as shown in FIG. 3 . In Figure 3, "*" means that the p-value is less than 0.05 compared with the control group, "**" means that the p-value is less than 0.01 when compared with the control group, and "***" means that the p-value is less than 0.01 when compared with the control group Its p-value is less than 0.001, and "▲▲▲" means that its p-value is less than 0.001 compared with the control group.

Relative melanin content (%) = (OD450 sample/OD450 control) × 100% (2)

Among them, OD450 sample represents the absorbance value of the group to be converted, and OD450 control represents the absorbance value of the control group.

Referring to FIG. 3 , compared with the control group, the melanin content of the experimental group A was significantly reduced, and it could reduce the production of melanin by 21.58%. Moreover, compared with the corresponding concentration of kojic acid, the melanin content of the experimental group A was also significantly reduced, and it could reduce the production of melanin by 21.58%. Compared with experimental group B, experimental group A can also reduce melanin production by 1.0%. It can be seen that the mangosteen fermented liquid can effectively inhibit the production of melanin, that is, effectively inhibit the formation of tyrosinase, and has the effect of whitening.

Example 5: Fat accumulation detection

Here, the pre-adipocyte expansion medium used is the minimum essential medium α (MinimumEssential Medium Alpha, MEMα) supplemented with 20vol% FBS (brand: Gibco) and 1vol% penicillin-streptomycin , Brand: Gibco). The differentiation medium used was MEMα (brand: Gibco) supplemented with 20 vol% FBS (brand: Gibco) and 1 vol% penicillin-streptomycin. And, Oil-Red O staining reagent (brand name: Sigma) was thoroughly dissolved in 100% isopropanol (supplier: ECHO) to prepare a stock solution of Oil-Red O staining reagent at 3 mg/mL. In order to obtain the available oil-red O working solution (oil-red O working solution), the stock solution of the oil-red O staining reagent was diluted with secondary water (ddH ₂ O) to a concentration of 1.8 mg/ mL is the stock solution of 60% Oil-Red O staining reagent.

First, the mouse bone marrow stromal cell line OP9 (purchased from ATCC, number CRL-2749) was inoculated in each well of a ²⁴ -well culture plate containing 500 μL of preadipocyte proliferation medium at a cell number of 8×104 cells per well. wells and cultured at 37°C for 7 days. During the 7-day culture period, fresh 500 μL differentiation medium was replaced every 3 days. After 7 days of culture, use a microscope (brand: ZEISS) to observe the formation of lipid droplets in the cells in each well to confirm that the cells are completely differentiated into adipocytes for use in subsequent experiments.

After culturing for 24 hours, the adipocytes were divided into three groups: two experimental groups (ie, experimental group A and experimental group B) and a control group. The differentiation medium of each group was removed and replaced with 500 μL of experimental medium per well, and then cultured at 37° C. for 7-10 days. During the culture period of 7-10 days, fresh 500 μL experimental medium was replaced every 3 days. Wherein, the experimental medium of the experimental group A is a differentiation medium containing 0.0625 vol% of the mangosteen fermented liquid obtained in Example 1. The experimental medium of the experimental group B is a differentiation medium containing 0.0625 vol % of the mangosteen culture solution obtained in Example 1. The experimental medium of the control group is a simple differentiation medium (that is, no mangosteen fermentation broth).

Next, the assay medium in each well was removed and rinsed twice with 1xPBS. Then, 1 mL of 10% formaldehyde (formaldehyde, supplier: ECHO) was added to each well and incubated at room temperature for 30 minutes to fix the cells. Afterwards, the formaldehyde in each well was removed and each well was rinsed twice with 1 mL of PBS. After rinsing again, 1 mL of 60% isopropanol was added to each well for 1 minute. Then, isopropanol was removed, and 1 mL of oil-red O working solution was added and allowed to react at room temperature for 1 hour.

After 1 hour of exposure, the Oil-Red O working solution was removed and quickly destained with 1 mL of 60% isopropanol for 5 seconds. After destaining, the stained cells were rinsed with 1xPBS, 100% isopropanol was added to each well, and placed on a shaker for 10 minutes to dissolve the dye. Then, take 100 μL of the aforementioned lysing agent-isopropanol solution from each well to a 96-well culture plate and read the absorbance (OD510) of each well with an ELISA reader (brand: BioTek) at a wavelength of 510 nm. .

After the measurement, the relative fat oil droplet amount (%) was calculated by substituting the measured absorbance value into the following formula (3). In other words, here, the relative fat droplet volume (%) of each group is calculated by considering the fat droplet volume of the control group as 1 (ie, the relative fat droplet volume of the control group is 100%). And, statistically significant differences among the groups were statistically analyzed by Student's t-test, as shown in FIG. 4 . In Fig. 4, "**" means that the p-value is less than 0.01 compared with the control group.

Relative fat droplet amount (%)=(OD510 sample/OD510 control)×100% (3)

Among them, OD510 sample represents the absorbance value of the group to be converted, and OD510 control represents the absorbance value of the control group.

Referring to FIG. 4, compared with the control group, the relative amount of fat oil droplets in the experimental group A was significantly reduced, and it could reduce the amount of oil droplets by 16.9%. Compared with experimental group B, the relative amount of fat oil droplets in experimental group A also decreased significantly, and it could reduce the amount of oil droplets by 8.7%. It can be seen that the mangosteen fermented liquid can effectively inhibit fat accumulation and has the effect of reducing fat. Moreover, mangosteen may produce more fat-reducing active ingredients than mangosteen culture fluid through microbial fermentation.

Example 6: Human Body Test for Fat Reduction and Slimming

Make 8 obese subjects (that is, their body fat percentage greater than 25% or BMI value greater than 24) drink a bottle of 6mL mangosteen fermented beverage (which contains 12vol% mangosteen fermented liquid obtained in example 1 and 88vol% water) every day , Drinking continuously for 4 weeks. And, before drinking (i.e. the 0th week) and after drinking for 4 weeks (i.e. the 4th week), the body fat percentage of these subjects was measured with a body fat meter (brand: TANITA BC-601FS), and the body fat percentage was measured with a cloth Measure the waist circumference of these subjects with a ruler. Moreover, the statistically significant difference between the measurement results at week 0 and the measurement results at week 4 was statistically analyzed by Student's t-test, as shown in FIG. 5 and FIG. 6 . In Figure 5 and Figure 6, "*" means that the p-value is less than 0.05 compared with week 0.

Referring to Figures 5 and 6, compared with before drinking (week 0), drinking mangosteen fermented beverage for 4 weeks can significantly reduce the whole body fat rate by about 0.4%, and significantly reduce the waist circumference by about 2.0 cm. It can be known that long-term use of mangosteen fermented liquid can improve the fat accumulation index of obese people, that is, mangosteen fermented liquid has the effect of reducing body fat.

Example 7: Human body detection for beautiful skin

Make 8 obese subjects drink a bottle of 6mL mangosteen fermented beverage (which contains 12vol% mangosteen fermented liquid obtained in Example 1 and 88vol% water) every day for 6 weeks. And, before drinking (i.e. the 0th week) and after drinking for 6 weeks (i.e. the 6th week), the skin surface moisture tester (Corneometer CM825, Purchased from German C+K company) to detect skin condition. Among them, the VISIA skin detector is used to measure the deep spots (brown spots), skin wrinkles and skin texture of the skin, and the skin moisture content is measured by the skin surface humidity tester.

After the measurement, the skin condition at week 0 was taken as a reference (ie, the relative skin condition at week 0 was 100%), and the relative skin condition (%) at week 6 was calculated. And, the statistically significant difference between the relative skin condition at week 0 and the relative skin condition at week 6 was statistically analyzed by Student's t-test, as shown in FIGS. 7 to 10 . In Figures 7 to 10, "*" means that the p-value is less than 0.05 compared with week 0.

Referring to Figures 7 to 10, compared with before drinking (week 0), drinking mangosteen fermented beverage for 6 weeks can significantly reduce the deep spots of the skin by about 8.5%, reduce the skin wrinkles by about 8.3%, and reduce the skin texture by about 6.7% %, and significantly increase the moisture content of the skin by about 18.6%. It can be known that long-term use of mangosteen fermented liquid can improve skin condition, that is, mangosteen fermented liquid has the effect of beautifying skin.

In summary, according to any embodiment of the mangosteen fermented liquid of the present invention, it can prepare a composition for beautifying skin and/or reducing fat. In other words, the aforementioned composition has one or more of the following functions: enhancing antioxidant activity, inhibiting the formation of glycation end products, inhibiting the formation of tyrosinase, inhibiting fat accumulation, reducing body fat of recipients, and improving skin condition.

Certainly, the present invention also can have other various embodiments, without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes All changes and modifications should belong to the protection scope of the claims of the present invention.

Claims (8)

1. the purposes of a kind of mangosteen fermented liquid, it is characterized in that, it is used for preparing the composition for improving antioxidant capacity, wherein this mangosteen fermented liquid is to carry out a fermentation procedure by a mangosteen culture liquid and a plurality of strains obtained, wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01 %-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 2. The use of a mangosteen fermentation liquid, characterized in that it is used to prepare a composition for inhibiting the formation of saccharification end products, wherein the mangosteen fermentation liquid is fermented by a mangosteen culture liquid and a plurality of strains The procedure is obtained, wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 3. the purposes of a kind of mangosteen fermented liquid, it is characterized in that, it is used for preparing the composition for inhibiting the formation of tyrosinase, wherein this mangosteen fermented liquid is carried out by a mangosteen culture liquid and a plurality of strains Obtained from a fermentation process, wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the plurality of strains include 0.01%-0.5% of yeast , 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 4. The use of a mangosteen fermented liquid, which is characterized in that it is used to prepare a composition for inhibiting fat accumulation, wherein the mangosteen fermented liquid is obtained by a mangosteen culture liquid and a plurality of strains through a fermentation procedure , wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01% -0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 5. The use of a mangosteen fermented liquid, characterized in that it is used to prepare a composition for reducing body fat of a receptor, wherein the mangosteen fermented liquid is produced by a mangosteen culture liquid and a plurality of strains Obtained from a fermentation procedure, wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the plurality of strains include 0.01%-0.5% of yeast Bacteria, 0.01%-0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 6. The use of a mangosteen fermented liquid, which is characterized in that it is used to prepare a composition for improving skin conditions, wherein the mangosteen fermented liquid is obtained by a mangosteen culture liquid and a plurality of strains through a fermentation process , wherein the mangosteen culture solution includes mangosteen and water, the ratio of the mangosteen to the water is 1:2-5, and relative to the mangosteen culture solution, the multiple strains include 0.01%-0.5% yeast, 0.01% -0.25% lactic acid bacteria and 1%-15% acetic acid bacteria, improving the skin condition is to increase skin moisture content, reduce deep spots, reduce wrinkles, reduce skin texture or a combination thereof, the fermentation process includes: adding the yeast to the mangosteen culture solution; Fermenting the mangosteen culture broth and the yeast for 1 to 3 days to form a first primary fermentation broth, wherein the yeast is brewer's yeast; adding the lactic acid bacteria into the first primary fermentation broth; Fermenting the first primary fermentation liquid with the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria are Lactobacillus plantarum; adding the acetic acid bacteria into the second primary fermentation broth; Fermenting the second primary fermentation liquid with the acetic acid bacteria for 3 to 10 days to form a third primary fermentation liquid, wherein the acetic acid bacteria are acetic acid bacteria; and Filter the third initial fermentation liquid to obtain the mangosteen fermentation liquid. 7. The use according to any one of claims 1 to 6, characterized in that the pH value of the mangosteen fermentation liquid is 3.7±1.0, and the sugar content of the mangosteen fermentation liquid is 40±2. 8. The use according to any one of claims 1 to 6, characterized in that the polyphenol content of the mangosteen fermentation broth is 691ppm.

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