CN114099586A

CN114099586A - Application of muskmelon and eggplant fermented juice

Info

Publication number: CN114099586A
Application number: CN202011147988.6A
Authority: CN
Inventors: 吴佩宜; 林咏翔
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-08-27
Filing date: 2020-10-23
Publication date: 2022-03-01
Anticipated expiration: 2040-10-23
Also published as: TWI774046B; TWI757850B; CN114099586B; CN112442457B; CN112442457A; CN112442458B; TW202108759A; TWI749734B; TW202108124A; TW202108758A; CN112442458A

Abstract

The invention discloses application of muskmelon and eggplant fermented juice in preparing a composition. The composition has one or more of the following functions: increasing glutathione synthesis, inhibiting formation of glycosylation final products, increasing anti-aging related gene, inhibiting melanin generation, increasing moisturizing related gene secretion, inhibiting fat accumulation, promoting lipolysis, reducing body fat of receptor, and improving skin condition. The muskmelon-eggplant fermentation juice is obtained by performing a fermentation process on a muskmelon culture solution and a plurality of strains.

Description

Application of muskmelon and eggplant fermented juice

Technical Field

The invention relates to a muskmelon and eggplant fermented juice, in particular to an application of the muskmelon and eggplant fermented juice in preparing a composition for improving skin conditions 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.

Solanum melongena (Pepino Dulce, a scientific name of Solanum muricatum), also called Herminium, is a perennial herb of Solanum of Solanaceae. The muskmelon is native to south America and becomes one of natural plants researched and developed in recent years.

Disclosure of Invention

In one embodiment, the use of a solanum melongena fermented juice for preparing a composition for improving oxidation resistance is disclosed, wherein the solanum melongena fermented juice is obtained by performing a fermentation process on a solanum melongena culture solution and a plurality of strains, the solanum melongena culture solution comprises solanum melongena juice and water, and the ratio of the solanum melongena juice to the water is 1: 2-5. Relative to the muskmelon eggplant 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 melon eggplant fermentation broth for the preparation of a composition for improving skin condition. Wherein the muskmelon eggplant fermentation juice is obtained by fermenting a muskmelon eggplant culture solution and a plurality of strains. The muskmelon and eggplant culture solution comprises muskmelon and eggplant juice and water, the ratio of the muskmelon and the water is 1:2-5, and relative to the muskmelon and eggplant culture solution, a plurality of strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria.

In an embodiment, the use of a melon eggplant fermentation broth for the preparation of a composition for reducing fat. Wherein the muskmelon eggplant fermentation juice is obtained by fermenting a muskmelon eggplant culture solution and a plurality of strains. The muskmelon eggplant culture solution comprises muskmelon eggplant juice and water, and the ratio of the muskmelon eggplant to the water is 1: 2-5. Relative to the muskmelon eggplant 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 yet another embodiment, the melon eggplant fermented juice has an increased antioxidant Capacity of Total Anti-oxidative Capacity (Total Anti-oxidative Capacity), a reduced peroxidation product mda (malonadialdehyde), an increased glutathione, or a combination thereof.

In yet another embodiment, the solanum melongena fermented juice improves skin condition by increasing skin anti-aging ability, increasing anti-glycosylation ability, decreasing melanin content, increasing skin moisturization, decreasing skin texture, decreasing skin wrinkles, decreasing skin redness, or a combination thereof.

In another embodiment, the capability of improving skin anti-aging capability of the solanum melongena fermented juice is regulating and controlling a CCT5 gene, a FOXO gene, an MRPS5 gene, a Pink1 gene or a UBL5 gene.

In yet another embodiment, the solanum melongena fermented juice promotes skin moisturization to regulate HAS2 gene or KRT1 gene.

In yet another embodiment, the solanum melongena fermented juice reduces fat to inhibit fat accumulation, promote fat decomposition, or a combination thereof.

In yet another embodiment, the cigar fermentation juice is prepared by a fermentation procedure, wherein the fermentation procedure comprises adding the yeast to the cigar broth; fermenting the eggplant culture solution with the yeast for 1 to 3 days to form a first primary fermentation liquid, wherein the yeast is preferably Saccharomyces cerevisiae; adding the lactic acid bacteria to the first primary fermented juice; fermenting the first primary fermented juice and the lactic acid bacteria for 1 to 5 days to form a second primary fermented juice, wherein the lactic acid bacteria is preferably Streptococcus thermophilus; adding the acetic acid bacteria into the second primary fermented juice; fermenting the second primary fermented juice with the acetic acid bacteria for 3-10 days to form a third primary fermented juice, wherein the acetic acid bacteria is preferably Acetobacter aceti; and filtering the third primary fermented juice.

In yet another embodiment, the pH of the melon eggplant fermented juice is 4.0 ± 1.0, and the sugar degree of the melon eggplant fermented juice is 40 ± 2.

In yet another embodiment, the melon eggplant fermentation broth has a polyphenol content of 88 ppm.

In summary, according to any embodiment of the present invention, the fermented juice of solanum melongena can be used for preparing a composition for improving skin condition and/or reducing fat. In other words, the aforementioned composition has one or more of the following functions: increasing glutathione synthesis, inhibiting formation of glycosylation end products, increasing anti-aging related genes, inhibiting melanogenesis, increasing moisturizing related gene secretion, inhibiting fat accumulation, promoting lipolysis, reducing body fat of receptor, and improving skin condition.

The following examples are presented to illustrate the present invention and are not intended to limit the scope of the invention, which is not to be construed as limiting the scope of the invention since various modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention.

Drawings

Fig. 1 is a histogram showing the change in total polyphenol content of a c.melo-eggplant culture solution (before fermentation) and a c.melo-eggplant fermentation broth (after fermentation).

FIG. 2 is a histogram showing the relative glutathione content of each group.

FIG. 3 is a histogram showing the relative amounts of formation of end products of glycosylation for each group.

FIG. 4 is a histogram showing the relative anti-aging gene expression amounts of each group.

Fig. 5 is a histogram showing the relative melanin content of each group.

FIG. 6 is a histogram showing the relative moisturizing gene expression of each group.

Fig. 7 is a histogram showing the effect of suppressing fat accumulation for each group.

Fig. 8 is a histogram showing the promotion of lipolysis for each group.

FIG. 9 is a histogram showing the relative expression of lipid-burning genes of each group.

Fig. 10 is a histogram showing the change in body weight before and after use of an exemplary fermented juice of solanum melongena.

Fig. 11 is a histogram showing the change in body fat percentage of the entire body before and after the use of an exemplary fermented juice of solanum melongena.

Fig. 12 is a histogram showing changes in body fat percentage before and after use of an exemplary fermented juice of solanum melongena.

Fig. 13 is a histogram showing the change in hip circumference before and after use of an exemplary fermented juice of solanum melongena.

Fig. 14 is a histogram showing changes in the total antioxidant capacity of an exemplary fermented juice of solanum melongena before and after use.

Fig. 15 is a histogram showing changes in oxidation product MDA before and after use of an exemplary solanum melongena fermented juice.

Fig. 16 is a histogram showing changes in relative skin texture (roughness) before and after use of an exemplary solanum melongena fermented juice.

Fig. 17 is a histogram showing changes in relative skin wrinkles before and after use of an exemplary fermented juice of solanum melongena.

Fig. 18 is a histogram showing the change in relative skin redness (redness) before and after use of an exemplary fermented juice of solanum melongena.

Fig. 19 is a histogram showing the change in relative skin moisture content before and after use of an exemplary fermented juice of solanum melongena.

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, "melon eggplant" refers generally to melon eggplant fruits.

As used herein, the terms "culture solution of solanum melongena" and "water extract of solanum melongena" are used interchangeably, and refer to a mixture of solanum melongena juice and water after being mixed uniformly.

The procedures and parameters for the fermentation of microorganisms according to the present invention are within the skill of those skilled in the art.

In some embodiments, the fermented juice of the muskmelon is obtained by performing a fermentation process on a muskmelon culture solution and a plurality of strains. Wherein the culture solution comprises fructus Benincasae and water. In the muskmelon eggplant culture solution, the ratio of the muskmelon eggplant to water is 1: 2-5. In the fermentation process, relative to the muskmelon eggplant culture solution, the strains comprise 0.01-0.5% of yeast, 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 can be Streptococcus thermophilus (Streptococcus thermophilus) or lactobacillus plantarum. In some embodiments, the Acetobacter aceti can be Acetobacter aceti (Acetobacter aceti).

In some embodiments, in the fermentation process, 0.01% -0.5% of yeast is added to the culture liquid of the solanum melongena, and the culture liquid of the solanum melongena and the yeast are fermented for 1 day to 3 days to form the first primary fermentation juice. In other words, 0.01% -0.5% of yeast is fermented for 1 to 3 days with the first mixed solution of the culture solution of the eggplant to form the first primary fermented juice. In some embodiments, the first mixture is fermented at 28 ℃ to 37 ℃.

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

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

After the third primary fermented juice is formed, filtering the third primary fermented juice to obtain a melon eggplant fermented juice. In a first exemplary embodiment, the step of filtering the third primary fermented juice includes filtering the third primary fermented juice with a sieve having 200-400 mesh openings. In a second exemplary embodiment, the filtering step of the third primary fermented juice includes concentrating under reduced pressure at 40 ℃ to 70 ℃ and filtering the third primary fermented juice with a sieve having 200 mesh to 400 mesh. In a third exemplary embodiment, the filtering step of the third primary fermented juice includes concentrating under reduced pressure at 40 ℃ to 70 ℃ and filtering the third primary fermented juice with a sieve having 200 mesh to 400 mesh openings to obtain a fermentation raw liquid, and adjusting the sugar degree (Degrees Brix) of the fermentation raw liquid to form the solanum melongena fermented juice.

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 brix may be isomalto-oligosaccharide.

In some embodiments, the pH of the c.melo-eggplant fermented juice is 3.7 ± 1.0, and the brix of the c.melo-eggplant fermented juice is 40 ± 2.

In some embodiments, the polyphenol content of the c.melo-eggplant fermentation juice is 88 ppm.

The muskmelon eggplant culture solution is prepared by mixing muskmelon eggplant juice and water in a weight ratio of 1:2-5 to obtain. In a first exemplary embodiment, the c.melo-eggplant culture solution is prepared by mixing c.melo-eggplant juice selected from a source in south america with water in a ratio of 1:2-5 to obtain. In a second example, the culture solution of solanum melongena is prepared by mixing solanum melongena juice selected from peru Agro international business a & c sac company with water in a ratio of 1: mixing at a ratio of 2-5, and adding glucose solution to adjust sugar degree to 9. In a third exemplary embodiment, the culture solution of solanum melongena is prepared by mixing solanum melongena juice selected from peru Agro international business a & c sac company with water in a ratio of 1:5, and adding a glucose solution to adjust the sugar degree to 9.

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

In some embodiments, the melon eggplant juice has the effect of improving skin condition and/or reducing fat. In some examples, improving the skin condition of the subject can be increasing skin anti-aging capacity, increasing anti-glycation capacity, decreasing melanin content, increasing skin moisturization, decreasing skin texture, decreasing skin wrinkles, decreasing skin redness, or a combination thereof. In some embodiments, the fermented juice of solanum melongena can achieve the effects of improving skin condition and/or reducing fat through the effects of one or more of the following cell layers: increasing glutathione synthesis, inhibiting formation of glycosylation final products, increasing anti-aging related gene, inhibiting melanin generation, increasing moisturizing related gene secretion, inhibiting fat accumulation, promoting lipolysis, reducing body fat of receptor, and improving skin condition. Wherein, the receptor can be human.

In some embodiments, the solanum melongena fermented juice can be used to prepare a composition for enhancing antioxidant Capacity, wherein enhancing antioxidant Capacity may be enhancing Total Anti-oxidative Capacity (Total Anti-oxidative Capacity), reducing peroxidation products mda (malonodialdehyde), enhancing glutathione, or a combination thereof.

In some embodiments, the solanum melongena fermented juice can be used to prepare a composition for improving skin condition, wherein the skin condition can be improved by enhancing skin anti-aging ability, enhancing anti-glycosylation ability, reducing melanin content, enhancing skin moisturizing, reducing skin texture, reducing skin wrinkles, reducing skin redness, or a combination thereof, wherein the skin anti-aging ability is regulated by at least one of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene, or UBL5 gene, and the skin moisturizing is regulated by at least one of HAS2 gene or KRT1 gene.

In some embodiments, the solanum melongena fermented juice can be used to prepare a composition for reducing fat, wherein reducing fat is inhibiting fat accumulation, promoting fat breakdown, or a combination thereof.

In some embodiments, any of the compositions described above can be a pharmaceutical. In other words, the pharmaceutical comprises an effective amount of the fermented juice of solanum melongena.

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

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 preparations), or the like. In some embodiments, the administration of the injectate can be subcutaneous (subcutaneous), intradermal (intraepithelial injection), or intralesional (intrafocal injection).

In some embodiments, the aforementioned pharmaceuticals may comprise pharmaceutically acceptable carriers that are 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), suspending agents (suspending agent), disintegrating agents (disintegrant), 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), wetting agents (wetting agent), lubricants (lubricating agent), absorption delaying agents (absorption accelerating agent), liposomes (liposome) and the like. The type and amount of carrier selected for use is within the skill and expertise of one skilled in the art. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline (normal saline), Phosphate Buffered Saline (PBS), or an aqueous solution containing alcohol (alcohol stabilizing 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 the melon eggplant fermented juice. 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) or dietary supplements (dietary supplements).

In some embodiments, the aforementioned edible composition can be administered orally to a subject. The edible composition can be in the form of powder, granule, solution, colloid 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 fermented juice of solanum melongena.

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 can 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 muskmelon and eggplant fermented juice

First, melon and eggplant juice (product name Pepino Dulce, Nature pure, original place of melon and eggplant is Peru Anderson mountain) purchased from Peru, agricultural international business a & c sac company and water are uniformly mixed in a ratio of 1:5 (namely, the melon and eggplant juice is one time weight, and the water is five times weight), and then glucose solution is added to adjust the sugar degree to 9.0, so that the melon and eggplant culture solution is obtained. However, the present invention is not limited to the source of the juice, such as the juice provided by a supplier in south America, Peruvian Nature company or Naike company.

Next, 0.1% Saccharomyces cerevisiae (purchased from the center for conservation and research of biological resources (BCRC) of the institute for food industry development, accession No. BCRC20271) was inoculated into the culture broth of Solanum melongena and fermented at about 30 ℃ for 1 day to obtain a first primary fermentation broth. Next, 0.05% Streptococcus thermophilus (purchased from BCRC under accession number BCRC910636) was inoculated into the first primary fermented juice and fermented at about 30 ℃ for 1 day to obtain a second primary fermented juice. Finally, 10% acetic acid bacteria (Acetobacter aceti) (purchased from BCRC, accession number BCRC11688) was inoculated into the second primary fermented juice and fermented at about 30 ℃ for 5 days to obtain a third primary fermented juice.

And concentrating the third primary fermented juice at about 60 deg.C under reduced pressure and filtering with 200 mesh sieve to obtain fermentation stock solution. Finally, 60% isomalto-oligosaccharide was added to the fermentation stock solution and then sterilized at about 100 ℃ for 2 hours to obtain a melon eggplant fermentation broth.

In this case, the sugar degree of the culture solution, the fermentation stock solution and the fermented juice of the muskmelon eggplant were measured. Wherein the sugar degree of the mixed liquid of the muskmelon and eggplant raw materials is about 9.0, the sugar degree of the fermentation stock solution is about 4.0, and the sugar degree of the muskmelon and eggplant fermentation juice is about 40.

Example two: preparation of muskmelon eggplant water extract (without fermentation)

The method comprises the steps of uniformly mixing the muskmelon and eggplant juice purchased from the Agro international business a & c sac company (product name Pepino Dulce, Nature Pulp, and the origin place of the muskmelon and eggplant is Peru Andes mountain range) with water in a ratio of 1:5, adding a glucose solution to adjust the sugar degree to 9, and obtaining the muskmelon and eggplant water extract with the pH value of 6.3.

Example three: total polyphenol content test

10.0mg of Gallic acid (Gallic acid) was weighed out in a 10mL volumetric flask and then quantified to 10mL with water (H2O) 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 stock solution of gallic acid was added with 900. mu.L water, to obtain an initial solution of 100. mu.g/mL gallic acid (i.e., containing 1000ppm 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 a gallic acid standard solution was 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 (available from Merck) to each glass tube, mix well with the standard solution and 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 curve was obtained by taking 200. mu.L of the standard reaction solution into a 96-well plate and measuring its absorbance at 750 nm.

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 test sample of the experimental group (i.e. the fermented juice of the solanum melongena in the example one) and the test sample of the control group (i.e. the water extract of the solanum melongena in the example two) were diluted 10 times with water, respectively. 100mL of each sample was taken into a glass tube. Then, 500. mu.L of the forinophenol reagent was added to the glass test tube, mixed with the sample uniformly and left to stand for 3 minutes, and then 400. mu.L of 7.5% sodium carbonate was added, mixed uniformly and reacted for 30 minutes to obtain a reaction solution to be measured. 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 of the reaction solution to be measured at 750nm was measured.

And then, the light absorption value of the reaction solution to be detected corresponding to each sample is divided by the sugar degree of the sample, and then the total polyphenol content is converted by utilizing a standard curve and an interpolation method. In this way, the total polyphenol content of the resulting water extract of c.melo-eggplant was 27ppm and the total polyphenol content of the fermented c.melo-eggplant juice was 88ppm, as shown in fig. 1. Therefore, the total polyphenol content of the muskmelon eggplant can be increased by 3.2 times after the muskmelon eggplant is fermented by the microorganisms. Namely, the muskmelon eggplant fermentation juice can improve antioxidant activity relative to the muskmelon eggplant water extract.

Example four: assessment of cellular antioxidant efficacy-glutathione content assay

Experimental Material

Cell lines: human peripheral blood mononuclear cells (hPBMC).

Culture medium: X-VIVO10 medium (purchased from Lonza, model 04-380Q)

GSH detection reagent (purchased from abcam, model Ab 112132).

Liquid extract of muskmelon and eggplant: the melon eggplant water extract used in this experiment was obtained as described in example two above.

The muskmelon and eggplant fermentation juice: the melon eggplant water extract used in this experiment was obtained as described in example one above.

Experimental procedure

1. In 6-well culture plates, 2X10 medium was planted per well⁶Individual human peripheral blood mononuclear cells (which will be abbreviated as cells in the later steps).

2. Incubated at 37 ℃ for 24 hours.

3. Cells were divided into three groups: blank group, experimental group A, experimental group B. The first group is a blank group (Mock); the second group (experiment group A) is added with the water extract of the muskmelon eggplant, so that after the muskmelon eggplant fermentation juice is added, the concentration of the muskmelon eggplant fermentation juice in the whole solution is 0.0625%; the third group (experiment group B) is added with the muskmelon and eggplant fermented juice, so that after the muskmelon and eggplant fermented juice is added, the concentration of the muskmelon and eggplant fermented juice in the whole solution is 0.0625%; the N three groups of cells were then cultured at 37 ℃ for 24 hours.

4. The cells were collected.

5. Washed once with Phosphate Buffered Saline (PBS) (from Gibco).

6. The cells were resuspended (Resuspend cells) in 1ml PBS.

7. Cells were stained with GSH reagent (1: 1000) for 15 min.

8. Washed once with PBS.

9. The cells were resuspended (Resuspend cells) in 200. mu.l PBS.

10. The relative Fluorescent Isocyanate (FITC) signals were analyzed by flow cytometry (purchased from BD Accuri, model C6 Plus).

11. The statistical significance of the values was analyzed by Student's t-test using microsoft EXCEL software, and the results are shown in fig. 1. Wherein, compared to the blank group, indicates p < 0.001.

As can be seen from the results in fig. 2, the GSH content was not increased in the second group (experimental group a) of cells added with the solanum melongena aqueous extract, but was reduced by 7.41% compared to the blank group; the GSH content of the third group (experiment group B) of cells added with the fermented juice of the muskmelon eggplant is obviously increased by 8.34 percent compared with that of the blank group, so that the relative GSH content (relative GSH content) of the third group (experiment group) of cells is 1.08 times that of the first group (blank group) of cells and 1.17 times that of the second group (experiment group) of cells. GSH is a polypeptide composed of glutamic acid, cystine, and glycine, whose thiol (-SH) group is related to redox, and its main function is the intracellular antioxidant defense, which can protect against the oxidative damage of ROS. Therefore, the results shown in fig. 1 also demonstrate that the addition of the fermented juice of solanum melongena obtained in the first embodiment of the present invention can actually increase the GSH content in the cells and increase the intracellular redox ability, so as to achieve the effect of cell oxidation resistance.

Example five: anti-glycation test

200mM Sodium phosphate buffer (pH 7.4), Sodium azide (NaN)₃) And Bovine serum albumin (Bovine serum albumin, BSA, brand: gibco) was prepared as a 60mg/mL BSA solution containing 0.06% NaN 3.

200mM sodium phosphate buffer solution and D Fructose (D- (-) -Fructose, C)₆H₁₂O₆) Preparing 1.5M D-fructose solution.

200mM sodium phosphate buffer and Aminoguanidine hydrochloride (amino guanidine hydrochloride, AG, CH)₆N₄HCl) 3mM AG solution.

0.25mL of the extract of Solanum melongena obtained in example II was added with 0.25mL of BSA solution and D-fluctose solution and mixed uniformly to obtain the solution to be tested of experiment group A.

0.25mL of the fermented juice of Solanum melongena obtained in example one was added to 0.25mL of BSA solution and D-fluctose solution and mixed uniformly to obtain a solution to be tested for test group B.

0.25mL of 3mM AG solution was added to 0.25mL of BSA solution and D-fluctose solution and mixed uniformly to obtain a blank group of solutions to be tested.

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 solutions were measured with excitation light of 360nm and emission light of 460nm using a fluorescence spectrophotometer (Thermo Fisher Scientific) to obtain fluorescence values 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 the end point of the reaction was obtained by taking 0.1mL of each group of end point solutions and measuring the fluorescence value of 0.1mL of each group of end point solutions with excitation light of 360nm and emission light of 460nm using a spectrofluorometer.

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

Wherein, the Fluorescence sample_24hrFluorescence values representing the end points of the experimental groups, Fluorescence sample_0hrFluorescence values representing the zero point of the experimental group, Fluorescence control_24hrFluorescence value representing the end point of the blank set, Fluorescence control_0hrFluorescence values representing the zero points of the blank set.

Referring to fig. 3, experimental group a (water extract) did not affect the formation of the glycosylated end product, whereas experimental group B (fermented juice) significantly reduced the formation of 24.24% of the glycosylated end product compared to the blank. Therefore, the fermented juice of the muskmelon eggplant can effectively inhibit the formation of the glycosylation final product, namely has the function of resisting glycosylation.

Example six: anti-aging gene detection

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent set is combined with a quantitative PCR instrument to determine the change of the anti-aging related genes in human fibroblasts after being treated by the muskmelon water extract or the muskmelon fermentation juice.

For example, the skin anti-aging CCT5 Gene (Gene ID:22948), FOXO Gene (Gene ID:2308), MRPS5 Gene (Gene ID:64969), Pink1 Gene (Gene ID:65018), Ubl-5 Gene (Gene ID: 59286).

Wherein, the CCT5 gene is a member of CCT gene family and is related to the process of restoring the mature cells to the young state; the Pink1 gene is related to the process of restoring the young state of the aged mitochondria; FOXO gene, MRPS5 gene and Ubl-5 gene are all related to the activation process of mitochondria, and the mitochondria are in a power plant in a synbiotic and are easy to cause the cell aging phenomenon if the activity of the mitochondria is insufficient.

Therefore, in the sixth example, CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene and Ubl-5 gene were used as the anti-aging gene analysis targets.

Materials and instruments

Cell line (hereinafter referred to as cell): human fibroblasts (CCD-996SK, from ATCC).

Culture medium: minimum Essential Medium (MEM) (Gibco; model: 11095080) was added with 10% Fetal Bovine Serum (FBS) (Gibco; model: 10437-.

RNA extraction reagent set (from Geneaid corporation, Taiwan, type:. FC24015-G)

Reverse transcriptase (A)

III Reverse Transcriptase) (Invitrogen, USA, No. 18080-.

Target gene primers were measured, including the FOXO gene, MRPS5 gene, Pink1 gene, Ubl-5 gene, and internal control group (GAPDH gene).

KAPA

FAST qPCR reagent set (purchased from Sigma, usa, No. 38220000000).

ABI StepOnePlus^TMReal-time PCR System (ABI StepOnePlus)^TMReal-Time PCR system (Thermo Fisher Scientific, USA)).

The muskmelon and eggplant fermentation juice: the melon eggplant fermentation broth used in this experiment was obtained as described in example one above.

First, human fibroblasts (simply referred to as cells in this example) were added at 1X 10 per well⁵The individual cells were cultured in a six-well plate containing 2mL of the above-mentioned culture solution and cultured at 37 ℃ for 16 hours, and then the cells were divided into the following three groups: experimental group a, experimental group B and blank group.

Test conditions

In detail, in the experimental group a, 2ml of the medium containing 2.0% of the extract of solanum melongena prepared in the above example was cultured for 24 hours. Experimental group B human fibroblasts were cultured in 2ml of 2.0% strength medium of the fermented juice of Cissus angustifolia prepared as described in the above example for 24 hours.

The blank group was used as experimental blank group by culturing human fibroblasts with 2ml of culture medium without any additional components and for 24 hours.

The above experimental group a and experimental group B and blank groups were performed in quadruplicates per group.

The treated human fibroblasts (i.e., experimental group a and experimental group B) and the blank human fibroblasts were disrupted with cell lysates to form three cell solutions. Next, RNA in the three cell solutions was extracted using an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Then, 1000 nanograms (ng) of extracted RNA was taken as template for each group, and the RNA was permeated

III reverse transcriptase (from Invitrogene, USA, No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription pol) was performed on six sets of cDNAs by using ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA)), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000) and primers of Table 1 (SEQ ID NO:1 to SEQ ID NO:12)ymerase chain reaction) to observe expression amounts of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene, UBL5 gene in three groups of human fibroblasts. The quantitative real-time reverse transcription polymerase chain reaction instrument sets the conditions of reaction at 95 ℃ for 20 seconds, then reaction at 95 ℃ for 3 seconds, reaction at 60 ℃ for 30 seconds, and repeats 40 cycles, and performs gene quantification using the 2-delta Ct method. In this case, the expression levels of mRNAs of the CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene and UBL5 gene can be indirectly quantified by quantitative real-time reverse transcription polymerase chain reaction using cDNA, and the expression levels of proteins encoded by the CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene and UBL5 gene can be inferred.

Watch 1

It should be noted that the relative gene expression of each gene shown in the figures mentioned below is presented in relative magnification, wherein the standard deviation is calculated using the STDEV formula of Excel software, and whether there is a statistically significant difference is analyzed in the Excel software by single Student t test (Student t-test). In the figures, the expression "indicates a p value of less than 0.05, the expression" indicates a p value of less than 0.01, and the expression "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.

Please refer to fig. 4. When the expression level of the CCT5 gene in the blank group is 1 (i.e., 100%), the expression level of the CCT5 gene in the experiment group a relative to the CCT5 gene in the blank group is 0.22 (i.e., 22%) and the expression level of the CCT5 gene in the experiment group B relative to the CCT5 gene in the blank group is 1.46 (i.e., 146%), which means that the expression level of the CCT5 gene is increased to 1.46 times that in the blank group after the cells are treated by the solanum melongena fermentation juice.

Please refer to fig. 4. When the expression amount of the FOXO gene in the blank group was regarded as 1 (i.e., 100%), the expression amount of the FOXO gene in the experimental group a was 0.25 (i.e., 25%) relative to the FOXO gene in the blank group, and the expression amount of the FOXO gene in the experimental group B was 1.58 (i.e., 158%) relative to the FOXO gene in the blank group, which represents that the expression amount of the FOXO gene was increased to 1.58 times that in the blank group after the cells were treated with the fermented juice of solanum melongena.

Please refer to fig. 4. When the expression level of the MRPS5 gene in the blank group is 1 (i.e., 100%), the expression level of the MRPS5 gene in the test group A relative to the blank group is 0.17 (i.e., 17%), and the expression level of the MRPS5 gene in the test group B relative to the blank group is 1.12 (i.e., 112%), which represents that the expression level of the MRPS5 gene is increased to 1.12 times that of the blank group after the cells are treated with the fermented juice of Solanum melongena.

Please refer to fig. 4. When the expression level of the Pink1 gene in the blank group is 1 (i.e. 100%), the expression level of the Pink1 gene in the experimental group a is 0.30 (i.e. 30%) relative to the blank group, and the expression level of the Pink1 gene in the experimental group B is 1.35 (i.e. 135%) relative to the blank group, which represents that the expression level of the Pink1 gene is increased to 1.35 times of the blank group after the cells are treated with the solanum melongena fermentation juice.

Please refer to fig. 4. When the expression level of the UBL5 gene in the blank group was 1 (i.e., 100%), the expression level of the UBL5 gene in the test group a was 0.25 (i.e., 25%) and the expression level of the UBL5 gene in the test group B was 1.48 (i.e., 148%) in the test group B, which represent that the expression level of the UBL5 gene in the test group was increased to 1.48 times that in the blank group after the cells were treated with the solanum melongena fermentation broth.

Therefore, after the human fibroblast is treated by the fermented juice of the solanum melongena, the expression quantity of various genes related to the anti-aging and rejuvenation in the cell is improved, the cell is returned to the state before non-aging from the aging state, and the aging of the cell is effectively delayed.

Example seven: melanin content detection

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

First, at 1.5X10 per hole⁵Cell 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 plate containing 3mL of cell culture medium and cultured at 37 ℃ for 24 hours.

After 24 hours of culture, B16F10 cells were divided into 3 groups: one experimental group (i.e. experimental group (solanum melongena fermented juice group)), one control group, and one blank 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 experimental culture medium of the experimental group was a cell culture medium containing 0.03125 vol% of the fermented juice of solanum melongena obtained in example one. The control group was cultured in cell culture medium containing 0.03125 vol% kojic acid (kojic acid) (the blank group was cultured in cell culture medium without fermented juice of Solanum melongena and kojic acid).

After 48 hours of incubation, the experimental medium in each well was removed and rinsed twice with 1-fold (1 ×) phosphate buffer (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 repeating the cell pellet resuspension through 1xPBS and centrifugation twice, the 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 min, the supernatant was removed from each centrifuge tube and 120. mu.L of 1N NaOH (in ddH) was added₂O) to mix with the sediment in each centrifuge tube. After mixing well, each centrifuge tube containing the mixed solution was allowed 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 applied to ELISA (enzyme-linked immunosorbent assay) at a wavelength of 450nmAttachment method) reader (brand: BioTek) to read absorbance (OD450) of each well in a 96 well culture plate.

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 blank group as 1 (i.e., the relative melanin content of the blank group is 100%). Also, statistically significant differences among groups were statistically analyzed by student t-test (student t-test), as shown in FIG. 5. In fig. 5, the p value is less than 0.05 when compared with the blank group, the p value is less than 0.01 when compared with the blank group, and the p value is less than 0.001 when compared with the blank group.

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

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

Referring to fig. 5, the melanin content of the experimental group was significantly reduced compared to the blank group, and it decreased melanin production by 24.71%; the control group also had a significantly reduced melanin content, which reduced melanin production by 14.45%, and kojic acid was a known whitening ingredient, and thus it was found that the solanum melongena fermented juice was effective in inhibiting melanin production, i.e., tyrosinase formation, and had whitening effect.

Example eight: moisture-retaining gene detection

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent group is matched with a quantitative PCR instrument to determine the change of the moisture-retention related genes in the human primary skin keratinocytes after the human primary skin keratinocytes are treated by the water extract or fermented juice of the muskmelon eggplant.

For example, the moisturizing genes are Krt1 Gene (Gene ID:3848) and HAS2 Gene (Gene ID:3037)

Wherein, the Krt1 gene is related to keratin, which is a main structure of skin and has the water-locking effect of reducing water evaporation when being closely arranged, so that the Krt1 gene can be related to skin moisturizing when being promoted; (ii) a HAS2 gene is hyaluronic acid synthetase related gene, is related to hyaluronic acid synthesis, HAS2 gene promotion is related to hyaluronic acid content, and therefore skin moisture and elasticity can be affected.

Therefore, in example eight, Krt1 gene and HAS2 gene were used as targets for analysis.

Materials and instruments

Cell lines: cell lines: human primary skin keratinocytes (Human primary epidermal keratinocytes) (CELLnTEC, Switzerland, HPEK-50).

Culture medium: Keratinocyte-SFM (1X) (Thermo).

RNA extraction reagent set (from Geneaid corporation, Taiwan, Lot No. FC24015-G)

Reverse transcriptase (A)

III Reverse Transcriptase) (Invitrogen corporation, USA, No. 18080-

Target gene primers were measured, including the Krt1 gene and the HAS2 gene, and an internal control group (TBP gene).

KAPA

FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

ABI StepOnePlus^TMReal-Time PCR System (ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA)).

The muskmelon and eggplant fermentation juice: the melon eggplant fermentation broth used in this experiment was obtained as described above in example 1.

Liquid extract of muskmelon and eggplant: the melon eggplant water extract used in this experiment was obtained by the same method as that of example 2.

Experimental procedure

First, take 1.5x10⁵Human primary skin keratinocytes were cultured in a medium containing 2ml of the above culture per wellAfter culturing for 24 hours at 37 ℃ in a six-well cell culture plate of medium, human primary skin keratinocytes cultured in each well were treated in a blank group and an experimental group (two groups in total) according to the following test conditions.

Test conditions

Specifically, the blank group was used as experimental blank group by culturing human primary skin keratinocytes in 2ml of culture medium without adding any additional components for 6 hours.

Experimental group human primary skin keratinocytes were cultured for 6 hours in 2ml of a medium containing 1% of the fermented juice of solanum melongena prepared as in example 1 above.

The above blank and experimental groups were performed in quadruplicates per group.

The treated human primary skin keratinocytes (i.e., blank and experimental groups) were disrupted with cell lysates to form two cell solutions. Next, RNA in four groups of cell solutions was extracted using an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Then, 1000 nanograms (ng) of extracted RNA was taken as template for each group, and the RNA was permeated

III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Then use ABI StepOnePlus^TMReal-time PCR System (ABI StepOnePlus)^TMTwo sets of cDNAs were subjected to quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) on Real-Time PCR system (Thermo Fisher Scientific Co., U.S.), KAPA SYBR FAST (available from Sigma Co., U.S. Pat. No. 38220000000) and primers of Table 1 (SEQ ID NO:13 and SEQ ID NO:18) to observe two sets of human primary skinExpression levels of Krt1 gene and HAS2 gene in keratinocytes. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to react at 95 ℃ for 20 seconds, followed by 95 ℃ for 3 seconds, 60 ℃ for 30 seconds, and repeated for 40 cycles, and gene quantification was performed using the 2- Δ Ct method. In this case, the quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression levels of the Krt1 gene and HAS2 gene, and further infer the expression levels of the proteins encoded by the Krt1 gene and HAS2 gene.

TABLE 2

R is REVERSE, F is FORWARD.

It should be noted that the relative gene expression systems of the Krt1 gene and the HAS2 gene shown in the figures mentioned below are presented in relative magnification, wherein the standard deviation is calculated by using the STDEV formula of Excel software, and whether the statistically significant difference is found in the Excel software by single Student t-test (Student t-test) analysis. In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.

Referring to fig. 6, when the expression levels of HAS2 gene and KRT1 gene in the blank group are regarded as 1 (i.e., 100%), the expression level of HAS2 gene in the experimental group relative to the blank group is 1.43 (i.e., 143%) and the expression level of KRT1 gene is 2.27 (i.e., 227%), which means that the expression level of HAS2 gene in the experimental group is 1.43 times that in the blank group and the expression level of KRT1 gene is 2.27 times that in the blank group.

In other words, the expression level of HAS2 gene was 43% and the expression level of KRT1 gene was 127% higher in the experimental group compared to the blank group.

As shown in fig. 6, after the human primary skin keratinocytes were treated with the fermented juice of solanum melongena, the expression levels of HAS2 gene and KRT1 gene of the human primary skin keratinocytes were increased, which indicates that the fermented juice of solanum melongena HAS the ability to regulate keratin and hyaluronic acid contents and HAS the potential to improve the skin moisturizing ability.

Example nine: fat accumulation detection

Here, the preadipocyte expansion Medium (pre-adipocyte expansion Medium) used was Minimum Essential Medium α (MEM α, brand: Gibco) supplemented with 20 vol% FBS (brand: Gibco) and 1 vol% penicillin-streptomycin. The differentiation medium (differentiation medium) used was MEM α (brand: Gibco) supplemented with 20 vol% FBS (brand: Gibco) and 1 vol% 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. In order to obtain an oil-red O working solution (oil-red O working solution) which can be used, a stock solution of the oil-red O dyeing reagent is diluted to the concentration of 1.8mg/mL by secondary water (ddH2O) in real time before use, namely the stock solution of the 60% oil-red O dyeing reagent.

First, at 8 × 10 per hole⁴Cell number of individual cells, mouse bone marrow stromal cell strain OP9 (purchased from ATCC, accession number CRL-2749) was inoculated into each well of a 24-well culture plate containing 500. mu.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 changed 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, experimental group a (aqueous extract of c. melo-shaped eggplant) and experimental group B (fermented c. melo-shaped eggplant juice), and a blank group. The differentiation medium of each group was removed and replaced with 500. mu.L of the experimental medium per well, and then placed at 37 ℃ for a further 7 days. During the 7 day culture period, 500 μ L of fresh experimental medium was replaced every 3 days. Wherein the experimental medium of experimental group A is a differentiation medium containing 0.0625 vol% of the extract of Eisenia hanthoides obtained in example one. The experimental medium of experimental group B was a differentiation medium containing 0.0625 vol% of the fermented juice of solanum melongena obtained in example one. The blank experimental medium was a simple differentiation medium (i.e. containing neither the aqueous extract nor the fermented juice of c.

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) to react 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 (OD510) 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 assuming that the fat drop of the blank group was 1 (i.e., the relative fat drop of the blank 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 blank group.

Relative fatty oil drop (%) - (OD510 sample/OD510 control). times.100% (3)

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

Referring to fig. 4, the relative fat oil droplet amount of the experimental group a was significantly reduced compared to the blank group, and it was possible to reduce the oil droplet amount by 11.72%. The relative fatty oil droplet size was significantly reduced in experimental group B compared to the blank group, and it was reduced by 32.29% of the oil droplet size. Therefore, the muskmelon eggplant fermented juice can effectively inhibit fat accumulation and has the efficacy of reducing fat. Moreover, the muskmelon eggplant can produce more fat-reducing active ingredients after being fermented by the microorganisms than the muskmelon eggplant culture solution.

Example ten: test for efficacy in reducing fat

Fat reduction refers to the decomposition of fat, and Lipolysis (Lipolysis) refers to the process in which Triglyceride (TG) stored in fat cells is gradually degraded into Fatty Acid (FA) and Glycerol (Glycerol). Based on this, the present experiment analyzed the content of Glycerol (Glycerol) in adipocytes as a quantitative index to see if lipolysis was produced.

This experiment used mouse bone marrow stromal cells (abbreviated as OP9 cells hereinafter), purchased from the American Type Culture Collection (American Type Culture Collection,

) OP9 cell line (ATCC CRL-2749).

First, a 24-well plate was inoculated with 8X 10 cells per well⁴OP9 cells were cultured in 500. mu.L of Medium (Medium) containing 80% MEMAM (Minimum Essential Medium Alpha Medium, ex Gibco, USA) cell culture, 20% Fetal Bovine Serum (Fetal Bovine Serum, ex Gibco, USA, Cat # 10437-. The culture medium was changed every 3 days during the 7-day cell culture period. After 7 days, intracellular oil droplet formation was observed by a microscope (ZEISS; magnification 400X), whereby it was confirmed that the cells had completely differentiated into adipocytes.

Then, the differentiated adipocytes were divided into two groups: two experimental groups, experimental group a (with the addition of the aqueous extract of solanum melongena prepared in example two) and experimental group B (with the addition of the fermented juice of solanum melongena prepared in example one), and a blank group.

Experimental group a: the water extract of the melon eggplant was added to the medium containing the differentiated cells at a rate of 0.156. mu.L of the water extract of the melon eggplant (i.e., at a concentration of 0.03125%) per 500. mu.L of the medium per well, and cultured at 37 ℃ for 7 days. The medium was changed every 3 days during 7 days of cell processing.

Experimental group B: the fermented juice of the melon eggplant was added to the medium containing the differentiated medium at a rate of 0.156. mu.L of the fermented juice of the melon eggplant (i.e., at a concentration of 0.03125%) per 500. mu.L of the medium per well, and cultured at 37 ℃ for 7 days. The medium was changed every 3 days during 7 days of cell processing.

Blank group: no treatment, i.e., no additional addition of other components, was performed on the differentiated medium, and the medium was cultured at 37 ℃ for 7 days. The medium was changed every 3 days during 7 days of cell processing.

After 7 days of cell treatment, Glycerol content was measured using a cell Glycerol-based assay kit (available from Cayman, usa under product number 10011725) according to the following procedure. The supernatants from each group were collected and 25. mu.L of each was transferred to a new 96-well plate and 100. mu.L of Reconstituted free glycerol assay reagent (Reconstituted free glycerol assay reagent) was added to each well and after 15 minutes at room temperature, the plates were read for OD in each group using an ELISA reader_540nmTo quantify the amount of glycerol decomposed and released into the cell culture broth by each group of adipocytes, as shown in fig. 6. In this case, the amount of glycerol is proportional to the amount of fat decomposition. In this, the determination of whether statistically significant differences between the two sample populations were obtained by subjecting the test t-test to Excel software (in the figure, "x" represents a p value of less than 0.05, p value of less than 0.01, and "x" represents a p value of less than 0.001. when "x" is more, the statistical differences are more significant).

Refer to fig. 8. Considering the fat decomposition amount of the blank group as 100%, the fat decomposition amount of the experimental group a was 96.07%, and the fat decomposition amount of the experimental group B was 104.85%, that is, the fat decomposition amount of the melon and eggplant fermentation liquor was significantly increased by 4.85% after the treatment. The results show that the muskmelon eggplant fermentation juice can directly and effectively promote the decomposition of fat in fat cells so as to reduce the content of fat in the fat cells and achieve the effect of burning fat and losing weight.

EXAMPLE eleven-cell experiment of fat metabolism Gene

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent set is combined with a quantitative PCR instrument to determine the change of fat metabolism gene genes in the cells of the mouse bone marrow matrix cells after the treatment of the muskmelon water extract or the muskmelon fermentation juice.

For example, the fat metabolism Gene is UCP2 Gene (Gene ID: 7351).

Among them, the Protein encoded by UCP2 (unclipping Protein 2) gene is Uncoupling Protein (UCP), which is one of the members of the Mitochondrial Anion Carrier Protein (MACP) family, and mainly functions to reduce Adenosine Triphosphate (ATP), promote outward transfer of anions from the inner membrane of mitochondria, promote return transfer of protons from the outside to the inner membrane of mitochondria, and release the energy generated in the process as heat energy; the UCP2 gene is expressed in many tissues, and is most highly expressed in skeletal muscle, and is considered to be involved in non-shivering fever (nonsshivering fever). Therefore, the expression level of UCP2 gene is increased, and the decomposition of fat is promoted, so that the accumulation of fat is reduced.

Therefore, in example eleven, the UCP2 gene was used as an analysis target.

Materials and instruments

Cell lines: mouse bone marrow stromal cells OP9 (BCRC; accession number CRL-2749).

Culture medium: contains 80% of MEMAM (Minimum Essential Medium Alpha Medium, available from Gibco, USA) cell culture fluid, 20% of Fetal Bovine Serum (Total Bovine Serum, available from Gibco, USA, Cat # 10437-.

Reverse transcriptase (A)

III Reverse Transcriptase) (Invitrogen corporation, USA, No. 18080-

Target gene primers were measured, including the UCP2 gene, and including an internal blank (m-ACTB gene).

KAPA

FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

Experimental procedure

First, take 1.5x10⁵Mouse bone marrow stromal cells OP9 were cultured in six-well cell culture plates containing 2ml of the above-mentioned medium per well at 37 ℃ for 24 hours, and the cultured mouse bone marrow stromal cells OP9 were treated in each well in a blank group and an experimental group (three groups in total) according to the following test conditions.

Test conditions

Specifically, the blank group was used as an experimental blank group by simply culturing the bone marrow stromal cells of the mouse in 2ml of culture medium without adding other additives for 6 hours.

Experimental group A was prepared by culturing mouse bone marrow stromal cells in 2ml of the 0.0625% strength culture medium of the E.melo aqueous extract prepared in the above example for 6 hours.

Experimental group B was prepared by culturing mouse bone marrow stromal cells in 2ml of the culture medium containing 0.0625% of the fermented juice of Cissus angustifolia prepared in the above example for 6 hours.

The blank, experimental group a, experimental group B, each group was repeated four times.

The treated bone marrow stromal cells of the mice (i.e. blank group, experimental group A, experimental group B) were disrupted with cell lysate to form three groups of cell solutions. Next, RNA in the three cell solutions was extracted using an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Then, 1000 nanograms (ng) of extracted RNA was taken as template for each group, and the RNA was permeated

III reverse transcriptase (from Invitrogene, USA, No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) was then performed on the cDNAs of the three groups by the ABI StepOnePlusTM Real-Time PCR system (ABI StepOnePlusTM Real-Time PCR system, Thermo Fisher Scientific Co., U.S.), KAPA SYBR FAST (purchased from Sigma Co., U.S. No. 38220000000), and primers of Table 1 (SEQ ID NO:19 to SEQ ID NO:22) to observe the expression of UCP2 gene in bone marrow stromal cells of the mice of the three groups. The quantitative real-time reverse transcription polymerase chain reaction apparatus was set to react at 95 ℃ for 20 seconds, then at 95 ℃ for 3 seconds, at 60 ℃ for 30 seconds, and repeated for 40 cycles, and gene quantification was performed using the 2- Δ Ct method. Here, m of UCP2 gene can be indirectly quantified by quantitative real-time RT-PCR using cDNAThe expression level of RNA and the expression level of protein encoded by UCP2 gene were further estimated.

TABLE 1

R is REVERSE, F is FORWARD.

It should be noted that the relative gene expression of UCP2 shown in the figures described below is presented in relative magnification, wherein the standard deviation is calculated using the STDEV formula of Excel software, and analyzed in the Excel software by single Student t test (Student t-test) to determine whether there is a statistically significant difference. In the figures, the expression "indicates a p value of less than 0.05, the expression" indicates a p value of less than 0.01, and the expression "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.

Referring to fig. 9, when the expression level of UCP2 gene in blank group is 1 (i.e., 100%), the expression level of UCP2 gene in experimental group a is 1.26 (i.e., 126%) relative to blank group, the expression level of UCP2 gene in experimental group B is 4.75 (i.e., 475%) relative to blank group, which means that the expression level of UCP2 gene in experimental group a is 1.26 times that in blank group, and the expression level of UCP2 gene in experimental group B is 4.75 times that in blank group.

In other words, the expression level of UCP2 gene in the test group a was increased by 26% compared to the blank group, and the expression level of UCP2 gene in the test group B was increased by 375% compared to the blank group.

From this, it was found that the expression level of UCP2 in mouse bone marrow stromal cells was increased when the mouse bone marrow stromal cells were treated with the fermented juice containing solanum melongena.

As shown in fig. 9, the fermented juice of solanum melongena according to the present invention can increase the gene expression level of UCP2, and thus it is effective in promoting the decomposition of fat and reducing the accumulation of fat.

Example twelve: 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 50mL bottle of the fermented Cissus angustifolia beverage (containing 12 vol% of the fermented Cissus angustifolia juice obtained in example 1 and 88 vol% of water) daily for 4 weeks. Before drinking (i.e. week 0) and after drinking for 4 weeks (i.e. week 4), the body weights of the subjects were measured by a weighing machine, the body fat rates of the subjects were measured by a body fat meter (brand: TANITA BC-601FS), and the waist circumferences of the subjects were 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. 10-13. In fig. 10 to 13, "" indicates that the p-value thereof is less than 0.05 in comparison with week 0; and "", represents that the p value is less than 0.01 when compared with the blank group.

Referring to fig. 10, drinking the fermented solanum melongena beverage for 4 weeks significantly reduced body weight by about 1.0 kg compared to that before drinking (week 0).

Referring to fig. 11, drinking the fermented solanum melongena beverage for 4 weeks significantly reduced the whole body fat rate by about 1.0% compared to before drinking (week 0).

Referring to fig. 12, drinking the fermented solanum melongena beverage for 4 weeks significantly reduced the body fat percentage of the trunk by about 1.2% compared to before drinking (week 0).

Referring to fig. 13, drinking the fermented solanum melongena beverage for 4 weeks resulted in a significant reduction of the waist circumference by about 1.1 cm compared to before drinking (week 0).

Therefore, the long-term use of the muskmelon and eggplant fermentation juice can improve fat accumulation and weight of obese people, namely the muskmelon and eggplant fermentation juice has the effects of slimming and reducing fat.

Example thirteen: antioxidant human detection

8 obese subjects (i.e., subjects with body fat percentage greater than 25% or BMI greater than 24) were allowed to drink a 50mL bottle of the fermented Cissus angustifolia beverage (containing 12 vol% of the fermented Cissus angustifolia juice obtained in example 1 and 88 vol% of water) daily for 4 weeks. Blood was collected before drinking (i.e., week 0) and after drinking for 4 weeks (i.e., week 4), and Total Anti-oxidative Capacity (Total Anti-oxidative Capacity) and a peroxidation product (MDA) in the blood were examined by a clinical laboratory (taiwan).

In general, the sum of the oxidation resistance, abbreviated as TAC, represents the capacity to reduce oxides in vivo. Many studies have shown that free radicals cause some pathologies in the human body, such as: cardiovascular diseases and various inflammatory diseases, and the like, and antioxidants in vivo have the function of protecting cells and can prevent the attack of free radicals, so that the unbalanced state of the body can be monitored by detecting the state of the antioxidant capacity in vivo, and the higher the total antioxidant capacity is, the better the antioxidant capacity in vivo is; peroxidation products (MDA), which represent products of lipid peroxidation in vivo, are widely accepted as one of the targets for the determination of oxidative damage, the higher the peroxidation product (MDA), the higher the oxidative stress in vivo.

After the measurement, the relative antioxidant correlation (%) at week 4 was calculated using the value at week 0 as a reference (i.e., the antioxidant correlation at week 0 was 100%). 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. 14 and 15. In fig. 14 and 15, "") indicates that the p-value was less than 0.05 when compared with week 0; and "", represents that the p value is less than 0.001 when compared with the blank group.

Referring to fig. 14, drinking the fermented solanum melongena beverage for 4 weeks resulted in a significant increase in total antioxidant capacity of about 11.0% compared to before drinking (week 0); wherein the baseline value of the total antioxidant capacity at week 0 is 0.74nmol/L and the baseline value of the total antioxidant capacity at week 4 is 0.83 nmol/L.

Referring to fig. 15, drinking the solanum melongena fermented beverage for 4 weeks resulted in a significant reduction of the over oxidation products (MDA) by about 26.1% compared to before drinking (week 0); wherein the MDA prime value at week 0 is 1.1nmol/ml and the MDA prime value at week 4 is 0.9 nmol/ml.

Example fourteen: human detection to improve skin condition

8 obese subjects (i.e., subjects with body fat percentage greater than 25% or BMI greater than 24) were allowed to drink a 50mL bottle of the fermented Cissus angustifolia beverage (containing 12 vol% of the fermented Cissus angustifolia juice obtained in example 1 and 88 vol% of water) daily for 4 weeks. Before drinking (i.e., week 0) and after drinking for 4 weeks (i.e., week 4), the skin condition was measured by a VISIA skin measuring instrument (VISIA complex Analysis System, available from Canfield, usa) and a skin surface moisture measuring instrument (Corneometer CM825, available from C + K, germany). Here, the VISIA skin detector was used to measure skin texture, skin wrinkles, and skin redness (red pigment), and the skin surface humidity tester was used to measure skin moisture 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 4 was calculated. Also, the statistically significant difference between the relative skin condition at week 0 and the relative skin condition at week 4 was statistically analyzed by student t-test, as shown in fig. 16 to 19. In fig. 16 to 19, "", indicates that the p-value was less than 0.05 in comparison with week 0.

Referring to fig. 16 to 19, drinking the cantaloupe fermented beverage for 4 weeks significantly reduced skin texture by about 4.3%, skin wrinkles by about 5.3%, skin red pigment by about 7.6%, and skin moisture content by about 9.6% compared to before drinking (week 0). Therefore, the long-term use of the muskmelon and eggplant fermentation juice can improve the skin condition, namely the muskmelon and eggplant fermentation juice has the effect of improving the skin condition.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and modifications as fall within the true spirit and scope of the appended claims be embraced by the invention.

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Claims

1. The application of the muskmelon and eggplant fermentation juice is characterized in that the muskmelon and eggplant fermentation juice is obtained by performing a fermentation procedure on a muskmelon culture solution and a plurality of strains, wherein the muskmelon and eggplant culture solution comprises the muskmelon and eggplant juice and water, the ratio of the muskmelon and eggplant juice to the water is 1:2-5, and the plurality of strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria relative to the muskmelon and eggplant culture solution.

2. The use of claim 1, wherein the improvement in skin condition is an increase in skin anti-aging, an increase in anti-glycation, a decrease in melanin content, an increase in skin moisturization, a decrease in skin texture, a decrease in skin wrinkles, a decrease in skin redness, or a combination thereof.

3. The use according to claim 2, wherein the improvement of skin anti-aging ability is the regulation of at least one of CCT5 gene, FOXO gene, MRPS5 gene, Pink1 gene or UBL5 gene.

4. The use of claim 2, wherein the skin moisturization is modulated by at least one of the HAS2 gene or KRT1 gene.

5. The application of the muskmelon and eggplant fermentation juice is characterized in that the muskmelon and eggplant fermentation juice is used for preparing a composition for improving the antioxidant capacity, wherein the muskmelon and eggplant fermentation juice is obtained by performing a fermentation procedure on a muskmelon and eggplant culture solution and a plurality of strains, the muskmelon and eggplant culture solution comprises the muskmelon and eggplant juice and water, the ratio of the muskmelon and the melon and eggplant juice to the water is 1:2-5, and the plurality of strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria relative to the muskmelon and eggplant culture solution.

6. The use according to claim 5, wherein the increased antioxidant capacity is increased total antioxidant capacity, decreased peroxidation product MDA, increased glutathione or a combination thereof.

7. The application of the muskmelon and eggplant fermentation juice is characterized in that the muskmelon and eggplant fermentation juice is used for preparing a composition for reducing fat, wherein the muskmelon and eggplant fermentation juice is obtained by performing a fermentation process on a muskmelon and eggplant culture solution and a plurality of strains, the muskmelon and eggplant culture solution comprises the muskmelon and eggplant juice and water in a ratio of 1:2-5, and the plurality of strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 1-15% of acetic acid bacteria relative to the muskmelon and eggplant culture solution.

8. The use according to claim 7, wherein the fat reduction is inhibition of fat accumulation, promotion of fat breakdown, or a combination thereof.

9. Use according to any one of claims 1 to 8, characterized in that the fermentation procedure comprises:

adding the yeast into the muskmelon eggplant culture solution;

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

adding the lactic acid bacteria to the first primary fermented juice;

fermenting the first primary fermented juice with the lactic acid bacteria for 1 day to 5 days to form a second primary fermented juice, wherein the lactic acid bacteria is streptococcus thermophilus;

adding the acetic acid bacteria into the second primary fermented juice;

fermenting the second primary fermented juice and the acetic acid bacteria for 3 to 10 days to form a third primary fermented juice, wherein the acetic acid bacteria is acetic acid bacteria; and

filtering the third primary fermented juice to obtain the fermented juice of Eschka.

10. Use according to any one of claims 1 to 8, characterized in that the pH of the melon eggplant fermented juice is 4.0 ± 1.0 and the sugar degree of the melon eggplant fermented juice is 40 ± 2.

11. Use according to any one of claims 1 to 8, characterized in that the melon eggplant fermented juice has a polyphenol content of 88 ppm.