CN114099586B - Application of muskmelon eggplant fermentation juice - Google Patents

Abstract

The invention discloses an application of muskmelon eggplant fermentation juice, which is used for preparing a composition. The composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting formation of glycation end products, enhancing anti-aging related genes, inhibiting melanin production, enhancing secretion of moisture related genes, inhibiting fat accumulation, promoting lipolysis, reducing body fat of the subject, and improving skin conditions. The muskmelon eggplant fermentation juice is obtained by a fermentation procedure of a muskmelon eggplant culture solution and a plurality of strains.

Description

Application of muskmelon eggplant fermentation juice

Technical Field

The present invention relates to a muskmelon eggplant fermentation juice, in particular to the use of a muskmelon eggplant fermentation juice for the preparation of a skin condition improving and/or lipid reducing composition.

Background

After the rise of organic and natural diet concepts, the biotechnology companies and food operators are actively involved in the development of related products concerning natural plants. In order to enable the plant related products to have a scientific verification basis for helping physical health, active ingredient analysis and efficacy evaluation of plants become key projects for product development.

Muskmelon eggplant (Pepino Dulce, academic name Solanum muricatum), also known as ginseng fruit, is a perennial herb of the genus solanum of the family solanaceae. Muskmelon solanum torvum is produced in south america, and has become one of the natural plants under research and development in recent years.

Disclosure of Invention

In one embodiment, the use of a muskmelon tomato fermentation juice for preparing a composition for enhancing antioxidant capacity, wherein the muskmelon tomato fermentation juice is obtained by performing a fermentation process on a muskmelon tomato culture solution and a plurality of strains, the muskmelon tomato culture solution comprises muskmelon tomato juice and water, and the ratio of the muskmelon tomato juice to the water is 1:2-5. The multiple strains comprise 0.01% -0.5% of saccharomycete, 0.01% -0.25% of lactobacillus and 1% -15% of acetic acid bacteria relative to the muskmelon eggplant culture solution.

In one embodiment, the use of a muskmelon eggplant fermentation juice for the preparation of a composition for improving skin conditions. Wherein the muskmelon eggplant fermentation juice is obtained by a fermentation procedure of a muskmelon eggplant culture solution and a plurality of strains. The muskmelon eggplant culture solution comprises muskmelon eggplant juice and water, wherein the ratio of the muskmelon eggplant to the water is 1:2-5, and the strains comprise 0.01% -0.5% of saccharomycetes, 0.01% -0.25% of lactic acid bacteria and 1% -15% of acetic acid bacteria relative to the muskmelon eggplant culture solution.

In one embodiment, the use of a muskmelon eggplant fermentation juice for the preparation of a composition for reducing fat. Wherein the muskmelon eggplant fermentation juice is obtained by a fermentation procedure of 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. The multiple strains comprise 0.01% -0.5% of saccharomycete, 0.01% -0.25% of lactobacillus and 1% -15% of acetic acid bacteria relative to the muskmelon eggplant culture solution.

In yet another embodiment, the cantaloupe tomato fermented juice has increased antioxidant capacity to increase Total antioxidant capacity (Total Anti-oxidative Capacity), decreased peroxidation products MDA (Malondialdehyde), increased glutathione or a combination thereof.

In yet another embodiment, the muskmelon eggplant fermentation juice improves skin condition by enhancing skin anti-aging ability, enhancing anti-glycation ability, reducing melanin content, enhancing skin moisturization, reducing skin texture, reducing skin wrinkles, reducing skin redness, or a combination thereof.

In yet another embodiment, the muskmelon eggplant fermentation juice enhances skin anti-aging ability to regulate CCT5 gene, FOXO gene, MRPS5 gene, pink1 gene, or UBL5 gene.

In yet another embodiment, the muskmelon eggplant fermentation juice promotes skin moisturization to regulate the HAS2 gene or the KRT1 gene.

In yet another embodiment, the cantaloupe fermented juice reduces fat to inhibit fat accumulation, promote fat breakdown, or a combination thereof.

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

In yet another embodiment, the cantaloupe fermented juice has a pH of 4.0±1.0 and the cantaloupe fermented juice has a sugar degree of 40±2.

In yet another embodiment, the cantaloupe fermented juice has a polyphenol content of 88ppm.

In view of the above, the muskmelon eggplant fermentation juice according to any of the embodiments of the present invention may prepare a composition for improving skin conditions and/or reducing fat. In other words, the aforementioned composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting formation of glycation end products, enhancing anti-aging related genes, inhibiting melanin production, enhancing secretion of moisture related genes, inhibiting fat accumulation, promoting lipolysis, reducing body fat of the subject, and improving skin conditions.

The following examples are set forth to illustrate the present invention and are not intended to limit the scope of the invention, as many variations and modifications are possible without departing from the spirit and scope of the invention as defined in the appended claims.

Drawings

FIG. 1 is a histogram showing the change in total polyphenol content of a cantaloupe culture broth (before fermentation) and a cantaloupe fermentation juice (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 glycosylated end products formed for each group.

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

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

FIG. 6 is a histogram showing the relative moisture retention gene expression levels of each group.

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

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

FIG. 9 is a histogram showing the relative lipid gene expression levels of each group.

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

FIG. 11 is a graph showing the change in body fat rate of an exemplary cantaloupe fermented juice before and after use.

FIG. 12 is a histogram showing the variation of body fat rate of the sample tomato fermented juice before and after use.

Fig. 13 is a histogram showing the variation of hip circumference of an exemplary cantaloupe fermented juice before and after use.

FIG. 14 is a graph showing the change in total antioxidant capacity of an exemplary melon eggplant fermentation juice before and after use.

Fig. 15 is a graph showing the change of the peroxide product MDA before and after use of the exemplary muskmelon eggplant fermentation juice.

Fig. 16 is a histogram showing the change in relative skin texture (roughness) of an exemplary cantaloupe fermented juice before and after use.

FIG. 17 is a histogram of changes in relative skin wrinkles before and after use of an exemplary muskmelon eggplant fermentation juice.

Fig. 18 is a histogram of the change in relative skin redness (red pigment) before and after use of an exemplary cantaloupe fermented juice.

FIG. 19 is a histogram of the change in relative skin moisture content before and after use of an exemplary muskmelon eggplant fermentation juice.

Detailed Description

The concentration designation "%" as used herein generally refers to weight percent concentration, and the concentration designation "% by volume" generally refers to volume percent concentration. As used herein, "cantaloupe" generally refers to cantaloupe fruit.

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

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

In some embodiments, the muskmelon eggplant fermentation juice is obtained by performing a fermentation process on the muskmelon eggplant culture solution and a plurality of strains. Wherein the muskmelon eggplant culture solution comprises muskmelon eggplant and water. In the muskmelon eggplant culture solution, the ratio of the muskmelon eggplant to water is 1:2-5. In the fermentation procedure, the strains comprise 0.01% -0.5% of saccharomycetes, 0.01% -0.25% of lactobacillus and 1% -15% of acetic acid bacteria relative to the muskmelon eggplant culture solution.

In some embodiments, the yeast may be Saccharomyces cerevisiae (Saccharomyces cerevisiae). In some embodiments, the lactic acid bacteria may be streptococcus thermophilus (Streptococcus thermophilus) or lactobacillus plantarum. In some embodiments, the acetic acid bacteria may be acetic acid bacteria (Acetobacter aceti).

In some embodiments, in the fermentation procedure, 0.01% -0.5% yeast is added to the cantaloupe culture broth first, and the cantaloupe culture broth is fermented with yeast for 1 day to 3 days to form a first primary fermentation broth. In other words, the first mixed solution, in which 0.01% -0.5% of yeast is mixed with the cantaloupe culture solution, is fermented for 1 to 3 days to form the first primary fermentation juice. In some embodiments, the first mixed liquor is fermented at 28 ℃ to 37 ℃.

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

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

After the third primary fermentation juice is formed, the third primary fermentation juice is filtered to obtain the muskmelon eggplant fermentation juice. In a first example, the step of filtering the third primary fermentation broth includes filtering the third primary fermentation broth with a 200 mesh to 400 mesh screen. In a second example, the filtering step of the third primary fermentation broth includes concentrating under reduced pressure at 40 ℃ to 70 ℃ and filtering the third primary fermentation broth with a 200 mesh to 400 mesh sieve. In a third example, the filtering step of the third primary fermentation broth includes concentrating under reduced pressure at 40 ℃ to 70 ℃ and filtering the third primary fermentation broth with a 200 mesh to 400 mesh sieve to obtain a fermentation broth, and adjusting the sugar degree (degreees Brix) of the fermentation broth to form the cantaloupe fermentation broth.

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

In some embodiments, the cantaloupe fermented juice has a pH of 3.7±1.0 and a sugar degree of 40±2.

In some embodiments, the cantaloupe fermented juice has a polyphenol content of 88ppm.

The muskmelon eggplant culture solution is prepared by mixing muskmelon eggplant juice and water according to a ratio of 1: 2-5. In a first example, the cantaloupe tomato culture solution is prepared from cantaloupe tomato juice from south america and water at a ratio of 1: 2-5. In a second example, the cantaloupe juice is prepared from cantaloupe juice from Peru Agro international business a & c sac company and water at a ratio of 1:2-5, and adding glucose solution to adjust sugar degree to 9. In a third example, the cantaloupe juice is prepared from cantaloupe juice from Peru Agro international business a & c sac company and water at a ratio of 1:5, and adding a glucose solution to adjust the sugar degree to 9.

In some embodiments, the sterilization procedure of the raw material mixture may be to sterilize the raw material mixture at 80 ℃ to 100 ℃ for 0.2 hours to 1 hour, and cool the sterilized raw material mixture to room temperature (i.e., 25 ℃ to 30 ℃) to form the cantaloupe culture broth. In some embodiments, the sterilized raw material mixture may be cooled to room temperature by natural cooling.

In some embodiments, the cantaloupe fermented juice has an effect of improving skin condition and/or reducing fat. In some examples, improving the skin condition of the subject may be improving the skin's anti-aging ability, improving anti-glycation ability, reducing melanin content, improving skin moisturization, reducing skin texture, reducing skin wrinkles, reducing skin redness, or a combination thereof. In some embodiments, the cantaloupe fermented juice is capable of achieving an improvement in skin condition and/or lipid reduction through one or more of the following cell-level effects: enhancing glutathione synthesis, inhibiting formation of glycation end products, enhancing anti-aging related genes, inhibiting melanin production, enhancing secretion of moisture related genes, inhibiting fat accumulation, promoting lipolysis, reducing body fat of the subject, and improving skin conditions. Wherein the recipient may be a human.

In some embodiments, the cantaloupe fermented juice can be used to prepare a composition to enhance antioxidant capacity, which may be enhancing Total antioxidant capacity (Total Anti-oxidative Capacity), lowering peroxidation products MDA (Malondialdehyde), enhancing glutathione, or a combination thereof.

In some embodiments, the muskmelon eggplant fermentation juice can be used to prepare a composition for improving a skin condition, wherein improving the skin condition can be enhancing the skin anti-aging ability, enhancing the anti-glycosylation ability, reducing the melanin content, enhancing the skin moisturization, reducing the skin texture, reducing the skin wrinkles, reducing the skin redness, or a combination thereof, wherein enhancing the skin anti-aging ability is modulating at least one of the CCT5 gene, FOXO gene, MRPS5 gene, pink1 gene, or UBL5 gene and enhancing the skin moisturization is modulating at least one of the HAS2 gene or KRT1 gene.

In some embodiments, the cantaloupe fermented juice can be used to prepare a composition to reduce fat, wherein the reduction in fat is to inhibit fat accumulation, promote fat breakdown, or a combination thereof.

In some embodiments, any of the foregoing compositions may be a pharmaceutical. In other words, the pharmaceutical product comprises an effective amount of the muskmelon eggplant fermentation juice.

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

In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, a tablet, a buccal tablet, a pill, a capsule, a dispersible powder, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, or the like. In some embodiments, the parenteral or topical dosage form may be, but is not limited to, an injectable, sterile powder, an external preparation (external preparation), or the like. In some embodiments, the administration of the injectate can be subcutaneous injection (subcutaneous injection), intradermal injection (intraepidermal injection), intradermal injection (intradermal injection), or intralesional injection (intralesional injection).

In some embodiments, the aforementioned pharmaceuticals may comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) that is widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following: solvents (solvents), buffers (buffers), emulsifiers (suspending agents), disintegrants (decomponents), disintegrants (disintegrating agent), dispersants (dispersing agents), binders (binding agents), excipients, stabilizers (stabilizing agent), chelating agents (chelating agents), diluents (solvents), gelling agents, preservatives (preservatives), wetting agents (wetting agents), lubricants (absorption retarders), absorption retarders (absorption delaying agent), liposomes (lipo-agents), and the like. The type and number of carriers selected are within the expertise and routine skill of those skilled in the art. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline (normal saline), phosphate buffer (phosphate buffered saline, PBS), or an aqueous solution containing alcohol (alcohol containing aqueous solution).

In some embodiments, any of the foregoing compositions may be edible compositions. In other words, the edible composition comprises a specific content of the muskmelon eggplant fermentation juice. In some embodiments, the foregoing 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 (beverages), fermented foods (fermented foods), baked products (bakery products), health foods (health foods), or dietary supplements (dietary supplements).

In some embodiments, the foregoing edible composition may be administered orally to the subject. Wherein the edible composition may 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 comprises a specific content of the muskmelon eggplant fermentation juice.

In some embodiments, the aforementioned cosmetic or care product may be any of the following types: lotion, gel, jelly film, mud film, emulsion, cream, lipstick, foundation, cake, honey powder, cleansing oil, cleansing milk, facial cleanser, bath lotion, shampoo, hair care cream, sun block, hand cream, nail polish, perfume, essence, and facial mask. In some embodiments, the aforementioned cosmetic or care product may optionally further comprise an external acceptable ingredient. 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.

Embodiment one: preparation of muskmelon eggplant fermentation juice

Firstly, a muskmelon tomato juice (product name Pepino Dulce, nature Pulp, where the source of the muskmelon tomato is Peruvian and the mountain area of the ground), purchased from Peruvian, agro international business a & c sac, is uniformly mixed with water in a ratio of 1:5 (i.e., the muskmelon tomato juice is twice weight, and the water is five times weight), and then a glucose solution is added to adjust the sugar degree to 9.0, thereby obtaining a muskmelon tomato culture solution. However, the source of the melon tomato juice is not limited in the present invention, and for example, melon tomato juice is provided by Peruvian Nature company or Naike company as a supplier in south America.

Next, 0.1% Saccharomyces cerevisiae (Saccharomyces cerevisiae) (purchased from the institute of food industry development, bioresource Conservation and Research Center (BCRC), accession number BCRC 20271) was inoculated into a culture broth of Katsumadai and fermented at about 30℃for 1 day to obtain a first primary fermentation broth. Next, 0.05% Streptococcus thermophilus (Streptococcus thermophilus) (available from BCRC, accession number BCRC 910636) 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 (Acetobacter aceti) (available from BCRC, accession number BCRC 11688) were inoculated into the second primary fermentation broth and fermented at about 30 ℃ for 5 days to obtain a third primary fermentation broth.

The third primary fermentation broth was concentrated under reduced pressure at about 60℃and filtered through a 200 mesh sieve to obtain a fermentation broth. Finally, 60% isomaltooligosaccharides were added to the fermentation stock solution and then sterilized at about 100 ℃ for 2 hours to obtain a cantaloupe fermented juice.

Here, sugar degree detection was performed on the muskmelon eggplant culture solution, the fermentation stock solution, and the muskmelon eggplant fermentation juice, respectively. Wherein the sugar degree of the muskmelon eggplant raw material mixed solution is about 9.0, the sugar degree of the fermentation raw solution is about 4.0, and the sugar degree of the muskmelon eggplant fermentation juice is about 40.

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

The muskmelon tomato juice purchased from Peru Agro international business a & c sac company (product name Pepino Dulce, nature Pulp, where the muskmelon solanaceous origin is Peru Andi mountain) is mixed with water uniformly in a ratio of 1:5, and glucose solution is added to adjust the sugar degree to 9, so as to obtain the muskmelon tomato water extract with the pH value of 6.3.

Embodiment III: total polyphenol content test

10.0mg of Gallic acid (Gallicic acid) was weighed into a 10mL volumetric flask and then quantified to 10mL with water (H2O) to give a stock solution of Gallic acid. The stock solution of gallic acid was diluted 10-fold, i.e., 100. Mu.L of gallic acid stock solution was added with 900. Mu.L of water to give an initial solution of gallic acid (i.e., 1000ppm of gallic acid). Next, standard solutions of gallic acid of 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 were prepared according to the following Table, and 100. Mu.L of each standard solution was taken into a glass test tube. mu.L of Folin-Ciocalteu's phenol reagent (from Merck) was added to each glass tube and mixed with the standard solution uniformly and allowed to stand for 3 minutes, and then 400. Mu.L of 7.5% sodium carbonate was added to the mixture and reacted for 30 minutes to obtain a standard reaction solution. 200. Mu.L of the standard reaction solution was taken into a 96-well plate, and its absorbance at 750nm was measured to obtain a standard curve.

List one

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

The test samples of the experimental group (i.e., the muskmelon eggplant fermentation juice of example one) and the test samples of the control group (i.e., the muskmelon eggplant water extract of example two) were diluted 10 times with water, respectively. Samples were taken in 100mL portions into glass test tubes. Then, 500. Mu.L of a Fulin phenol reagent was added to the glass tube to be mixed uniformly with the sample and allowed to stand for 3 minutes, and then 400. Mu.L of 7.5% sodium carbonate was added to be 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 oscillated to ensure no bubbles, 200. Mu.L of the reaction solution to be measured was taken into a 96-well plate, and the absorbance of the reaction solution to be measured at 750nm was measured.

Then, the absorbance value of the reaction solution to be measured corresponding to each sample is divided by the sugar degree of the sample, and then the total polyphenol content is converted by interpolation method through a standard curve. In this case, the total polyphenol content of the muskmelon eggplant water extract was 27ppm and the total polyphenol content of the muskmelon eggplant fermentation juice was 88ppm, as shown in FIG. 1. Therefore, after the muskmelon eggplant is fermented by microorganisms, the total polyphenol content can be increased by 3.2 times. Namely, compared with the muskmelon eggplant water extract, the muskmelon eggplant fermentation juice can improve the antioxidant activity.

Embodiment four: evaluation of cell antioxidant efficacy-glutathione content detection

Experimental materials

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

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

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

Melon eggplant water extract: the muskmelon solanum torvum water extract used in this experiment was obtained as described in example two above.

Muskmelon eggplant fermentation juice: the muskmelon solanum torvum water extract used in this experiment was obtained as in example one above.

Experimental procedure

1. In a 6-well culture dish, 2X10 is implanted into each well of culture medium ⁶ Human peripheral blood mononuclear cells (will be referred to as cells in the latter steps).

2. Incubated at 37℃for 24 hours.

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

4. The cells were collected.

5. The cells were washed once with Phosphate-Buffered Saline (PBS) (from Gibco).

6. Cells (resuspension cells) were resuspended in 1 ml of PBS.

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

8. The cells were washed once with PBS.

9. Cells (resuspension cells) were resuspended in 200 microliters of PBS.

10. The relative Fluorescent Isothiocyanate (FITC) signal was analyzed by flow cytometry (available from BD Accuri, model C6 Plus).

11. The detected values were analyzed for statistical significance between the values using Student t test using microsoft EXCEL software, and the results are shown in fig. 1. Wherein p <0.001 is represented by x compared to the blank.

As can be seen from the results of fig. 2, there was a second group (experimental group a) of cells added with the solanum melo water extract, and the GSH content was not increased, but was reduced by 7.41% compared to the blank group; the cells of the third group (experimental group B) added with the muskmelon eggplant fermentation juice have the GSH content which is obviously improved by 8.34 percent compared with the blank group, so that the relative GSH content (relative GSH content) is 1.08 times of the cells of the first group (blank group) and 1.17 times of the cells of the second group (experimental group). GSH is a tripeptide composed of glutamic acid, cystine, and glycine, whose thiol (-SH) is redox-related, and whose primary function is the defense against oxidative damage by intracellular nature, and can combat oxidative damage by ROS. Therefore, as can be confirmed from the results of fig. 1, the addition of the muskmelon eggplant fermentation juice obtained in the first embodiment can indeed increase the GSH content in the cells and increase the intracellular oxidation-reduction capability, so as to achieve the oxidation-resistant effect of the cells.

Fifth embodiment: anti-glycation test

With 200mM Sodium phosphate buffer (Sodium phosphate buffer, pH 7.4), sodium azide (NaN) ₃ ) With bovine serum albumin (Bovine serum albumin, BSA, brand: gibco) was configured with a 60mg/mL BSA solution containing 0.06% NaN 3.

200mM sodium phosphate buffer and D Fructose (D- (-) -Fructose, C ₆ H ₁₂ O ₆ ) A1.5. 1.5M D-fructise solution was prepared.

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

0.25mL of the cantaloupe aqueous extract obtained in the second example was taken, added with 0.25mL of BSA solution and D-fructise solution and mixed uniformly to obtain the solution to be tested of the experimental group A.

0.25mL of the muskmelon eggplant fermentation juice obtained in the first example was taken, added with 0.25mL of BSA solution and D-fructise solution and mixed uniformly to obtain a test solution of experiment group B.

0.25mL of 3mM AG solution was taken, 0.25mL of BSA solution and D-fructise solution were added and mixed uniformly to obtain a blank set of test solutions.

0.1mL of each group of solutions to be tested was taken as each group of zero point solutions. The fluorescence value of each zero point solution was measured with a fluorescence spectrometer (Thermo Fisher Scientific) using excitation light of 360nm and emission light of 460nm for 0.1mL of each zero point solution 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 each end point solution was measured by taking 0.1mL of each end point solution and using a fluorescence spectrometer, and the fluorescence value was measured for each end point solution of 0.1mL by using excitation light of 360nm and emission light of 460 nm.

The relative glycosylation end products (Advanced Glycation End products, AGEs) formation (%) of each group were then calculated according to the following formula (1) to find the anti-glycosylation activity (Antiglycative activity). In other words, the relative AGEs formation (%) of each group was calculated by taking the AGEs formation of the blank group as 1 (i.e., the relative AGEs formation of the blank group was 100%).

Therein, fluorescence sample _24hr Fluorescence values representing the end-points of the experimental group Fluorescence sample _0hr Fluorescence values representing zero point of experimental group Fluorescence control _24hr Fluorescence values representing the end point of the blank, fluorescence control _0hr Fluorescence values representing the zero points of the blank group.

Referring to fig. 3, the experimental group a (aqueous extract) did not affect the formation of the glycosylated end product, while the experimental group B (fermented juice) significantly reduced the formation of the glycosylated end product by 24.24% compared to the blank group. Therefore, the muskmelon eggplant fermentation juice can effectively inhibit the formation of glycosylation end products, 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 matched with a quantitative PCR instrument to measure the change of anti-aging related genes in human fibroblasts after the human fibroblasts are treated by the muskmelon eggplant water extract or the muskmelon eggplant fermentation juice.

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

Wherein, the CCT5 gene is a member of a CCT gene family and is related to the process of restoring the young state of mature cells; the Pink1 gene is involved in the process of restoring the young state of aged granulosomes; the FOXO gene, MRPS5 gene and Ubl-5 gene are all related to the activation process of the granulosa line body, which is a power plant in the same organism, and if the activity of the granulosa line body is insufficient, the granulosa line body is easy to make a response to the cell aging phenomenon.

Thus, in the sixth example, CCT5 gene, FOXO gene, MRPS5 gene, pink1 gene and Ubl-5 gene were used as the targets for the anti-aging gene analysis.

Materials and instruments

Cell lines (hereafter referred to as cells): human fibroblasts (CCD-996 SK, from ATCC).

Culture medium: minimum essential broth (Minimum essential medium) (MEM) (Gibco; model: 11095080), 10% Fetal Bovine Serum (FBS) (Gibco; model: 10437-028), 1% penicillin-streptomycin (Gibco; model: 15140122), 1mM sodium pyruvate (sodium pyruvate) (Gibco; model: 11360-070), 1.5G/l sodium bicarbonate (sodium bicarbonate) (Sigma; model: S5761-500G), and 0.1mM non-essential amino acid (non-essential amino acids) (Gibco; cat.11140050) were added.

RNA extraction kit (from Geneaid corporation, taiwan, model:. FC 24015-G)

Reverse transcriptase [ ]

III Reverse Transcriptase) (Invitrogen corporation, U.S. Pat. No. 18080-051).

The target gene primer is measured and comprises FOXO gene, MRPS5 gene, pink1 gene, ubl-5 gene and internal control group (GAPDH gene).

KAPA

FAST qPCR kit (purchased from Sigma, usa, no. 38220000000).

ABI StepOnePlus ^TM Real-time PCR System (ABI StepOnEPlus) ^TM Real-Time PCR system (Thermo Fisher Scientific Co., U.S.).

Muskmelon eggplant fermentation juice: the muskmelon eggplant fermentation juice used in this experiment was obtained as in example one above.

Melon eggplant water extract: the muskmelon solanum torvum water extract used in this experiment was obtained as described in example two above.

First, human fibroblasts (abbreviated as cells as in this example) were grown at 1X 10 cells per well ⁵ Individual cell amounts were cultured in a six-well culture dish containing 2mL of the above 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, the experimental group A was prepared by culturing human fibroblasts in 2ml of a 2.0% concentration medium of a cantaloupe aqueous extract prepared in the above example for 24 hours. Experiment group B was prepared by culturing human fibroblasts in 2ml of a 2.0% concentration medium of the tomato fermentation broth prepared in the above example for 24 hours.

The blank group was prepared by culturing human fibroblasts using 2 ml of the culture medium alone, and culturing human fibroblasts for 24 hours without adding other additives, and was used as an experimental blank group.

The above experimental group a and experimental group B and blank group, each of which was repeated four times.

The treated human fibroblasts (i.e., experimental group A and B)And the blank group of human fibroblasts are used for respectively breaking cell membranes by using cell lysate to form three groups of cell solutions. Next, RNA was extracted from each of the three cell solutions using an RNA extraction reagent kit (available from Genaid corporation, taiwan, lot No. FC24015-G). Next, 1000 nanograms (ng) of the extracted RNA were used as templates for each group, and the template was passed through

III reverse transcriptase (available from Invitrogene, U.S. Pat. No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. The six cDNA groups were then subjected to quantitative real-Time reverse transcription polymerase chain reaction (quantitative real-Time reverse transcription polymerase chain reaction) by means of a ABI StepOnePlusTM real-Time PCR system (ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA)), KAPA SYBR FAST (purchased from Sigma, USA, accession number 38220000000) and primers (SEQ ID NO:1 to SEQ ID NO: 12) of Table 1 to observe the expression amounts of CCT5 gene, FOXO gene, MRPS5 gene, pink1 gene, UBL5 gene in the three groups of human fibroblasts. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to 95℃for 20 seconds, followed by 95℃for 3 seconds, 60℃for 30 seconds, and 40 cycles were repeated, and gene quantification was performed using the 2-DeltaCt method. In this case, the mRNA expression levels of the CCT5 gene, the FOXO gene, the MRPS5 gene, the Pink1 gene and the UBL5 gene can be indirectly quantified by quantitative real-time reverse transcription polymerase chain reaction using cDNA, and the expression levels of the proteins encoded by the CCT5 gene, the FOXO gene, the MRPS5 gene, the Pink1 gene and the UBL5 gene can be estimated.

List one

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

Please refer to fig. 4. When the expression level of CCT5 gene in the blank group was regarded as 1 (i.e., 100%), the expression level of CCT5 gene in the experiment group a was 0.22 (i.e., 22%) and the expression level of CCT5 gene in the experiment group B was 1.46 (i.e., 146%), which means that the expression level of CCT5 gene was increased 1.46 times as high as that in the blank group after the cells were treated with the muskmelon fermentation juice.

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

Please refer to fig. 4. When the expression level of the MRPS5 gene in the blank was regarded as 1 (i.e., 100%), the expression level of the MRPS5 gene in the experiment group a was 0.17 (i.e., 17%) and the expression level of the MRPS5 gene in the experiment group B was 1.12 (i.e., 112%) relative to the blank, which means that the expression level of the MRPS5 gene was increased 1.12 times as high as that in the blank after the cells were treated with the muskmelon fermentation broth.

Please refer to fig. 4. When the expression level of the Pink1 gene in the blank group was regarded as 1 (i.e., 100%), the expression level of the Pink1 gene in the experimental group A was 0.30 (i.e., 30%) and the expression level of the Pink1 gene in the experimental group B was 1.35 (i.e., 135%), which means that the expression level of the Pink1 gene was 1.35 times higher than that in the blank group after the cells were treated with the muskmelon fermentation broth.

Please refer to fig. 4. When the expression level of UBL5 gene in the blank group was regarded as 1 (i.e., 100%), the expression level of UBL5 gene in the test group a was 0.25 (i.e., 25%) and the expression level of UBL5 gene in the test group B was 1.48 (i.e., 148%) relative to the blank group, which means that the expression level of UBL5 gene in the test group was increased 1.48 times as high as that in the blank group after the cells were treated with the muskmelon fermentation sap.

Therefore, after the human fibroblasts are treated by the muskmelon eggplant fermentation juice, the expression level of various genes related to aging resistance and rejuvenation in the cells is improved, which means that the cells return to the state before aging from the aging state, and the aging of the cells is effectively delayed.

Embodiment seven: melanin content detection

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

First, 1.5X10 per well ⁵ Cell number of individual cells, mouse melanoma cell line B16F10 (purchased from American Type Culture Collection (ATCC), no. CRL-6475) was inoculated into each well of a 6-well culture dish containing 3mL of cell culture medium and incubated at 37℃for 24 hours.

After 24 hours of culture, B16F10 cells were divided into 3 groups: one experimental group (i.e., experimental group (muskmelon eggplant fermentation juice group)), one control group, and one blank group. The cell culture medium of each group was removed and replaced with 3mL of experimental medium per well, and then placed at 37 ℃ for subsequent culture for 48 hours. Wherein the experimental medium of the experimental group was a cell culture medium containing 0.03125vol% of the muskmelon eggplant fermentation juice obtained in example one. The experimental medium of the control group was a cell culture medium containing 0.03125vol% kojic acid (kojic acid) (the experimental medium of the blank group was a simple cell culture medium (i.e., no muskmelon eggplant fermentation broth nor kojic acid).

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

After the measurement, the relative melanin content (%) was calculated by substituting the measured absorbance value into the following formula (2). In other words, here, the relative melanin content (%) of each group was calculated by taking the melanin content of the blank group as 1 (i.e., the relative melanin content of the blank group was 100%). Also, the statistically significant differences between the groups were statistically analyzed by student t-test (student t-test), as shown in FIG. 5. In fig. 5, "x" represents that the p value is less than 0.05 when compared to the blank, and "x" represents that the p value is less than 0.01 when compared to the blank, and "x" represents that the p value is less than 0.001 when compared to the blank.

Relative melanin content (%) = (OD 450 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 could reduce melanin production by 24.71%; the melanin content of the control group is also obviously reduced, the melanin generation is reduced by 14.45%, and the kojic acid is a known whitening component, so that the muskmelon eggplant fermentation juice can effectively inhibit the melanin generation, namely the formation of tyrosinase, and has the whitening effect.

Example eight: moisture-preserving gene detection

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent set is matched with a quantitative PCR instrument to measure the change of the relevant genes in the cells after the primary skin keratinocytes of human beings are treated by the muskmelon eggplant water extract or the muskmelon eggplant fermentation juice.

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

Wherein, the Krt1 gene is related to keratin, the keratin is a main structural object of skin, and the keratin has the water locking effect of reducing water evaporation when being closely arranged, so that the Krt1 gene can influence the skin to moisturize; the method comprises the steps of carrying out a first treatment on the surface of the The HAS2 gene is a hyaluronic acid synthase related gene, is related to hyaluronic acid synthesis, and HAS2 gene promotion is related to hyaluronic acid content, so that skin moisture and elasticity can be influenced.

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

Materials and instruments

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

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

RNA extraction reagent kit (from Geneaid corporation, taiwan, lot No. FC24015-G)

Reverse transcriptase [ ]

III Reverse Transcriptase) (Invitrogen corporation, U.S. Pat. No. 18080-051)

The target gene primer was measured to include the Krt1 gene and HAS2 gene, and further included an internal control group (TBP gene).

KAPA

FAST qPCR kit (purchased from Sigma Co., U.S. Pat. No. 38220000000)

ABI StepOnePlus ^TM real-Time PCR System (ABI StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific Co., USA)).

Muskmelon eggplant fermentation juice: the muskmelon eggplant fermentation juice used in this experiment was obtained as described in example 1 above.

Melon eggplant water extract: the muskmelon solanum torvum water extract used in this experiment was obtained as described in example 2 above.

Experimental procedure

First, 1.5x10 is taken ⁵ Human primary skin keratinocytes were cultured in six-well cell culture dishes containing 2 ml of the above medium per well at 37℃for 24 hours, and the cultured human primary skin keratinocytes per well were divided into a blank group and an experimental group (total two groups) according to the following test conditions to treat the cultured human primary skin keratinocytes per well.

Test conditions

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

The experimental group was to culture human primary skin keratinocytes for 6 hours in 2ml of a medium containing 1% concentration of the muskmelon eggplant fermentation broth prepared in example 1 above.

The blank and experimental groups above were repeated four times for each group.

The treated human primary skin keratinocytes (i.e. blank group and experimental group) are respectively broken into cell membranes by cell lysate to form two groups of cell solutions. Next, the kit was used with RNA extraction reagent (available from Genaid, chinaTaiwan, lot No. fc 24015-G) extracts RNA in the four sets of cell solutions, respectively. Next, 1000 nanograms (ng) of the extracted RNA were used as templates for each group, and the template was passed through

III reverse transcriptase (available from Invitrogene, U.S. Pat. No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Then by ABI StepOneGlus ^TM Real-time PCR System (ABI StepOnEPlus) ^TM Real-Time PCR system (Thermo Fisher Scientific, USA)), KAPA SYBR FAST (purchased from Sigma, USA, accession No. 38220000000) and the primers of Table 1 (SEQ ID NO:13 and SEQ ID NO: 18) were used to quantify Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) of two groups of cDNAs to observe the expression amounts of Krt1 gene and HAS2 gene in two groups of human primary skin keratinocytes. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to 95℃for 20 seconds, followed by 95℃for 3 seconds, 60℃for 30 seconds, and 40 cycles were repeated, and gene quantification was performed using the 2-DeltaCt method. In this case, the mRNA expression levels of the Krt1 gene and the HAS2 gene can be indirectly quantified by quantitative real-time reverse transcription polymerase chain reaction using cDNA, and the expression levels of the proteins encoded by the Krt1 gene and the HAS2 gene can be estimated.

TABLE 2

* R is REVERSE direction and F is FORWARD direction.

It should be noted that the relative gene expression of the Krt1 gene and HAS2 gene shown in the drawings described below is presented at a relative magnification, wherein standard deviation was calculated using STDEV formula of Excel software, and whether there was a statistically significant difference was analyzed in the Excel software by single Student t-test (Student t-test). In the drawings, "p" means that the value of p is less than 0.05, "p" means that the value of p is less than 0.01, and "p" means that the value of p is less than 0.001. The more "x" the more significant the statistical difference.

Referring to fig. 6, when the expression levels of the HAS2 gene and the KRT1 gene in the panel were regarded as 1 (i.e., 100%), the expression level of the HAS2 gene in the panel was 1.43 (i.e., 143%), the expression level of the KRT1 gene was 2.27 (i.e., 227%), which means that the expression level of the HAS2 gene in the panel was increased by 1.43 times and the expression level of the KRT1 gene was increased by 2.27 times.

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

As a result, as shown in FIG. 6, when the human primary skin keratinocytes were treated with the muskmelon eggplant fermentation broth, the expression level of HAS2 gene and KRT1 gene of the human primary skin keratinocytes was increased, which represents that the muskmelon eggplant fermentation broth had the ability to regulate the contents of keratin and hyaluronic acid, and had the potential to promote the moisture retention ability of skin.

Example nine: fat accumulation detection

Here, the preadipocyte proliferation medium (pre-adipocyte expansion medium) used was the minimum essential medium α (Minimum Essential Medium Alpha, MEM α, brand: gibco) supplemented with 20vol% FBS (brand: gibco) and 1vol% penicillin-streptomycin. The differentiation medium (differentiation medium) used was MEM alpha (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% isopropyl alcohol (isopopanol, supplier: ECHO) to prepare a stock solution of 3mg/mL of the oil-red O staining reagent. To obtain a ready-to-use oil-red O working solution (oil-red O working solution), the stock solution of oil-red O staining reagent was diluted with secondary water (ddH 2O) to a concentration of 1.8mg/mL in real time before use, i.e. a stock solution of 60% oil-red O staining reagent.

First, at 8X 10 per well ⁴ Cell number of individual cells, mouse bone marrow stromal cell line OP9 (purchased fromATCC, no. CRL-2749) was inoculated into each well of a 24-well culture dish containing 500. Mu.L of preadipocyte proliferation medium, and incubated at 37℃for 7 days. During the 7 day incubation period, fresh 500 μl of differentiation medium was changed every 3 days. After 7 days of culture, intracellular oil droplet (lipid droplet) formation in each well was observed using a microscope (trade name: ZEISS) to confirm complete differentiation of cells into adipocytes for use in subsequent experiments.

After 24 hours of culture, adipocytes were divided into 3 groups: two experimental groups, i.e., experimental group a (cantaloupe water extract) and experimental group B (cantaloupe fermented 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 7 days in succession. During the 7 day incubation period, fresh 500 μl of the experimental medium was changed every 3 days. Wherein, the experiment medium of the experiment group A is a differentiation medium containing 0.0625vol% of the muskmelon solanum torvum water extract obtained in the example I. The experimental medium of experimental group B was a differentiation medium containing 0.0625vol% of the muskmelon eggplant fermentation juice obtained in example one. The experimental medium of the blank group is a simple differentiation medium (i.e. does not contain the muskmelon eggplant water extract or the muskmelon eggplant fermentation juice).

Next, the experimental medium in each well was removed and rinsed twice with 1 xPBS. Then, 1mL of 10% formaldehyde (formaldyde, supplier: ECHO) was added to each well and incubated at room temperature for 30 minutes, thereby fixing the cells. Thereafter, formaldehyde was removed from each well and each well was rinsed twice with 1mL PBS. After rewashing, 1mL of 60% isopropyl alcohol was added to each well and allowed to act for 1 minute. Subsequently, isopropanol was removed, and 1mL of oil-red O working solution was added and allowed to act 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 the destaining, the stained cells were rinsed with 1xPBS, 100% isopropyl alcohol was added to each well, and reacted on a shaker (shaker) for 10 minutes to dissolve the dye. Then, 100. Mu.L of the aforementioned solution of the dye-isopropanol was taken from each well to a 96-well culture plate and the absorbance of each well was read (OD 510) at a wavelength of 510nm by ELISA reader (trade name: bioTek).

After the measurement, the relative oil droplet amount (%) was calculated by substituting the measured absorbance value into the following formula (3). In other words, here, the relative fatty oil droplet amount (%) of each group was calculated by taking the fatty oil droplet amount of the blank group as 1 (i.e., the relative fatty oil droplet amount of the blank group is 100%). Also, statistically significant differences between groups were statistically analyzed by student t-test, as shown in fig. 4. In fig. 4, "x" represents that the p value is less than 0.01 when compared with the blank group.

Relative oil droplet amount (%) = (OD 510 sample/OD510 control) ×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 amount of fatty oil droplets of experimental group a was significantly reduced compared to the blank group, and it was possible to reduce the amount of oil droplets by 11.72%. The relative amount of fatty oil droplets was significantly reduced in experimental group B compared to the blank group, and it was possible to reduce the amount of oil droplets by 32.29%. Therefore, the muskmelon eggplant fermentation juice can effectively inhibit fat accumulation and has the efficacy of reducing fat. Moreover, the muskmelon eggplant can produce more fat-reducing active ingredients than the muskmelon eggplant culture solution after being fermented by microorganisms.

Example ten: fat reduction efficacy test

Fat reduction refers to the decomposition of fat, and fat decomposition (Lipolysis) refers to the process in which Triglycerides (TG) stored in fat cells are gradually degraded into Fatty Acids (FA) and Glycerol (Glycerol). Accordingly, the present test analyzes the content of Glycerol (glycol) in adipocytes as a quantitative index to see whether or not lipolytic action is occurring.

The test uses mouse bone marrow stromal cells (OP 9 cells for short, hereinafter) purchased from the american type culture collection (American Type Culture Collection,

) OP9 cell strain (ATCC CRL-2749).

First, a 24-well culture plate is taken for each wellInoculation of 8X 10 ⁴ OP9 cells and 500 μl of Medium (Medium) containing 80% MEMAM (Minimum Essential Medium Alpha Medium, available from Gibco, usa) cell culture broth, 20% fetal bovine serum (Fetal Bovine Serum, available from Gibco, usa, cat# 10437-028) and 0.1% Penicillin/streptomycin (available from Gibco, usa) were added and incubated at 37 ℃ for 7 days. The medium was changed every 3 days during this 7-day cell culture period. After 7 days, intracellular oil droplet formation was observed under a microscope (ZEISS; magnification 400 x), thereby confirming that the cells had been fully differentiated into adipocytes.

The differentiated adipocytes were then divided into two groups: two experimental groups (i.e., experimental group a (added with the muskmelon eggplant water extract prepared in example two) and experimental group B (added with the muskmelon eggplant fermentation juice prepared in example one)) and a blank group.

Experiment group a: the cantaloupe water extract was added to the differentiated medium at a ratio of 500. Mu.L medium per well containing 0.156. Mu.L cantaloupe water extract (i.e., at a concentration of 0.03125%) and cultured at 37℃for 7 days. The medium was changed every 3 days during the cell treatment period of 7 days.

Experimental group B: the muskmelon tomato fermentation broth was added to the differentiated medium at a rate of 0.156 μl/well of the medium (i.e. 0.03125% concentration) and incubated at 37deg.C for 7 days. The medium was changed every 3 days during the cell treatment period of 7 days.

Blank group: without any treatment, i.e. without additional addition of other components to the medium after differentiation, at 37℃for 7 days. The medium was changed every 3 days during the cell treatment period of 7 days.

After 7 days of cell treatment, glycerol content was measured according to the following procedure using a cell Glycerol-based assay kit (Glycerol cell-based assay kit, available from Cayman, U.S. A., product No. 10011725). The supernatants of each group were collected and 25. Mu.L of each were 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 allowed to react at room temperature for 15 minutesThe OD of each group was read from the culture dish by ELISA reader _540nm To quantify the amount of glycerol that was broken down and released into the cell culture broth by each group of adipocytes, as shown in fig. 6. The amount of glycerol is proportional to the amount of fat decomposed. The Excel software was used to determine whether there was a statistically significant difference between the two sample populations (in the figures, "x" represents a p-value of less than 0.05, "x" represents a p-value of less than 0.01, and "x" represents a p-value of less than 0.001. The more "x" the more "the difference was, the more significant the statistical difference was).

Refer to fig. 8. When the fat degradation amount of the blank group was regarded as 100%, the fat degradation amount of the experimental group a was 96.07% and the fat degradation amount of the experimental group B was 104.85%, that is, the fat degradation amount after the processing of the muskmelon eggplant fermentation juice was significantly improved by 4.85%. The result shows that the muskmelon eggplant fermentation juice can directly and effectively promote the lipolysis in fat cells so as to reduce the fat content in the fat cells and achieve the effects of fat burning and weight losing.

Example eleven-cell experiment of fat metabolism Gene

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent set is matched with a quantitative PCR instrument to measure the change of the fat metabolism gene in the cells of the mouse bone marrow stromal cells after being treated by the muskmelon eggplant water extract or the muskmelon eggplant fermentation juice.

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

Wherein, the protein coded by UCP2 (Uncoupling Protein 2) gene is a body uncoupling protein (UCP), which is one of the members of mitochondrial anion carrier protein (Mitochondrial anion carrier proteins, MACP) family, and has the main functions of reducing adenosine triphosphate (Adenosine triphosphate, ATP) and promoting the outward transfer of anions from the inner membrane of mitochondria and the return transfer of protons from outside to the inner membrane of mitochondria, and releasing the energy generated in the process as heat energy; UCP2 gene is expressed in many tissues and is most expressed in skeletal muscle, and is considered to be involved in non-shivering thermogenesis (Nonshivering thermogenesis). Therefore, the expression level of the UCP2 gene increases, and the decomposition of fat can be promoted and the accumulation level of fat can be reduced.

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

Materials and instruments

Cell lines: mouse bone marrow stromal cells OP9 (BCRC; accession No. CRL-2749).

Culture medium: contains 80% MEMAM (Minimum Essential Medium Alpha Medium, available from Gibco, U.S.) cell culture broth, 20% fetal bovine serum (Fetal Bovine Serum, available from Gibco, U.S. Cat # 10437-028), and 0.1% Penicillin/streptomycin (available from Gibco, U.S.) was added.

RNA extraction reagent kit (from Geneaid corporation, taiwan, lot No. FC24015-G)

Reverse transcriptase [ ]

III Reverse Transcriptase) (Invitrogen corporation, U.S. Pat. No. 18080-051)

The target gene primer was measured to contain the UCP2 gene and also included an internal panel (m-ACTB gene).

KAPA

FAST qPCR kit (purchased from Sigma Co., U.S. Pat. No. 38220000000)

ABI StepOnePlus ^TM real-Time PCR System (ABI StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific Co., USA)).

Muskmelon eggplant fermentation juice: the muskmelon solanum torvum water extract used in this experiment was obtained as in example one above.

Melon eggplant water extract: the muskmelon solanum torvum water extract used in this experiment was obtained as described in example two above.

Experimental procedure

First, 1.5x10 is taken ⁵ The cultured mouse bone marrow stromal cells OP9 were treated in each well in a six-well cell culture tray containing 2ml of the above medium per well at 37℃for 24 hours, and the cultured mouse bone marrow stromal cells OP9 per well were divided into a blank group and an experimental group (total of three groups) according to the following test conditions.

Test conditions

Specifically, the blank group was prepared by culturing the bone marrow stromal cells of the mice with 2ml of the culture medium alone, and culturing for 6 hours without adding any additional components.

Experiment group A was prepared by culturing mouse bone marrow stromal cells in 2ml of a 0.0625% concentration medium of a muskmelon eggplant water extract prepared in the above example for 6 hours.

Experiment group B was prepared by culturing mouse bone marrow stromal cells in 2ml of a medium having a concentration of 0.0625% of the tomato fermentation broth prepared in the above example for 6 hours.

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

The treated bone marrow stromal cells (i.e. blank, experimental group a, experimental group B) were disrupted with cell lysates to form three groups of cell solutions, respectively. Next, RNA was extracted from each of the three cell solutions using an RNA extraction reagent kit (available from Genaid corporation, taiwan, lot No. FC24015-G). Next, 1000 nanograms (ng) of the extracted RNA were used as templates for each group, and the template was passed through

III reverse transcriptase (available from Invitrogene, U.S. Pat. No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Then by ABI StepOnePlusTM real-time PCR system (ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific Co., ltd., U.S.), KAPA SYBR FAST (purchased from Sigma Co., ltd., U.S. No. 38220000000), primers (SEQ ID NO:19 to SEQ ID NO: 22) of Table 1 were subjected to quantitative real-Time reverse transcription polymerase chain reaction (quantitative real-Time reverse transcription polymerase chain reaction) on three groups of cDNAs to observe the expression amounts of UCP2 genes in three groups of mouse bone marrow stromal cells. The apparatus for quantitative real-time reverse transcription polymerase chain reaction was set to 95℃for 20 seconds, followed by 95℃for 3 seconds, 60℃for 30 seconds, and 40 cycles were repeated, and gene quantification was performed using the 2-DeltaCt method. In this case, the mRNA expression level of the UCP2 gene can be indirectly quantified by quantitative real-time reverse transcription polymerase chain reaction using cDNA, and the expression level of the protein encoded by the UCP2 gene can be estimated.

TABLE 1

* R is REVERSE direction and F is FORWARD direction.

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

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

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

From this, it was found that the expression level of UCP2 in the mouse bone marrow stromal cells increased when the mouse bone marrow stromal cells were treated with the fermentation sap containing muskmelon.

As a result, as shown in FIG. 9, the tomato fermented juice of the present invention can increase the gene expression level of UCP2, representing that it has an effect of promoting the decomposition of fat and reducing the accumulation of fat.

Embodiment twelve: human body detection for fat-reducing and slimming

8 obese subjects (i.e., having body fat greater than 25% or BMI greater than 24) were allowed to drink one bottle of 50mL of the cantaloupe fermented beverage (which contained 12vol% of the cantaloupe fermented juice obtained in example 1 with 88vol% of water) daily for 4 weeks. And, before drinking (i.e. week 0) and after drinking (i.e. week 4), the body weights of the subjects were measured by a body weight machine, the total body fat rate of the subjects was measured by a body fat meter (brand: TANITA BC-601 FS), and the waist circumference of the subjects was measured by a cloth scale. And, the statistically significant difference between the measurement results at week 0 and the measurement results at week 4 was statistically analyzed by student t-test, as shown in fig. 10 to 13. In fig. 10-13, "x" represents that the p-value is less than 0.05 when compared to week 0; "x" means that the p-value is less than 0.01 when compared to the blank.

Referring to fig. 10, drinking the cantaloupe fermented beverage for 4 weeks can significantly reduce the body weight by about 1.0 kg compared to before drinking (week 0).

Referring to fig. 11, drinking the cantaloupe fermented beverage for 4 weeks can significantly reduce the systemic body fat rate by about 1.0% compared to before drinking (week 0).

Referring to fig. 12, drinking the cantaloupe fermented beverage for 4 weeks can significantly reduce the body fat percentage by about 1.2% compared to before drinking (week 0).

Referring to fig. 13, drinking the cantaloupe fermented beverage for 4 weeks can significantly reduce the waist circumference by about 1.1 cm compared to before drinking (week 0).

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

Embodiment thirteen: antioxidant human detection

8 obese subjects (i.e., having body fat greater than 25% or BMI greater than 24) were allowed to drink one bottle of 50mL of the cantaloupe fermented beverage (which contained 12vol% of the cantaloupe fermented juice obtained in example 1 with 88vol% of water) daily for 4 weeks. And, blood collection was performed before drinking (i.e., week 0) and after drinking (i.e., week 4), and Total antioxidant capacity (Total Anti-oxidative Capacity) and peroxidation products (MDA) in the blood were measured, wherein the Total antioxidant capacity and peroxidation products were measured by the commission of the doctor laboratory (taiwan, china).

In general, the sum of antioxidant capacity, abbreviated as TAC, may represent the capacity to reduce oxides in vivo. Many studies have shown that free radicals can cause some lesions in humans, such as: cardiovascular diseases and various inflammatory diseases, and the like, and the in vivo antioxidants have the function of protecting cells and can defend against free radical attack, so that the unbalanced state of the body can be monitored by detecting the in vivo antioxidant capacity state, and the higher the total antioxidant capacity, the better the in vivo antioxidant capacity is represented; the product of peroxidation (MDA, malondialdehyde), which is a product of lipid peroxidation in vivo, is widely accepted as one of the measurement targets of oxidative damage, and the higher the product of peroxidation (MDA, malondialdehyde), the higher the oxidative pressure in vivo.

After the measurement, the relative oxidation resistance value (%) at week 4 was calculated using the value at week 0 as a reference (i.e., the oxidation resistance value at week 0 was 100%). And, the statistically significant difference between the measurement results at week 0 and the measurement results at week 4 was statistically analyzed by student t-test, as shown in fig. 14 and 15. In fig. 14 and 15, "x" represents that the p-value is less than 0.05 when compared to week 0; "p-value" is less than 0.001 when compared to the blank.

Referring to fig. 14, drinking the cantaloupe fermented beverage for 4 weeks can significantly increase the total antioxidant capacity by about 11.0% compared to before drinking (week 0); wherein the original value of the total antioxidant capacity at week 0 is 0.74nmol/L and the original value of the total antioxidant capacity at week 4 is 0.83nmol/L.

Referring to fig. 15, drinking the cantaloupe fermented beverage for 4 weeks can significantly reduce the peroxygenated product (MDA) by about 26.1% compared to before drinking (week 0); wherein the MDA initial value at week 0 is 1.1nmol/ml and the MDA initial value at week 4 is 0.9nmol/ml.

Fourteen examples: human detection for improving skin condition

8 obese subjects (i.e., having body fat greater than 25% or BMI greater than 24) were allowed to drink one bottle of 50mL of the cantaloupe fermented beverage (which contained 12vol% of the cantaloupe fermented juice obtained in example 1 with 88vol% of water) daily for 4 weeks. The skin condition was measured with a ViSIA skin tester (VISIA Complexion Analysis System, available from Canfield Inc. USA) and a skin surface humidity tester (Corneometer CM825, available from C+K, germany) before drinking (i.e., week 0) and after drinking (i.e., week 4). Here, skin texture, skin wrinkles, skin redness (red pigment) were measured with a VISIA skin detector, and skin moisture content was measured with a skin surface moisture tester.

After the measurement, the relative skin condition (%) at week 4 was calculated using the skin condition at week 0 as a reference (i.e., the relative skin condition at week 0 was 100%). 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. 16-19, "x" represents that the p-value is less than 0.05 when compared to week 0.

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

In view of the above, the muskmelon eggplant fermentation juice according to any of the embodiments of the present invention may prepare a composition for improving skin conditions and/or reducing fat. In other words, the aforementioned composition has one or more of the following functions: enhancing glutathione synthesis, inhibiting formation of glycation end products, enhancing anti-aging related genes, inhibiting melanin production, enhancing secretion of moisture related genes, inhibiting fat accumulation, promoting lipolysis, reducing body fat of the subject, and improving skin conditions.

Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims (5)

1. Use of a cantaloupe fermented juice for the preparation of a composition for reducing fat, wherein the cantaloupe fermented juice is obtained by a fermentation procedure of a cantaloupe culture solution and a plurality of strains, wherein the cantaloupe culture solution comprises cantaloupe juice and water, the ratio of the cantaloupe juice to the water is 1:2-5, and 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 relative to the cantaloupe culture solution.

2. The use according to claim 1, wherein the reduction of fat is inhibition of fat accumulation, promotion of fat breakdown or a combination thereof.

3. Use according to any one of claims 1 to 2, characterized in that the fermentation procedure comprises:

Adding the saccharomycete into the muskmelon eggplant culture solution;

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

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

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

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

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

filtering the third primary fermentation juice to obtain the muskmelon eggplant fermentation juice.

4. Use according to any one of claims 1 to 2, characterized in that the melon tomato fermented juice has a pH value of 4.0±1.0 and the melon tomato fermented juice has a sugar degree of 40±2.

5. Use according to any one of claims 1 to 2, characterized in that the polyphenol content of the muskmelon eggplant fermentation juice is 88ppm.

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