CN112438922B

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

Info

Publication number: CN112438922B
Application number: CN202010788238.0A
Authority: CN
Inventors: 林咏翔; 庄伟秀
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-08-27
Filing date: 2020-08-07
Publication date: 2023-04-07
Anticipated expiration: 2040-08-07
Also published as: TWI754345B; CN112438922A; TW202108160A

Abstract

The invention discloses an application of mangosteen fermentation liquor in preparing a composition for beautifying muscles and/or reducing fat, wherein the composition has one or more of the following functions: promoting antioxidant activity, inhibiting formation of glycation end products, inhibiting formation of tyrosinase, inhibiting fat accumulation, reducing body fat of receptor, and improving skin condition. The mangosteen fermentation liquid is obtained by performing a fermentation process on a mangosteen culture solution and a plurality of strains.

Description

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

Technical Field

The invention relates to a mangosteen related product, in particular to application of mangosteen fermentation liquor in preparing a composition for beautifying skin and/or reducing fat.

Background

Since the development of organic and natural dietary concepts, biotechnology companies and food manufacturers have actively invested in the development of products related to natural plants. In order to enable plant-related products to have a scientific verification basis for body health help, the analysis of active ingredients and the evaluation of efficacy of plants become key projects for product development. Mangosteen (Garcinia mangostana) native to southeast asia has also become one of the subjects of research and development.

Disclosure of Invention

In one embodiment, the use of mangosteen fermentation broth for preparing a composition for enhancing antioxidant activity. Wherein, the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In one embodiment, use of a mangosteen broth for the preparation of a composition for inhibiting formation of a saccharification end product. Wherein, the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In one embodiment, the use of a mangosteen fermentation broth for preparing a composition for inhibiting tyrosinase formation is provided. Wherein, the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In one embodiment, the use of a mangosteen fermentation broth for the preparation of a composition for inhibiting fat accumulation. Wherein, the mangosteen fermentation liquid is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In one embodiment, the use of a mangosteen broth for preparing a composition for reducing body fat of a recipient. Wherein, the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In one embodiment, the application of the mangosteen fermentation liquor in preparing the composition for improving skin conditions is disclosed. Wherein, the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains. The mangosteen culture solution comprises mangosteen and water, wherein the ratio of the mangosteen to the water is 1. Relative to the mangosteen culture solution, the multiple strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In summary, the mangosteen fermentation broth according to any one of the embodiments of the present invention can be used to prepare a composition for skin beauty and/or fat reduction. In other words, the aforementioned composition has one or more of the following functions: promoting antioxidant activity, inhibiting formation of glycation end products, inhibiting formation of tyrosinase, inhibiting fat accumulation, reducing body fat of receptor, and improving skin condition.

The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.

Drawings

Fig. 1 is a histogram showing the change in total polyphenol content before and after mangosteen fermentation.

FIG. 2 is a histogram showing the relative amounts of end product formation in 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 amounts of the respective groups.

FIG. 5 is a histogram showing the change in body fat percentage of an exemplary mangosteen fermentation broth before and after use.

Fig. 6 is a histogram showing the change in waist circumference before and after use of an exemplary mangosteen fermentation broth.

Fig. 7 is a histogram showing changes in skin deep spots before and after use of an exemplary mangosteen fermentation broth.

Fig. 8 is a histogram showing changes in relative skin wrinkles before and after use of an exemplary mangosteen fermentation broth.

Fig. 9 is a histogram showing changes in relative skin texture before and after use of an exemplary mangosteen fermentation broth.

Fig. 10 is a histogram showing changes in relative skin moisture content before and after use of an exemplary mangosteen fermentation broth.

Detailed Description

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

In some embodiments, the mangosteen broth is obtained by subjecting a mangosteen broth to a fermentation process with a plurality of bacterial species. Wherein the mangosteen culture solution comprises mangosteen and water. In the mangosteen culture solution, the ratio of mangosteen to water is 1-5. In the fermentation process, relative to the mangosteen culture solution, the strains comprise 0.01-0.5% of saccharomycetes, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

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

In some embodiments, in the fermentation process, 0.01% -0.5% of yeast is added into the mangosteen culture solution, and the mangosteen culture solution and the yeast are fermented for 1 day to 3 days to form a first primary fermentation broth. In other words, 0.01% -0.5% of the first mixed solution of yeast and the mangosteen culture solution is fermented for 1 to 3 days to form a first primary fermentation solution. In some embodiments, the first mixture is fermented at 28 ℃ to 37 ℃.

After the first primary fermentation liquid is formed, 0.01-0.25% of lactic acid bacteria are added into the first primary fermentation liquid, and the first primary fermentation liquid and the lactic acid bacteria are fermented for 1-5 days to form a second primary fermentation liquid. In other words, the second mixed solution of 0.01% -0.25% lactobacillus and the first primary fermentation solution is fermented for 1 to 5 days to form the second primary fermentation solution. In some embodiments, the second mixture is fermented at 28 ℃ to 37 ℃.

And after the second primary fermentation liquid is formed, adding 1% -15% of acetic acid bacteria into the second primary fermentation liquid, and fermenting the second primary fermentation liquid and the acetic acid bacteria for 3-10 days to form third primary fermentation liquid. In other words, the third mixed solution formed by mixing 1% -15% of acetic acid bacteria and the second primary fermentation liquid is fermented for 3 days to 10 days to form the third primary fermentation liquid. In some embodiments, the third mixture is fermented at 28 ℃ to 37 ℃.

And after the third primary fermentation liquid is formed, filtering the third primary fermentation liquid to obtain the mangosteen fermentation liquid. In a first exemplary embodiment, the step of filtering the third primary fermentation broth comprises filtering the third primary fermentation broth through a sieve having a mesh size of 200 mesh to 400 mesh. In a second exemplary embodiment, the filtering step of the third primary fermentation liquid comprises concentrating the third primary fermentation liquid at 40 ℃ to 70 ℃ under reduced pressure and filtering the third primary fermentation liquid through a filter mesh with 200 meshes to 400 meshes. In a third exemplary embodiment, the filtering step of the third primary fermentation liquid includes concentrating under reduced pressure at 40 ℃ to 70 ℃ and filtering the third primary fermentation liquid with a filter screen having 200 mesh to 400 mesh to obtain a fermentation raw liquid, and adjusting the sugar degree of the fermentation raw liquid to form the mangosteen fermentation liquid.

In some embodiments, the brix of the fermentation broth can be adjusted by adding 55% -70% excipient (exipient). In some embodiments, the excipient used to adjust the sugar degree may be isomalto-oligosaccharide.

In some embodiments, the pH of the mangosteen broth is 3.7 ± 1.0, and the sugar degree of the mangosteen broth (Degrees Brix) is 40 ± 2.

In some embodiments, the mangosteen broth has a polyphenol content of 691ppm.

In some embodiments, the mangosteen broth is prepared as follows. First, mangosteen is broken up to form mangosteen particles, which are then mixed with water at a ratio of 1:2-5 to obtain a raw material mixed solution. Then, the obtained raw material mixture is subjected to a sterilization procedure to obtain a mangosteen culture solution (also called mangosteen juice).

In some embodiments, the mangosteen selected for use herein can be a shelled whole fruit. In other words, the whole mangosteen fruit along with the shell are broken into mangosteen particles. In some embodiments, the mangosteen selected for use herein can be mature edible mangosteen fruit. In some embodiments, the particle size of the mangosteen particles can be 12mm (millimeters) or less.

In some embodiments, the raw mixture may be sterilized at 80 ℃ to 100 ℃ for 0.2 hour to 1 hour, and the sterilized raw mixture is cooled to room temperature (i.e., 25 ℃ to 30 ℃) to form the mangosteen broth. In some embodiments, the sterilized raw material mixture may be cooled to room temperature by natural cooling.

In some embodiments, the mangosteen broth has muscle beautifying and/or fat reducing effects. In some embodiments, the mangosteen broth is capable of effecting fat reduction by reducing body fat in a recipient. In some embodiments, the mangosteen fermentation broth can achieve skin-beautifying effects by improving the condition of skin of a subject. In some examples, improving the skin condition of the subject may be increasing the moisture content of the skin of the subject, decreasing deep spots of the subject, decreasing wrinkles of the subject, decreasing skin texture of the subject, or a combination thereof. In some embodiments, the mangosteen broth is capable of achieving a skin-beautifying and/or lipid-reducing effect through one or more of the following cell-level effects: promoting antioxidant activity, inhibiting formation of glycation end products, inhibiting formation of tyrosinase, and inhibiting accumulation of fat. Wherein, the receptor can be human.

In some embodiments, the mangosteen fermentation broth can be used to prepare a composition to enhance antioxidant activity.

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

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

In some embodiments, the mangosteen fermentation broth can be used to prepare a composition to inhibit fat accumulation.

In some embodiments, the mangosteen broth can be used to prepare a composition for reducing body fat of a recipient.

In some embodiments, the mangosteen broth can be used to prepare a composition for improving skin condition. Wherein the skin condition is improved by increasing the moisture content of the skin, reducing deep spots, reducing wrinkles, reducing skin texture, or a combination thereof.

In some embodiments, any of the compositions described above can be a pharmaceutical. In other words, the pharmaceutical comprises the mangosteen fermentation broth with effective content.

In some embodiments, the foregoing pharmaceuticals may be formulated into dosage forms suitable for enteral, parenteral (parenteral), oral, or topical (topically) administration using techniques well known to those skilled in the art.

In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, a lozenge (tablet), a tablet (troche), a buccal tablet (dosage), a pill (pill), a capsule (capsule), a dispersible powder (dispersible powder) or fine granules (granules), a solution, a suspension (suspension), an emulsion (emulsion), a syrup (syrup), an elixir (elixir), a slurry (syrup), or the like. In some embodiments, parenteral or topical administration dosage forms may be, but are not limited to, injectables (injections), sterile powders (sterile powders), external preparations (external preparation), or the like. In some embodiments, the administration of the injectate can be subcutaneous (subcutaneous), intradermal (intraepithelial), or intralesional (intralesional).

In some embodiments, the aforementioned pharmaceuticals may comprise pharmaceutically acceptable carriers widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvents (solvent), buffers (buffer), emulsifiers (emulsifying agent), suspending agents (suspending agent), disintegrating agents (decongestant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients, stabilizing agents (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving agent), wetting agents (wetting agent), lubricants (lubricant), absorption delaying agents (adsorption delaying agent), liposomes (lipofectamine), and the like. The type and amount of carrier selected for use is within the skill of one of ordinary skill in the art. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, physiological saline (normal saline), phosphate Buffered Saline (PBS), or an aqueous solution containing alcohol (alcohol containing aqueous solution).

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

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

In some embodiments, any of the foregoing compositions may be a cosmetic or a care product. In other words, the cosmetic or care product contains a specific content of the mangosteen fermentation broth.

In some embodiments, the cosmetic or care product may be any of the following types: lotions, gels, jellies, mud masks, lotions, creams, lipsticks, foundations, pressed powders, honey powders, make-up removers, facial cleansers, shower gels, shampoos, hair tonics, sun blocks, hand creams, nail polishes, perfumes, essences, and facial masks. In some embodiments, the cosmetic or care product may further comprise an external acceptable ingredient, if desired. In some embodiments, the topical acceptable ingredient may be, for example, an emulsifier, a penetration enhancer, a softener, a solvent, an excipient, an antioxidant, or a combination thereof.

The first embodiment is as follows: preparation of mangosteen fermentation liquor

Firstly, smashing the whole mangosteen (containing shells) into mangosteen particles with the diameter of less than 12 mm. The mangosteen particles are uniformly mixed with water in a ratio of 1. Then, the raw material mixture was sterilized at about 100 ℃ for 0.5 hour, and the sterilized raw material mixture was cooled to about 30 ℃ to obtain a mangosteen culture solution.

0.1% brewer's yeast (purchased from center for biological resource conservation and research (BCRC) of institute of food industry development, deposited number BCRC 20271) was inoculated into the mangosteen culture solution and fermented at about 30 ℃ for 1 day to obtain a first primary fermentation broth. Next, 0.05% Lactobacillus plantarum (purchased from BCRC, accession number BCRC 910760) was inoculated into the first primary fermentation broth and fermented at about 30 ℃ for 1 day to obtain a second primary fermentation broth. Finally, 10% acetic acid bacteria (purchased from BCRC, accession number BCRC 11688) was inoculated into the second primary fermentation broth and fermented at about 30 ℃ for 5 days to obtain a third primary fermentation broth.

And then carrying out reduced pressure concentration on the third primary fermentation liquid at the temperature of 3200460 ℃ and filtering by a filter screen with meshes to obtain a fermentation stock solution. And finally, adding 60% isomalto-oligosaccharide into the fermentation stock solution, and then sterilizing to obtain the mangosteen fermentation liquor.

In this case, the mangosteen culture solution, the fermentation stock solution, and the mangosteen fermentation solution were subjected to respective detection. Wherein, the sugar degree of the mangosteen culture solution is about 10.6, the sugar degree of the fermentation stock solution is about 3.9, and the sugar degree of the mangosteen fermentation solution is about 40.

Example two: total polyphenol content test

Weigh 10.0mg of Gallic acid (Gallic acid) into a 10mL volumetric flask, and add water (H) ₂ O) was quantified to 10mL to obtain a stock solution of gallic acid (stock solution). The stock solution of gallic acid was diluted 10-fold, i.e., 100. Mu.L of the stock solution of gallic acid was added to 900. Mu.L of water, to obtain an initial solution of 100. Mu.g/mL of gallic acid (i.e., containing 1000ppm of gallic acid). Then, 0. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL, and 100. Mu.g/mL of the gallic acid standard solutions were prepared according to the following Table I, and 100. Mu.L of each concentration of the standard solution was put into a glass tube. Add 500. Mu.L of Folin-Ciocalteu's phenol reagent (from Merck) to each glass tube, mix well with the standard solution and let stand for 3 minutes, add 400. Mu.L of 7.5% sodium carbonate, mix well and react for 30 minutes to obtain the standard reaction solution. A standard reaction solution of 200. Mu.L was taken in a 96-well plate, and its absorbance at 750nm was measured to obtain a standard curve.

Watch 1

Standard solution (μ g/mL)	0	20	40	60	80	100
							Initial solution (μ L)	0	20	40	60	80	100
Water (mu L)	100	80	60	40	20	0

The mangosteen culture solution and the fermentation stock solution obtained in example one were used as samples, respectively. 100mL of each sample was taken into a glass tube. Then, 500 μ L of forlin phenol reagent is added into a glass test tube to be uniformly mixed with the sample and kept stand for 3 minutes, and then 400 μ L of 7.5% sodium carbonate is added to be uniformly mixed and reacted for 30 minutes to obtain a reaction solution to be detected. After the glass test tube containing the reaction solution to be measured was shaken to ensure that no air bubbles were present, 200. Mu.L of the reaction solution to be measured was placed in a 96-well plate, and the absorbance at 750nm of the reaction solution to be measured was measured.

And then, dividing the absorbance value of the reaction solution to be detected corresponding to each sample by the sugar degree of the sample, and converting the absorbance value into the total polyphenol content by an interpolation method by using a standard curve. Thus, the total polyphenol content of the obtained mangosteen culture solution was 334ppm and the total polyphenol content of the mangosteen fermentation solution was 691ppm, as shown in FIG. 1. Therefore, the mangosteen can increase the total polyphenol content by 2.1 times after being fermented by microorganisms. That is, the mangosteen enzyme solution can enhance antioxidant activity relative to the mangosteen culture solution.

Example three: anti-glycation test

200mM Sodium phosphate buffer (pH 7.4), sodium azide (Sodium azide, naN) ₃ ) And Bovine serum albumin (Bovine serum albumin, BSA, brand: gibco) configuration contained 0.06% NaN ₃ 60mg/mL BSA solution.

200mM sodium phosphate buffer solution and D Fructose (D- (-) -Fructose, C) ₆ H ₁₂ O ₆ ) Preparing 1.5M D-fluctose solution.

200mM sodium phosphate buffer and Aminoguanidine hydrochloride (amino guanidine hydrochloride, AG, CH) ₆ N ₄ HCl) 3mM AG solution.

And adding 0.25mL of BSA solution and 0.25mL of D-fluctose solution into 0.25mL of mangosteen fermentation liquor obtained in the first step, and uniformly mixing to obtain a solution to be detected of an experimental group.

0.25mL of 3mM AG solution was added to 0.25mL of BSA solution and D-fluctose solution and mixed uniformly to obtain a control test solution.

0.1mL of each group of test solutions was taken as a zero point solution for each group. Fluorescence values of 0.1mL of each group of the zero point solution were measured with excitation light at 360nm and emission light at 460nm using a spectrofluorometer (Thermo Fisher Scientific) to obtain the fluorescence value of the zero point before the reaction.

0.45mL of each group of test solutions was incubated at 50 ℃ for 24 hours to obtain each group of end-point solutions. The fluorescence value of 0.1mL of each end point solution was measured by a fluorescence spectrophotometer with excitation light of 360nm and emission light of 460nm to obtain the fluorescence value of the end point of the reaction.

Then, the formation amount (%) of each group of Advanced Glycation End products (AGEs) was calculated according to the following formula (1) to obtain the anti-Glycation activity (anti activity). In other words, the relative AGEs formation (%) of each group was calculated by considering the AGEs formation of the control group as 1 (i.e., the relative AGEs formation of the control group was 100%).

Wherein, the Fluorescence sample _24hr Fluorescence values representing the end points of the experimental groups, fluorescence sample _0hr Fluorescence values representing the zero point of the experimental group, fluorescence control _24hr Fluorescence value representing the endpoint of the control group, fluorescence control _0hr Fluorescence values representing the zeros of the control group.

Referring to fig. 2, the experimental group significantly reduced the formation of the final saccharification product by 70% compared to the control group. Therefore, the mangosteen fermentation liquor can effectively inhibit the formation of the saccharification end product, namely has the function of resisting glycosylation.

Example four: melanin content detection

Here, the cell culture Medium used was Dulbecco's Modified Eagle's Medium (DMEM, brand: gibco) supplemented with 1vol% penicillin-streptomycin (brand: gibco) and 10vol% fetal bovine serum (brand: gibco).

First, at 1.5X 10 per hole ⁵ Cell number of individual cells, mouse melanoma cell line B16F10 (purchased from American Type Culture Collection (ATCC), accession number CRL-6475) was inoculated into each well of a 6-well culture plate containing 3mL of cell culture medium and cultured at 37 ℃ for 24 hours.

After 24 hours of culture, B16F10 cells were divided into 4 groups: two experimental groups (experimental group a and experimental group B), one control group and one control group. The cell culture medium was removed from each group and replaced with 3mL of experimental medium per well, followed by incubation at 37 ℃ for a further 48 hours. Wherein the culture medium of experiment group A is a cell culture medium containing 0.125vol% of the mangosteen fermentation broth obtained in example one. The test medium of test group B was a cell culture medium containing 0.125vol% of the mangosteen broth obtained in example one. The experimental medium for the control group was cell culture medium containing 0.125mg/mL kojic acid. The experimental culture medium of the control group is a simple cell culture medium (namely, the experimental culture medium does not contain mangosteen fermentation liquor, mangosteen culture solution or kojic acid).

After 48 hours of incubation, the experimental medium in each well was removed and rinsed twice with 1-fold (1 ×) phosphate buffered saline (PBS, brand: gibco). After washing, trypsin (trypsin) was added to each well to treat the cells for 3 minutes. After 3 minutes of treatment, the suspended cells from each well were individually collected in a 15mL centrifuge tube and then centrifuged at 400Xg for 5 minutes to separate the cell pellet (cell pellet) from the supernatant. After cell pellet resuspension by 1xPBS and centrifugation were repeated twice, cell pellet was resuspended in 200. Mu.L of 1xPBS to obtain a cell solution. Subsequently, the cell solution was left in liquid nitrogen for 10 minutes, and then left to stand at room temperature for 30 minutes for thawing. After thawing was complete, each tube was centrifuged at 12,000xg for 30 minutes. After centrifugation for 30 minutes, the supernatant was removed from each tube and 120. Mu.L of 1N NaOH (in ddH) was added ₂ O) to mix with the sediment in each centrifuge tube. After mixing well, each centrifuge tube containing the mixed solution was left to stand in a dry bath at 60 ℃ for 1 hour. Thereafter, 100. Mu.L of the mixed solution was taken out of each centrifuge tube into a 96-well plate and the absorbance (OD 450) of each well in the 96-well plate was read at a wavelength of 450nm with an ELISA (enzyme-linked immunosorbent assay) reader (brand: bioTek).

After the measurement, the relative melanin content (%) was calculated by substituting the measured absorbance into the following formula (2). In other words, the relative melanin content (%) of each group was calculated by considering the melanin content of the control group as 1 (i.e., the relative melanin content of the control group is 100%). Also, statistically significant differences between groups were statistically analyzed by student t-test (student t-test), as shown in fig. 3. In fig. 3, the "x" represents a p-value of less than 0.05 when compared to the control group, the "x" represents a p-value of less than 0.01 when compared to the control group, the "x" represents a p-value of less than 0.001 when compared to the control group, and the "a-tangle-solidup" represents a p-value of less than 0.001 when compared to the control group.

Relative melanin content (%) = (OD 450 sample/OD450 control). Times.100% (2)

Wherein OD450 sample represents the absorbance of the group to be converted, and OD450 control represents the absorbance of the control group.

Referring to fig. 3, the melanin content of the experimental group a was significantly reduced compared to the control group, and it could reduce melanin production by 21.58%. Also, the melanin content of experimental group a was significantly reduced compared to the corresponding concentration of kojic acid, and it reduced melanin production by 21.58%. The group A also reduced melanin production by 1.0% compared to the group B. Therefore, the mangosteen fermentation liquid can effectively inhibit melanin generation, namely tyrosinase formation, and has whitening effect.

Example five: fat accumulation detection

Here, the preadipocyte expansion Medium (pre-adipocyte expansion Medium) used was Minimum Essential Medium α (MEM α, brand: gibco) supplemented with 20vol% FBS (brand: gibco) and 1vol% penicillin-streptomycin. The differentiation medium (differentiation medium) used was MEM α (brand: gibco) supplemented with 20vol% FBS (brand: gibco) and 1vol% penicillin-streptomycin. Also, the oil-red O staining reagent (brand: sigma) was thoroughly dissolved in 100% isopropanol (isopropanol, supplier: ECHO) to prepare a stock solution of the oil-red O staining reagent at 3 mg/mL. To obtain an oil-red O working solution (oil-red O working solution) ready for use, a stock solution of oil-red O staining reagent is treated with secondary water (ddH) in real time prior to use ₂ O) is diluted to the concentration of 1.8mg/mL, and the solution is the stock solution of 60% oil-red O staining reagent.

First, at 8 × 10 per hole ⁴ Cell number of individual cells, mouse bone marrow stromal cell line OP9 (purchased from ATCC)Number CRL-2749) were inoculated into each well of a 24-well culture plate containing 500 μ L of preadipocyte proliferation medium and cultured at 37 ℃ for 7 days. During the 7 day culture period, 500. Mu.L of fresh differentiation medium was replaced every 3 days. After 7 days of culture, intracellular oil droplet (lipid drop) formation in each well was observed using a microscope (brand: ZEISS) to confirm complete differentiation of the cells into adipocytes for subsequent experiments.

After 24 hours of culture, adipocytes were divided into 3 groups: two experimental groups (i.e., experimental group a and experimental group B) and a control group. The differentiation medium of each group was removed and replaced with 500. Mu.L of the experimental medium per well, and then left at 37 ℃ for continuous culture for 7-10 days. During the 7-10 day culture period, the fresh 500. Mu.L of experimental medium was replaced every 3 days. Wherein, the experimental culture medium of the experimental group A is a differentiation culture medium containing 0.0625vol% of the mangosteen fermentation liquor obtained in the first example. The culture medium of experiment group B was a differentiation medium containing 0.0625vol% of the culture solution of mangosteen obtained in example one. The experimental medium of the control group is a simple differentiation medium (i.e. does not contain mangosteen fermentation broth).

Next, the assay medium in each well was removed and rinsed twice with 1 xPBS. Then, 1mL of 10% formaldehyde (Formaldehyde, supplier: ECHO) was added to each well and cultured at room temperature for 30 minutes, thereby fixing the cells. Thereafter, the formaldehyde in each well was removed and each well was rinsed twice with 1mL PBS. After another rinse, 1mL of 60% isopropanol was added to each well and allowed to act for 1 minute. Next, the isopropanol was removed and 1mL of oil-red O working solution was added and allowed to react at room temperature for 1 hour.

At 1 hour of action, the oil-red O working solution was removed and rapidly destained with 1mL 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 (shaker) for 10 minutes to dissolve the stain. Then, 100. Mu.L of the aforementioned dye-isopropyl alcohol solution was taken out from each well to a 96-well culture plate and the absorbance (OD 510) of each well was read at a wavelength of 510nm with an ELISA reader (brand: bioTek).

After the measurement, the relative fat oil drop (%) was calculated by substituting the measured absorbance into the following formula (3). In other words, the relative fat drop (%) of each group was calculated by considering the fat drop of the control group as 1 (i.e., the relative fat drop of the control group was 100%). Also, statistically significant differences between groups were statistically analyzed by student t-test, as shown in fig. 4. In fig. 4, "+" indicates that the p-value was less than 0.01 when compared to the control group.

Relative fatty oil drop amount (%) = (OD 510 sample/OD510 control). Times.100% (3)

Wherein OD510 sample represents the absorbance of the group to be scaled, and OD510 control represents the absorbance of the control group.

Referring to fig. 4, the relative fatty oil droplet size of the experimental group a was significantly reduced compared to the control group, and it was possible to reduce the oil droplet size by 16.9%. Compared with the experimental group B, the relative fatty oil drop of the experimental group A is also obviously reduced, and the oil drop can be reduced by 8.7%. Therefore, the mangosteen fermentation liquid can effectively inhibit fat accumulation and has the efficacy of reducing fat. Moreover, the mangosteen may produce more fat-reducing active ingredients than the mangosteen culture solution after microbial fermentation.

Example six: body test for fat reduction

8 obese subjects (i.e., subjects with body fat percentage greater than 25% or BMI greater than 24) were allowed to drink a 6mL bottle of mangosteen fermented drink (containing 12vol% of the mangosteen fermented liquid obtained in example 1 and 88vol% of water) daily for 4 weeks. And, the whole body fat rate of the subjects was measured by a body fat meter (brand: TANITA BC-601 FS) before drinking (i.e., week 0) and after drinking for 4 weeks (i.e., week 4), and the waist circumference of the subjects was measured by a cloth ruler. Also, statistically significant differences between the measurement results of week 0 and week 4 were statistically analyzed by student t-test, as shown in fig. 5 and 6. In fig. 5 and 6, "-indicates that the p-value was less than 0.05 in comparison with week 0.

Referring to fig. 5 and 6, drinking the mangosteen fermented beverage for 4 weeks significantly reduced the body fat percentage of the whole body by about 0.4% and the waist circumference by about 2.0 cm, compared to before drinking (week 0). Therefore, the fat accumulation index of obese people can be improved by using the mangosteen fermentation liquor for a long time, namely the mangosteen fermentation liquor has the effects of slimming and reducing fat.

Example seven: human detection of beautiful muscles

8 fat subjects were allowed to drink one bottle of 6mL of mangosteen fermented beverage (containing 12vol% of the mangosteen fermented liquid obtained in example 1 and 88vol% of water) daily for 6 weeks continuously. Before drinking (i.e., week 0) and after drinking for 6 weeks (i.e., week 6), the skin condition was measured by using a VISIA skin measuring instrument (VISIA Complexion Analysis System, available from Canfield, usa) and a skin surface moisture measuring instrument (Corneometer CM825, available from C + K, germany). Wherein, VISIA skin detector is used for measuring skin deep speckle (brown spot), skin wrinkle and skin texture, and skin surface humidity tester is used for measuring skin water content.

After the measurement, the skin condition at week 0 was taken as a reference (i.e., the relative skin condition at week 0 was 100%), and the relative skin condition (%) at week 6 was calculated. Also, statistically significant differences between the relative skin condition at week 0 and the relative skin condition at week 6 were statistically analyzed by student t-test, as shown in fig. 7-10. In fig. 7 to 10, "", indicates that the p-value was less than 0.05 in comparison with week 0.

Referring to fig. 7 to 10, drinking the mangosteen fermented beverage for 6 weeks significantly reduced skin deep spots by about 8.5%, skin wrinkles by about 8.3%, skin texture by about 6.7%, and skin moisture content by about 18.6%, compared to before drinking (week 0). Therefore, the mangosteen fermentation liquor can improve the skin condition after being used for a long time, namely the mangosteen fermentation liquor has the effect of beautifying the skin.

In summary, the mangosteen fermentation broth according to any embodiment of the present invention can be used to prepare a skin-beautifying and/or fat-reducing composition. In other words, the aforementioned composition has one or more of the following functions: promoting antioxidant activity, inhibiting formation of glycation end products, inhibiting formation of tyrosinase, inhibiting fat accumulation, reducing body fat of receptor, and improving skin condition.

The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the mangosteen fermentation liquor is characterized in that the mangosteen fermentation liquor is used for preparing a composition for improving the antioxidant capacity, wherein the mangosteen fermentation liquor is obtained by performing a fermentation procedure on a mangosteen culture solution and a plurality of strains, the mangosteen culture solution comprises mangosteen and water, the ratio of the mangosteen to the water is 1:

adding the yeast into the mangosteen culture solution;

fermenting the mangosteen culture solution and the yeast for 1 to 3 days to form a first primary fermentation liquid, wherein the yeast is beer yeast;

adding the lactic acid bacteria to the first primary fermentation broth;

fermenting the first primary fermentation liquid and the lactic acid bacteria for 1 to 5 days to form a second primary fermentation liquid, wherein the lactic acid bacteria is lactobacillus plantarum;

adding the acetic acid bacteria into the second primary fermentation liquid;

fermenting the second primary fermentation liquid and 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

filtering the third primary fermentation liquid to obtain the mangosteen fermentation liquid.

2. Use of a mangosteen broth for the preparation of a composition for inhibiting the formation of a saccharification end product, wherein the mangosteen broth is obtained by subjecting a mangosteen broth and a plurality of bacteria to a fermentation process, wherein the mangosteen broth comprises mangosteen and water in a ratio of 1 to 2 to 5, and the plurality of bacteria comprise 0.01 to 0.5% yeast, 0.01 to 0.25% lactic acid bacteria, and 1 to 15% acetic acid bacteria, relative to the mangosteen broth, the fermentation process comprising:

adding the yeast into the mangosteen culture solution;

adding the lactic acid bacteria to the first primary fermentation broth;

adding the acetic acid bacteria into the second primary fermentation liquid;

3. An application of a mangosteen fermentation liquid is characterized in that the mangosteen fermentation liquid is used for preparing a composition for inhibiting formation of tyrosinase, wherein the mangosteen fermentation liquid is obtained by performing a fermentation process on a mangosteen culture liquid and a plurality of strains, the mangosteen culture liquid comprises mangosteen and water, the ratio of the mangosteen to the water is 1:

adding the yeast into the mangosteen culture solution;

adding the lactic acid bacteria to the first primary fermentation broth;

fermenting the first primary fermentation liquid and 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 liquid;

4. Use of a mangosteen broth for the preparation of a composition for inhibiting fat accumulation, wherein the mangosteen broth is obtained by subjecting a mangosteen broth and a plurality of bacteria to a fermentation process, wherein the mangosteen broth comprises mangosteen and water in a ratio of 1:

adding the yeast into the mangosteen culture solution;

adding the lactic acid bacteria to the first primary fermentation broth;

adding the acetic acid bacteria into the second primary fermentation liquid;

5. Use of a mangosteen broth for the preparation of a composition for reducing body fat of a recipient, wherein the mangosteen broth is obtained by subjecting a mangosteen broth to a fermentation procedure with a plurality of bacteria species, wherein the mangosteen broth comprises mangosteen and water in a ratio of 1 to 2 to 5, and the plurality of bacteria species comprise 0.01 to 0.5% yeast, 0.01 to 0.25% lactic acid bacteria, and 1 to 15% acetic acid bacteria, relative to the mangosteen broth, the fermentation procedure comprising:

adding the yeast into the mangosteen culture solution;

adding the lactic acid bacteria to the first primary fermentation broth;

adding the acetic acid bacteria into the second primary fermentation liquid;

6. An application of a mangosteen fermentation liquid is characterized in that the mangosteen fermentation liquid is used for preparing a composition for improving skin conditions, wherein the mangosteen fermentation liquid is obtained by performing a fermentation process on a mangosteen culture solution and a plurality of strains, the mangosteen culture solution comprises 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 comprise 0.01-0.5% of saccharomycetes, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria, the skin conditions are improved by increasing the water content of skin, reducing deep-layer spots, reducing wrinkles, reducing skin textures or a combination thereof, and the fermentation process comprises the following steps:

adding the yeast into the mangosteen culture solution;

adding the lactic acid bacteria to the first primary fermentation broth;

adding the acetic acid bacteria into the second primary fermentation liquid;

7. Use according to any one of claims 1 to 6, wherein the pH of the mangosteen broth is 3.7 ± 1.0 and the sugar degree of the mangosteen broth is 40 ± 2.

8. Use according to any one of claims 1 to 6, wherein the mangosteen broth has a polyphenol content of 691ppm.