TW202106326A - A preparation method of a guarana fermentation liquid and an use for guarana fermentation liquid - Google Patents

Abstract

A preparation method of guarana fermentation liquid includes providing a culture medium, fermenting multiple strains with the culture medium for 7 days to obtain a basic fermentation, and adjusting the basic fermentation to obtain a guarana fermentation liquid. The culture medium includes a guarana extract made by 1 weight guarana (Paullinia Cupana ) and 10 weight water, and a glucose which weight is 10% of the total weight of the guarana fermentation liquid. The multiple strains includes 0.1% yeast relative to the culture medium, 0.05% Lactobacillus relative to the culture medium and 0.5% Acetobacter relative to the culture medium.

Description

Preparation method of guarana fermentation broth and use of guarana fermentation broth

The invention relates to a fermentation broth, in particular to a preparation method and use of a guarana fermentation broth.

Since the rise of the concept of organic and natural diets, biotech companies and food companies have actively invested in the research and development of products related to natural plants. In order to make plant-related products have a basis for scientific verification of health benefits, plant active ingredient analysis and efficacy evaluation have become key items in product development. Guarana (Paullinia cupana), which is native to the Amazon Basin in Brazil, has also become one of the objects of research and development.

Guarana (Scientific name Paullinia cupana), also known as Brazilian cocoa, is a climbing plant belonging to the genus Paullinia (also known as the genus Paullinia) of the Sapindus family. Guarana is distributed in the Amazon Basin such as Brazil, Peru, Colombia, and Venezuela. Currently, it is mainly grown in the Brazilian state of Amazonas and the Brazilian state of Bahia.

Guarana has three leaves, its flowers are small and white, and its fruit has a red shell. When the fruit is mature, the white flesh and seeds will be exposed. The fruit of Guarana contains a lot of caffeine, which is often used to make syrup, food, and then processed into beverages.

Since the 15th century, Guarana has been used as a traditional medicinal material used by the Amazons, and it is mainly used as a physical enhancer. In Portugal, guarana has been used as a beverage since the late 1990s.

In some embodiments, a method for preparing a guarana fermentation broth includes providing a culture broth, fermenting the culture broth and multiple strains for 7 days to obtain a fermentation stock solution, and adjusting the fermentation stock solution to form a guarana fermentation broth. The above-mentioned culture liquid includes a guarana extract formed by 1 part by weight of Paullinia cupana (Paullinia cupana) and 10 parts by weight of water and 10% glucose of the total weight of the guarana extract. The plurality of bacterial species include 0.1% yeast relative to the culture solution, 0.05% lactic acid bacteria relative to the culture solution, and 5% acetic acid bacteria relative to the culture solution.

In some embodiments, a guarana fermentation broth prepared by a method for preparing a guarana fermentation broth is used to prepare a composition for reducing the fat formation of a receptor, wherein the guarana fermentation broth is used to increase lipolytic enzymes (ATGL) Express volume to reduce fat formation.

In some embodiments, a guarana fermentation broth prepared by a method for preparing a guarana fermentation broth is used to prepare a composition for reducing the fat formation of the recipient, wherein the guarana fermentation broth is used to reduce lipid oil droplets To reduce fat formation.

In some embodiments, a guarana fermentation broth prepared by the above-mentioned preparation method of guarana fermentation broth is used to prepare a composition for reducing the fat formation of the receptor, wherein the guarana fermentation broth is used to reduce The activity of amylolytic enzymes and α-glucosidase as well as reducing the heat absorption of starch into sugars to reduce fat formation.

In summary, according to the preparation method of guarana fermentation broth according to any embodiment, a guarana fermentation broth can be prepared. In some embodiments, guarana fermentation broth can be used to prepare a composition that reduces fat formation. In some embodiments, the preparation method of guarana fermentation broth can increase the total polyphenol content of the prepared guarana fermentation broth. In some embodiments, the guarana fermentation broth is used to increase the expression level of lipolytic enzymes, thereby reducing the fat formation of the recipient. In some embodiments, the guarana fermentation broth is used to reduce the formation of lipid oil droplets, thereby reducing fat formation. In some embodiments, the guarana fermentation broth is used to reduce the activity of amylolytic enzymes and reduce the heat absorption after the starch is decomposed into sugars, thereby reducing fat formation.

Regarding the symbol "%" used in this article usually refers to weight percent concentration, and the symbol "vol%" usually refers to volume percent concentration. About the "Guarana" used in this article usually refers to the fruit (including seeds) of Guarana (Paullinia cupana ), the fruit size is 1 to 1.5 cm, and it is a fully ripe fruit.

Please refer to Figure 1. In some embodiments, the preparation method of the guarana extract includes the following steps: providing a culture solution (step S100), fermenting the culture solution and multiple strains for 7 days to obtain a fermentation stock (step S300), and adjusting the fermentation stock to A guarana fermentation broth is formed (step S500). Wherein, the culture liquid includes a guarana extract formed by 1 part by weight of Paullinia cupana (Paullinia cupana) and 10 parts by weight of water and 10% glucose of the total weight of the guarana extract. The plural bacterial species include 0.1% yeast relative to the culture solution, 0.05% lactic acid bacteria relative to the culture solution, and 5% acetic acid bacteria relative to the culture solution.

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

Please refer to Figure 2. In some embodiments, step S100 includes the following steps: mixing guarana and water to form a guarana base liquid (step S110), and extracting the guarana base liquid at 95° C. for 1 hour to obtain a guarana extract (Step S120), and mixing the guarana extract with glucose to obtain a culture solution (Step S130). Here, the ratio of guarana to water in the guaranaki liquid is 1:10.

In some embodiments of step S110, the guarana mixed with water is granular. In other words, before mixing the guarana and water, the guarana needs to be crushed to form guarana particles so that it can be fully mixed with the water.

In some embodiments, the guarana selected here may be a whole fruit including a red shell, white flesh and seeds. The whole fruit has a size of 1 to 1.5 cm and is a fully ripe fruit. In other words, the whole guarana fruit is crushed into guarana particles together with the husk and seeds.

In some embodiments of step S120, the extraction method of the guarana extract is by immersing the guarana in water at a constant temperature of 95° C. for 1 hour. In other embodiments of step S120, the method of extracting the guarana extract is by immersing the guarana in water and then placing the guarana and water in an environment of 95° C. for 1 hour.

In some embodiments, the guarana extract is adjusted to a sugar content of 10°Bx to 10.4°Bx with 10% of its total weight of glucose to form a culture broth. Here, the culture broth with a sugar content of 10°Bx to 10.4°Bx can ensure the smooth progress of subsequent fermentation and ensure that the strain has sufficient nutrient consumption.

In some embodiments, the obtained culture solution can be cooled to room temperature by natural cooling, so as to facilitate the subsequent fermentation of the culture solution with various strains.

In some embodiments, the pH of the culture solution is 6.3±2.

Please refer to Figure 3. In some embodiments, step S300 includes the following steps: first, the yeast is fermented in the culture broth for 1 day to form a first initial fermentation broth (step S310). In other words, the first mixed liquid formed by mixing 0.1% of the yeast and the culture liquid is fermented for 1 day to form the first initial fermentation liquid. Among them, the added amount of yeast is 1% relative to the total weight of the culture solution. In some embodiments, the first mixed liquor is fermented at 28°C to 37°C. In addition, in an embodiment of step S310, the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae).

After the first primary fermentation broth is formed, lactic acid bacteria are added to ferment in the first primary fermentation broth for 1 day to form a second primary fermentation broth (step S330). In other words, the second mixed liquid formed by mixing 0.05% of the lactic acid bacteria and the first primary fermentation liquid is fermented for 1 day to form the second primary fermentation liquid. Among them, the added amount of lactic acid bacteria is 0.05% relative to the total weight of the culture solution. In some embodiments, the second mixed liquor is fermented at 28°C to 37°C. In addition, in an embodiment of step S330, the lactic acid bacteria is Lactobacillus plantarum (Lactobacillus plantarum).

After the second primary fermentation broth is formed, acetic acid bacteria are added to ferment in the second primary fermentation broth for 5 days to form a third primary fermentation broth (step S350). In other words, the third mixed liquor formed by mixing 5% of acetic acid bacteria and the second primary fermentation broth is fermented for 5 days to form the third primary fermentation broth. Among them, the added amount of acetic acid bacteria is 5% relative to the total weight of the culture solution. In some embodiments, the third mixed liquor is fermented at 28°C to 37°C. In addition, in an embodiment of step S350, the acetic acid bacterium is Acetobacter aceti (Acetobacter aceti). In addition, the pH of the third primary fermentation broth is 3 to 4. In some embodiments, the pH of the third primary fermentation broth is 3.4.

After the third primary fermentation broth is formed, the third primary fermentation broth is filtered to obtain the fermentation stock (step S370). In an embodiment of step S370, the filtering step of the third primary fermentation broth includes concentrating under reduced pressure at 60°C, and filtering the third primary fermentation broth with a 200-mesh mesh filter to obtain fermentation Stock solution. Here, the pH of the fermentation stock solution is 3 to 4. In some embodiments, the pH of the fermentation stock solution is 3.4.

Please refer to Figure 1 again. After obtaining the fermentation stock solution, the fermentation stock solution is adjusted to form the guarana fermentation broth (step S500). For example, the guarana fermentation broth can be formed by adjusting the sugar content or/and concentration of the fermentation stock solution. In an implementation aspect of step S500, the sugar content of the fermentation stock solution is adjusted to form the guarana fermentation broth. In some embodiments, the guarana fermentation broth is formed by adding 60% of the oligosaccharide relative to the total weight of the fermentation stock to the fermentation stock. Here, the sugar content of the guarana fermentation broth is 40°Bx.

In some embodiments, the total polyphenol content of the guarana fermentation broth is more than 1600 micrograms/ml (µg/ml). In other words, every 1 ml of guarana fermentation broth contains more than 1600 micrograms of total polyphenols.

In some embodiments, the guarana fermentation broth has the effect of increasing the expression level of lipolytic enzyme genes. Among them, the lipolytic enzyme may be adipose triglyceride lipase (ATGL). In some embodiments, the guarana fermentation broth has the effect of reducing the formation of lipid oil droplets. In some embodiments, the guarana fermentation broth has the effect of reducing the activity of amylase. In some embodiments, the guarana fermentation broth has the effect of reducing the activity of α-glucosidase (α-glucosidase). In some embodiments, the guarana fermentation broth has the effect of reducing the heat absorption after the starch in the recipient body is decomposed into sugars. In some embodiments, guarana fermentation broth can reduce fat and/or reduce calorie absorption through one or more of the following cell-level effects: increase the expression of lipolytic enzyme (ATGL) genes and reduce lipid oil droplets Produce and reduce the activity of amylolytic enzymes and reduce the activity of α-glucosidase.

In some embodiments, the guarana fermentation broth can be used to prepare a composition that enhances the expression of lipolytic enzyme (ATGL) genes.

In some embodiments, the guarana fermentation broth can be used to prepare a composition that reduces the formation of lipid oil droplets.

In some embodiments, the guarana fermentation broth can be used to prepare a composition that reduces the activity of amylase and/or α-glucosidase.

In some embodiments, the guarana fermentation broth can be used to prepare a composition that reduces the heat absorption of starch in a recipient's body after decomposing into sugars.

In some embodiments, the guarana fermentation broth can be used to prepare a composition that reduces the fat formation of the recipient.

In some embodiments, any of the aforementioned compositions may be pharmaceuticals. In other words, this medicine contains an effective amount of guarana fermentation broth.

In some embodiments, the aforementioned medicines can be manufactured to be suitable for enteral, parenterally, oral, or topically using techniques well known to those skilled in the art. Dosage type.

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

In some embodiments, the aforementioned pharmaceutical products may include a 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 carriers: solvent (solvent), buffer (buffer), emulsifier (emulsifier), suspending agent (suspending agent), decomposing agent ( decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. Regarding the type and quantity of the selected carrier, it falls within the scope of professionalism and routine technology of those who are familiar with this technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline (normal saline), phosphate buffered saline (PBS), or aqueous solution containing alcohol (aqueous solution containing alcohol).

In some embodiments, any of the aforementioned compositions may be edible compositions. In other words, the edible composition contains a specific content of guarana fermentation broth. In some embodiments, the aforementioned edible composition may be a food product or a food additive. In some embodiments, the food product may be, but is not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

In some embodiments, any of the aforementioned compositions may be cosmetics or skin care products. In other words, cosmetics or skin care products contain a specific content of guarana fermentation broth.

In some embodiments, the aforementioned cosmetics or skin care products can be any of the following types: toner, gel, jelly film, mud mask, lotion, cream, lipstick, foundation, powder, powder, makeup remover, makeup remover Milk, facial cleanser, body wash, shampoo, hair conditioner, sunscreen, hand cream, nail polish, perfume, essence and facial mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, the external product acceptable ingredient may be, for example, an emulsifier, a penetration enhancer, a softener, a solvent, an excipient, an antioxidant, or a combination thereof.

Example 1: Preparation of Guarana Fermentation Broth

After the whole guarana fruit (including the shell, pulp and core) is crushed into guarana particles, 1 part by weight of guarana particles and 10 parts by weight of water are mixed to form a guarana base liquid. Next, the guarana base solution was extracted at 95°C for 1 hour to obtain a guarana extract solution. The guarana extract is mixed with 10% glucose of the total weight to form a culture broth for fermentation. In addition, the sugar content of the culture solution was 10.4°Bx and the pH value was 6.3.

The culture solution was cooled to room temperature (25°C) and the cooled culture solution was placed in a fermenter, and then 0.1% of the total weight of the culture solution was added with Saccharomyces cerevisiae (purchased from the Food Industry Development Institute). Resource Conservation and Research Center (BCRC) and deposit number BCRC20271) into the culture solution in the fermenter to form the first mixed solution. The first mixed liquor is fermented at 30°C for 1 day to form the first primary fermentation liquor.

Add 0.05% Lactobacillus plantarum TCI378 to the first initial fermentation broth in the fermenting tank with respect to the total weight of the culture broth, purchased from the Biological Resources Conservation and Research Center (BCRC) of the Food Industry Development Institute and the deposit number is BCRC910760 ) To form a second mixed liquid. The second mixed liquor was fermented at 30°C for 1 day to form a second primary fermentation liquor.

Add 5% acetic acid acetic acid bacteria ( Acetobacter aceti , purchased from the Biological Resources Conservation and Research Center (BCRC) of the Food Industry Development Institute (BCRC) and deposit number 11688) in the second primary fermentation broth in the fermentation tank. To form a third mixed solution. The third mixed liquor was fermented at 30°C for 5 days to form a third primary fermentation liquor. Next, confirm whether the pH value of the third primary fermentation broth is pH 3.4 to confirm the end time of the fermentation.

The third primary fermentation broth after the fermentation was carried out at 60° C. and concentrated under reduced pressure with a decompression concentrator, and the third primary fermentation broth was filtered with a 200-mesh mesh filter to obtain a fermentation raw liquid. In addition, 60% of the total weight of the fermentation stock is added to the fermentation stock to adjust the sugar content of the fermentation stock to 40°Bx to form a guarana fermentation broth.

Example 2: Preparation of Guarana Water Extract

After the whole guarana fruit (including the shell, pulp and core) is crushed into guarana particles, 1 part by weight of guarana particles and 10 parts by weight of water are mixed to form a guarana base liquid. Then, the guarana base liquid was extracted at 95° C. for 1 hour to obtain the guarana water extract stock solution. The guarana water extract stock solution is mixed with 10% glucose of the total weight to form the guarana water extract solution. In addition, the sugar content of the guarana water extract was 10.4°Bx and the pH was 6.3.

Experiment 1: Total polyphenol content test

1-1. Standard curve

Weigh 10.0 milligrams (mg) of gallic acid (mg) into a 10mL volumetric flask, and then quantify to 10mL with water (H2O) to obtain a stock solution of gallic acid. Dilute the stock solution of gallic acid 10-fold, that is, add 900 μL of water to 100 μL of the stock solution of gallic acid to obtain an initial solution of 100 μg/mL gallic acid (that is, containing 1000 ppm of gallic acid). Then, prepare standard solutions of gallic acid of 0μg/mL, 20μg/mL, 40μg/mL, 60μg/mL, 80μg/mL, and 100μg/mL according to Table 1 below, and take 100μL of the standard solutions of each concentration. Into a glass test tube. Add 500μL of Folin-Ciocalteu's phenol reagent (purchased from Merck) into each glass test tube and mix it with the standard solution and let it stand for 3 minutes, then add 400μL of 7.5% sodium carbonate (sodium carbonate) and mix well React for 30 minutes to obtain a standard reaction solution. Take 200 μL of the standard reaction solution into a 96-well plate, and measure its absorbance at 750 nm to obtain a standard curve.

Table 1 Standard solution (μg/mL) 0 20 40 60 80 100 Initial solution (μL) 0 20 40 60 80 100 Water (μL) 100 80 60 40 20 0

1-2. Experimental results

Dilute the test sample of the experimental group (i.e. the guarana fermentation broth of Example 1) and the test sample of the control group (i.e. the guarana water extract of Example 2) respectively with water 10 times, and take 100 microliters (μL) To the centrifuge tube. Add 500μL of Folin's reagent to the centrifuge tube containing the test sample diluted 10 times and mix it with the test sample and let it stand for 3 minutes, then add 400μL of 7.5% sodium carbonate, mix well, and react for 30 minutes to obtain the test sample. Reaction solution. After shaking the centrifuge tube containing the reaction solution to be tested to ensure that there are no bubbles, take 200 μL of the reaction solution to be tested into a 96-well plate, and measure the absorbance of the reaction solution to be tested at 750 nm.

Then, the absorbance value of the reaction solution to be tested is converted into the total polyphenol content using the standard curve and interpolation method. Here, it can be obtained that the total polyphenol content of the experimental group (i.e. guarana fermentation broth) is 1617.45μg/mL, and the total polyphenol content of the control group (i.e. guarana water extract) is 728.09μg/mL, such as Shown in Figure 4.

It can be seen that after guarana is fermented by microorganisms, the total polyphenol content can be increased by 2.2 times. That is, compared with the guarana water extract, the guarana fermentation broth can enhance the antioxidant activity.

Experiment 2: Enzyme activity test of amylolytic enzyme

2-1. Solvent configuration

Sodium phosphate buffer solution (hereinafter referred to as NaCl-Pi buffer solution) with 0.02 molar concentration (M) containing 6mM sodium chloride: 0.7356 grams of sodium monohydrogen phosphate (purchased from JTBaker, number 3828-01) , 0.5492 grams of sodium dihydrogen phosphate (purchased from Sigma, number 04270) and 1.7532 grams of sodium chloride (purchased from First Chemical Industry, number C4B07) were mixed and dissolved in 500 ml of water (H ₂ O) to prepare A NaCl-Pi buffer solution containing 6 mM sodium chloride and a pH of 6.3.

2 equivalent (N) sodium hydroxide (NaOH) solution: Dissolve 8 g of sodium hydroxide (purchased from Macron, No. 7708-10) in 100 ml of water to prepare a 2N sodium hydroxide solution.

Dinitrosalicylic acid color reagent (Dinitrosalicylic acid color reagent, hereinafter referred to as terminator): Dissolve 1 g of 3,5-dinitrosalicylic acid (purchased from Sigma, No. D0550) in 50 ml of deionized water, and then Slowly add 30 g of sodium potassium tartrate tetrahydrate (purchased from Sigma, No. 32312) and slowly add 20 ml of 2N sodium hydroxide solution, and quantify to 100 ml with deionized water. Here, a terminator can be obtained. Among them, the shelf life of the terminator is within two weeks.

1% starch solution: weigh 1 gram of starch to 100 ml of NaCl-Pi buffer solution and slowly heat it to completely dissolve the starch in the NaCl-Pi buffer solution. After the heated NaCl-Pi buffer solution containing starch drops to room temperature , Quantify to 100 ml with water. Here, a 1% starch solution can be obtained and stored at 4°C. In addition, at least the 1% starch solution should be placed at room temperature for 4 to 5 minutes before the enzyme activity test.

α-amylase solution (5 units/ml): Dissolve 0.0096 g of α-amylase in 25 ml of NaCl-Pi buffer solution. From this, 5 units per milliliter of α-amylase solution can be obtained. Generally speaking, the α-amylase solution is stored at 4°C and can be stored for 2 to 3 days.

2-2. Test process

The enzyme activity test of the amylolytic enzymes of each test group was performed according to Table 2 below. Among them, the experimental group (0 minutes), the control group (0 minutes) and the control group (0 minutes) in the test group represent the test groups where α-amylase does not react with starch (hereinafter referred to as the reaction starting point (0 minutes) Minutes)), while the experimental group (10 minutes), the control group (10 minutes) and the control group (10 minutes) represent the test group where α-amylase and starch react for 10 minutes (hereinafter referred to as the reaction termination point (10 minutes) )).

Table 2 Test group testing sample Reaction enzyme Reaction matrix Experimental group (0 minutes) 5 times diluted guarana fermentation broth alpha-amylase starch Experimental group (10 minutes) 5 times diluted guarana fermentation broth alpha-amylase starch Control group (0 minutes) 5 times diluted guarana water extract alpha-amylase starch Control group (10 minutes) 5 times diluted guarana water extract alpha-amylase starch Control group (0 minutes) NaCl-Pi buffer solution alpha-amylase starch Control group (10 minutes) NaCl-Pi buffer solution alpha-amylase starch

In Table 2, the guarana fermentation broth and the guarana water extract were all diluted with water, and each test group was subjected to a triple experiment. The reaction enzyme is 5 units/ml of α-amylase solution. The reaction substrate of reaction enzyme action is 1% starch solution. In other words, the substrate on which the enzyme (that is, α-amylase) acts is starch.

According to Table 2, firstly, 200 μL of test samples (ie, 5-fold diluted guarana fermentation broth, 5-fold diluted guarana water extract and NaCl-Pi buffer solution) were taken into centrifuge tubes, and then separately Add 200μL of α-amylase solution (5 units/ml) to each centrifuge tube, and shake the centrifuge tube containing the test sample and α-amylase solution to mix the test sample and the α-amylase solution evenly To form a solution to be reacted, the centrifuge tube containing the solution to be reacted is placed in an environment of 25° C. for 10 minutes.

Next, add 400μL of terminator to the centrifuge tube and mix well with the solution to be reacted in the three test groups at the reaction start point (0 minutes), and then add 200μL of 1% starch solution and let stand at 25°C. 10 minutes to form an unreacted solution. In other words, in the test group at the starting point of the reaction (0 minutes), α-amylase does not react with starch.

For the three test groups at the reaction termination point (10 minutes), 200 μL of 1% starch solution was added to the centrifuge tube to mix the 1% starch solution with the solution to be reacted to form a mixed solution. The centrifuge tube containing the mixed solution was placed for reaction at 25°C for 10 minutes to form a reaction solution, and then 400 μL of terminator was added and mixed with the reaction solution to stop the reaction between starch and α-amylase.

Then, the three test groups at the reaction start point (0 minutes) (that is, the centrifuge tube with unreacted solution) and the three test groups at the reaction end point (10 minutes) (that is, the centrifuge with the reaction solution) The tube) is placed in boiling water (100°C) to react for 5 minutes, and the centrifuge tubes of the 6 groups of test groups are cooled to room temperature (25°C) to form the test solutions of the 6 groups of test groups.

Take out 150μL of the test solution from the centrifuge tube and mix with 850μL of water to dilute the test solution. Then take 200 μL of the diluted solution to be tested into a 96-well plate, and measure its absorbance at 540 nm.

2-3. Experimental results

According to the following formula (1), calculate the enzyme activity percentage (%) of the amylolytic enzyme of each test group relative to the control group, as shown in Figure 5. In other words, the percentage of enzyme activity of the amylolytic enzyme in the control group is regarded as 100% to calculate the percentage (%) of the enzyme activity of the amylolytic enzyme in the experimental group and the control group.

×100%  （1）Formula (1)% α-Amylase activity =

×100% (1)

Among them,% α-Amylase activity represents the percentage of amylolytic enzyme enzyme activity (%); A _540nm (Sample _10min -Sample _0min ) represents the end point (10 minutes) of the test group. The absorbance at 540nm and the start point of the reaction (0 minutes) The difference between the absorbance values of the test group at 540nm, and this test group is the experimental group or the control group. A _540nm (Control _10min -Control _0min ) represents the difference between the absorbance at 540nm of the control group at the end of the reaction (10 minutes) and the absorbance at 540nm at the start of the reaction (0 min).

Please refer to Figure 5. Compared with the control group (enzyme activity is regarded as 100%), the amylolytic enzyme enzyme activity percentage of the experimental group was 20.9%. In other words, the percentage of enzyme activity of amylolytic enzymes in the experimental group was significantly lower than that in the control group by 79.1% (about 80%). Moreover, compared with the control group, the percentage of enzyme activity of amylolytic enzymes in the control group was 77.7%. In other words, the percentage of enzyme activity of amylolytic enzymes in the control group decreased by 22.3% compared to the control group. It can be seen that the inhibitory ability of guarana fermentation broth on the enzyme activity of amylolytic enzymes is significantly higher than the inhibitory ability of guarana water extract on the enzyme activity of amylolytic enzymes.

Experiment 3: α-Glucosidase enzyme activity inhibition test

3-1. Solvent configuration

0.1 mol concentration (M) sodium phosphate buffer (Pi buffer solution): 4.7283 grams of sodium monohydrogen phosphate (purchased from JTBaker, number 3828-01) and 2.028 grams of sodium dihydrogen phosphate (Purchased from Sigma, No. 04270) was mixed and dissolved in 400 ml of reverse osmosis water (RO water), and quantified to 500 ml with a quantitative bottle to obtain a Pi buffer solution with a pH of 7.0.

2.5mM p-Nitrophenyl β-D-glucopyranoside (PNPG): weigh 0.0377 g of PNPG and quantify it with reverse osmosis water (RO water) to 100 ml.

0.2M Sodium Carbonate (Na ₂ CO ₃ ): Weigh 2.1198 g of sodium carbonate and quantify it to 100 ml with RO water as a terminator for α-glucosidase.

0.2 units/ml (units/ml) α-glucosidase (α-glucosidase, purchased from sigma Chemical Co. (St. Louis, MO, G5003-100UN)) solution: Take 3.85 mg of solid α-glucosidase to Dissolve 2.0 ml of 0.1M Pi buffer solution to obtain 50 U/ml α-glucosidase stock solution. Then, take 0.1 ml of 50 U/ml α-glucosidase stock solution and quantify to 25 ml with RO water to obtain 0.2 U/ml α-glucosidase solution. Among them, the activity of α-glucosidase per milligram of solid is 26 units (units).

3-2. Test process

The enzyme activity test of the amylolytic enzymes of each test group was performed according to Table 3 below. Among them, the experimental group (0 minutes), the control group (0 minutes) and the control group (0 minutes) in the test group represent the test groups where α-glucosidase does not react with PNPG (hereinafter referred to as the reaction starting point (0 minutes) Minutes)), while the experimental group (5 minutes), the control group (5 minutes) and the control group (5 minutes) represent the test group where α-glucosidase reacts with PNPG for 5 minutes (hereinafter referred to as the reaction termination point (5 minutes) )).

table 3 Test group testing sample Buffer Reaction enzyme Reaction matrix Terminator Experiment (0 minutes) 5 times diluted guarana fermentation broth Pi buffer solution Pi buffer solution PNPG 0.2M sodium carbonate Experimental group (5 minutes) 5 times diluted guarana fermentation broth Pi buffer solution alpha-glucosidase PNPG 0.2M sodium carbonate Control group (0 minutes) 5 times diluted guarana water extract Pi buffer solution Pi buffer solution PNPG 0.2M sodium carbonate Control group (5 minutes) 5 times diluted guarana water extract Pi buffer solution alpha-glucosidase PNPG 0.2M sodium carbonate Control group (0 minutes) Pi buffer solution Pi buffer solution Pi buffer solution PNPG 0.2M sodium carbonate Control group (5 minutes) Pi buffer solution Pi buffer solution alpha-glucosidase PNPG 0.2M sodium carbonate

In Table 3, the guarana fermentation broth of Example 1 and the guarana aqueous extract of Example 2 were all diluted with water, and each test group was subjected to a triple experiment. The reaction enzyme is 0.2 unit/ml α-glucosidase solution. The reaction substrate for reaction enzyme action is 2.5mM PNPG.

According to Table 3, first, take 160 μL of test samples (ie, 5-fold diluted guarana fermentation broth, 5-fold diluted guarana aqueous extract and Pi buffer solution) into 96-well plates, and then place them in each Add 20 μL of Pi buffer solution to the well to form a solution to be reacted.

Next, add 20 μL of Pi buffer solution to the three test groups at the reaction starting point (0 minutes) to the corresponding wells, mix them with the solution to be reacted, and react at 25°C for 10 minutes. After reacting for 10 minutes, add 20 μL of 2.5 mM PNPG to each well, mix the reaction solution, Pi buffer solution, and PNPG uniformly, and place the reaction solution at 37° C. for 5 minutes to form a 0-minute group reaction solution. 80 μL of terminator was added to the reaction solution of the 0 minute group to stop the activity of α-glucosidase. Then, after the reaction solution and the terminator were mixed uniformly in the 0 minute group, the absorbance at 405 nm was measured.

For the three test groups at the reaction termination point (5 minutes), add 20 μL of 0.2 unit/ml α-glucosidase solution to the corresponding wells and mix them with the solution to be reacted. Then, set the reaction termination point (5 minutes). ) The three test groups were placed at 25°C for 10 minutes to activate α-glucosidase. After reacting for 10 minutes, add 20 μL of 2.5 mM PNPG to each well to allow the activated α-glucosidase to interact with PNPG. The reaction solution, α-glucosidase solution and PNPG are mixed uniformly, and then placed at 37° C. for reaction for 5 minutes to form a 5-minute group reaction solution. 80 μL of terminator was added to the 5-minute group reaction solution to stop the activity of α-glucosidase. Then, after the reaction solution and the terminator were mixed uniformly in the group of 5 minutes, the absorbance value at 405 nm was measured.

3-3. Experimental results

Calculate the enzyme activity percentage (%) of α-glucosidase in the experimental group and the control group according to the following formula (2), as shown in Figure 6. In other words, the percentage of enzyme activity of α-glucosidase in the control group is regarded as 100% to calculate the percentage (%) of the enzyme activity of α-glucosidase in the experimental group and the control group.

×100%  （2）Formula (2)% α-Glucosidase activity =

×100% (2)

Among them,% α-Glucosidase activity represents the enzyme activity percentage (%) of α-glucosidase; A _405nm (Sample _5min -Sample _0min ) represents the end point of the reaction (5 minutes) and the absorbance and reaction of the test group at 405nm The difference between the absorbance values of the test group at the starting point (0 minutes) at 405nm, and this test group is the experimental group or the control group. A _405nm (Sample _5min -Sample _0min ) represents the difference between the absorbance at 405nm of the control group at the reaction termination point (5 minutes) and the absorbance at 405nm of the control group at the reaction start point (0 min).

Please refer to Figure 6. Compared with the control group (enzyme activity is regarded as 100%), the enzyme activity percentage of α-glucosidase in the experimental group is 0%. In other words, the percentage of enzyme activity of α-glucosidase in the experimental group was significantly lower than that of the control group by 100%, indicating that the fermentation broth of guarana completely inhibited the enzyme activity of α-glucosidase. And, compared to the control group, the percentage of α-glucosidase enzyme activity in the control group was 10.2%. In other words, the percentage of α-glucosidase enzyme activity in the control group decreased by 89.8% relative to the control group. It can be seen that the inhibitory ability of guarana fermentation broth on the enzyme activity of α-glucosidase is significantly higher than the inhibitory ability of guarana water extract on the enzyme activity of α-glucosidase. Here, the guarana fermentation broth has the ability to inhibit the activity of α-glucosidase.

Experiment 4: Detection of lipolytic enzyme gene expression

4-1. Test target and solution configuration

The adipose triglyceride lipase (ATGL) gene is used as the target of lipolytic enzyme gene detection.

Here, the conditioned medium (Condition Medium) used is the minimum essential medium α (Minimum Essential Medium Alpha, MEMα, brand: Gibco) supplemented with 20 vol% FBS (brand: Gibco) and 1 vol% penicillin-streptomycin.

4-2. Testing process

First, the mouse bone marrow stromal cell line OP9 (purchased from ATCC ^® , number CRL-2749 ^™ ) was inoculated into each well of a 6-well culture plate containing 2 mL of conditioned medium at a cell number of 1×10 ^{5 cells per well} , And incubate at 37°C for 48 hours. Then, after 48 hours of culture, the 6-well culture plate containing OP9 cells was placed at 37°C for continuous culture for 7 days. And during the 7-10 day culture period, fresh 2mL conditioned medium was replaced every 2 days. After 7-10 days of culture, use a microscope (brand: ZEISS) to observe the formation of lipid droplets in the cells in each well to confirm that OP9 cells are completely differentiated into adipocytes.

Divide fat cells into 3 groups: experimental group, control group and control group. The conditioned medium of each group was removed and replaced with 2mL experimental medium per well, and then placed at 37°C for 48 hours. Among them, the experimental medium of the experimental group was a conditioned medium containing 0.031vol% of the guarana fermentation broth obtained in Example 1. The experimental medium of the control group was a conditioned medium containing 0.031vol% of the aqueous guarana extract obtained in Example 2. The experimental medium of the control group was a simple conditioned medium (that is, no guarana fermentation broth or guarana water extract).

After culturing for 48 hours, the experimental medium in each well was removed and washed once with PBS. Remove PBS, and then use RNA purification kit (brand: GeneMark) to extract RNA from fat cells of each group. Then, the RNA extracted from each group was reverse transcribed into cDNA through the reverse transcriptase kit (SuperScript™ Reverse Transcriptase kit, brand: Invitrogen), and the ABI system (ABI StepOne Plus™ System, brand: Applied Biosystems) was used to cooperate with the two Primer (as shown in Table 4, SEQ ID NO: 1 and SEQ ID NO: 2) quantified the expression of ATGL gene in adipocytes, and used the 2 ^-ΔΔCt method for gene quantification, as shown in Figure 7. It is important to note that the gene performance in Figure 7 is presented in terms of relative performance magnifications, in which the STDEV formula of Excel software is used to calculate the standard deviation, and the statistically significant differences between the groups are calculated by the student t-test. analysis. In Figure 7, "*" represents that the p value is less than 0.05 when compared with the control group.

Table 4 Primer name Serial number sequence ATGL-F SEQ ID NO:1 GGATGGCGGCATTTCAGACA ATGL-R SEQ ID NO: 2 CAAAGGGTTGGGTTGGTTCAG

4-3. Experimental results

Refer to Figure 7. The expression level of the ATGL gene in the control group was regarded as 1.00 (that is, the expression level of the ATGL gene in the control group was 100%). Compared with the control group, the expression level of ATGL gene in the experimental group was 3.30, while the expression level of ATGL gene in the control group was 1.57. Here, the expression level of the ATGL gene in the experimental group was significantly improved compared to the control group, and the expression level of the ATGL gene in the experimental group was also significantly improved compared to the control group. It can be seen that the guarana fermentation broth can effectively improve the expression of the lipolytic enzyme gene, thereby increasing the lipolytic enzyme. In other words, guarana fermentation broth has the function of decomposing fat, thereby avoiding fat accumulation, and achieving the effect of reducing the fat formation and fat reduction of the recipient. In addition, guarana may produce more active ingredients that decompose fat than guarana water extract after fermentation by microorganisms.

Experiment 5: Fat accumulation detection

5-1. Solvent configuration

Here, the pre-adipocyte expansion medium used is a minimum essential medium α (Minimum Essential Medium Alpha, MEMα, supplemented with 20vol% FBS (brand: Gibco)) and 1vol% penicillin-streptomycin. Brand: Gibco). The differentiation medium used was MEMα (brand: Gibco) supplemented with 20vol% FBS (brand: Gibco) and 1vol% penicillin-streptomycin. In addition, the oil-red O staining reagent (brand: Sigma) was completely dissolved in 100% isopropanol (isopropanol, supplier: ECHO) to prepare a 3 mg/mL oil-red O staining reagent stock solution. In order to obtain a usable oil-red O working solution, immediately before use _{, dilute the stock solution of the oil-red O staining reagent with secondary water (ddH 2} O) to a concentration of 1.8 mg/ mL is the stock solution of 60% oil-red O staining reagent.

5-2. Inspection process

First, the mouse bone marrow stromal cell line OP9 (purchased from ATCC ^® , number CRL-2749 ^™ ) was inoculated on a 24-well culture plate containing 500 μL of pre-adipocyte proliferation medium at a cell number of 8×10 ^{4 cells per well.} In each well, incubate at 37°C for 7 days. During the 7-day culture period, fresh 500 μL differentiation medium was replaced every 3 days. After 7 days of culture, use a microscope (brand: ZEISS) to observe the formation of lipid droplets in the cells in each well to confirm that the cells are fully differentiated into adipocytes for subsequent experiments.

After 24 hours of culture, the adipocytes were divided into 3 groups: experimental group, control group and control group. The differentiation medium of each group was removed and replaced with 500μL of experimental medium per well, and then placed at 37°C for continuous cultivation for 7 days. During the 7-day culture period, fresh 500 μL experimental medium was replaced every 3 days. Among them, the experimental medium of the experimental group was a differentiation medium containing 0.062vol% of the guarana fermentation broth obtained in Example 1. The experimental medium of the control group was a differentiation medium containing 0.062vol% of the aqueous guarana extract obtained in Example 2. The experimental medium of the control group was a simple differentiation medium (that is, no guarana fermentation broth or guarana water extract).

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

After acting for 1 hour, remove the oil-red O working solution and quickly destain it with 1 mL of 60% isopropanol for 5 seconds. After destaining, the stained cells were photographed with a microscope to obtain a micrograph of the stained cells, as shown in FIG. 8.

Next, add 100% isopropanol to each well, and place on a shaker to react for 10 minutes to dissolve the dye. Then, take 100 μL of the aforementioned dye-isopropanol solution from each well to a 96-well culture plate and read the absorbance (OD ₅₁₀ ) of each well with an ELISA reader (brand: BioTek) at a wavelength of 510 nm .

After the measurement, the relative content (%) of lipid oil droplets is calculated by substituting the measured absorbance value into the following formula (3). In other words, here, the lipid oil droplet content of the control group is regarded as 1 (that is, the relative lipid oil content of the control group is 100%) to calculate the relative lipid oil droplet content (%) of each group. Moreover, the statistically significant differences between the groups are statistically analyzed by the student t-test, as shown in Figure 9. In Figure 9, "*" means that the p value is less than 0.05 when compared with the control group.

Formula (3) Relative content of lipid oil droplets (%) = (OD ₅₁₀ sample/OD ₅₁₀ control)×100% (3)

Among them, OD ₅₁₀ sample represents the absorbance value of the group to be converted, and OD ₅₁₀ control represents the absorbance value of the control group.

5-3. Experimental results

Please refer to Figure 8. Compared with the control group and the control group, the fat cells of the experimental group have significantly reduced oil droplets. Please refer to Figure 9. Compared with the control group, the relative content of lipid oil droplets in the experimental group is 77.96%, and it can reduce the amount of oil droplets by 22.04%. Therefore, the relative content of lipid oil droplets in the experimental group was significantly lower than that in the control group. Compared with the control group, the relative content of lipid oil droplets in the control group was 95.05%, and it could reduce the amount of oil droplets by 4.95%. Here, the relative content of lipid oil droplets in the experimental group was also significantly lower than that in the control group. It can be seen that the guarana fermentation broth can effectively inhibit fat accumulation, has the function of reducing the fat formation of the receptor, and then achieves the effect of fat reduction. In addition, guarana may produce more fat-reducing active ingredients than guarana water extract after microbial fermentation.

Experiment 6: Human detection

6-1. Inspection process

8 subjects were asked to drink 5 mL of guarana fermented beverage (which contains 12 vol% of the guarana fermentation broth of Example 1 and 88 vol% water) daily for 4 weeks. And, before drinking (that is, the 0th week) and 4 weeks after drinking (that is, the 4th week), measure the weight of these subjects with a weight scale (brand: TANITA BC545N ten-in-one composition) and measure with a cloth ruler The waist circumference of these subjects. In addition, the BMI of 8 subjects (aged between 20-55 years old) was greater than or equal to 24 and less than 27, and the body fat rate of male subjects was greater than 25%, and the body fat rate of female subjects was greater than 30% .

It should be noted that the statistically significant difference between the measurement results of week 0 and the measurement results of week 4 is statistically analyzed by the student t-test, as shown in Figure 10 and Figure 11. In Figure 10 and Figure 11, "*" represents that the p value is less than 0.05 when compared with week 0, and "**" represents that the p value is less than 0.01 when compared with week 0.

6-2. Test results

Please refer to Figure 10 and Figure 11. The average weight of the eight subjects dropped from 68 kg to 67.3 kg (as shown in Figure 10), and the average waist circumference of these subjects decreased from 85.8 cm to 82.9 cm (as shown in Figure 10). 11). In other words, compared with the pre-drinking (week 0), drinking guarana fermented beverage containing guarana fermentation broth for 4 weeks can significantly reduce the average weight of these subjects by 0.7 kg, and make these subjects suffer. The participants’ average waist circumference was significantly reduced by 2.9 cm. It can be seen that long-term use of guarana fermentation broth can improve the body weight and waist circumference of the recipient, that is, guarana fermentation broth has the effect of slimming and reducing fat.

In summary, the method for preparing guarana fermentation broth according to any embodiment of the present invention can prepare a guarana fermentation broth. In some embodiments, the prepared guarana fermentation broth can be used to prepare a composition that reduces the fat formation of the recipient. In other words, the aforementioned composition has one or more of the following functions: enhancing antioxidant activity, inhibiting amylolytic enzyme activity, inhibiting α-glucosidase activity, improving lipolytic enzyme gene expression, inhibiting fat accumulation, and reducing receptor fat Formation and weight loss.

Although the technical content of the present invention has been disclosed in the preferred embodiments as above, it is not intended to limit the present invention. Anyone who is familiar with this technique and makes some changes and modifications without departing from the spirit of the present invention should be covered by the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

S100~S500: steps

Figure 1 is a flow chart of the preparation of guarana fermentation broth; FIG. 2 is a detailed flowchart of step S100 in FIG. 1; FIG. 3 is a detailed flowchart of step S300 in FIG. 1; Figure 4 is a graph showing the measurement results of polyphenol content in guarana fermentation broth; Figure 5 is a graph showing the relative measurement results of the enzyme activity of amylolytic enzymes; Figure 6 is a graph showing the relative measurement results of the enzyme activity of α-glucosidase; Figure 7 is a graph showing the magnification results of the ATGL gene; Figure 8 is a photograph of Oil Red O staining; Figure 9 is a graph showing the relative measurement results of lipid and oil content; Figure 10 is a graph showing the results of weight data at week 0 and week 4; and Figure 11 is a graph showing the results of waist circumference data in the 0th week and the 4th week.

S100~S500: steps

Claims (9)

A method for preparing guarana fermentation broth, comprising: providing a culture broth, the culture broth comprising a guarana extract formed by 1 part by weight of guarana (Paullinia cupana ) and 10 parts by weight of water and the Glucose of 10% of the total weight of the guarana extract; ferment the culture broth and plural strains for 7 days to obtain a fermentation stock solution, wherein the strains include 0.1% yeasts relative to the culture broth, relative 0.05% lactic acid bacteria in the culture solution and 5% acetic acid bacteria in the culture solution; and adjusting the fermentation stock solution to form the guarana fermentation solution. The method for preparing guarana fermentation broth according to claim 1, wherein the step of fermenting the culture broth and the strains comprises: fermenting the yeast in the culture broth for 1 day to form a first initial Fermentation broth, wherein the yeast is Saccharomyces cerevisiae ; adding the lactic acid bacteria in the first primary fermentation broth to ferment for 1 day to form a second primary fermentation broth, wherein the lactic acid bacteria is Lactobacillus plantarum ; adding The acetic acid bacteria are fermented in the second primary fermentation broth for 5 days to form a third primary fermentation broth, wherein the acetic acid bacteria are Acetobacter aceti ; and the third primary fermentation broth is filtered to obtain the fermentation stock. The method for preparing guarana fermentation broth according to claim 2, wherein the step of filtering the third primary fermentation broth includes: Concentrate under reduced pressure at 60°C and filter the third primary fermentation broth with 200 mesh. The method for preparing guarana fermentation broth according to claim 1, wherein the step of providing the culture broth includes: Mix the guarana and the water to form a guarana base liquid; Extract the guarana base liquid at 95°C for 1 hour to obtain the guarana extract; and The guarana extract is mixed with the glucose to obtain the culture broth. The method for preparing guarana fermentation broth according to claim 1, wherein the step of adjusting the fermentation stock to form the guarana fermentation broth is to adjust the sugar content of the fermentation stock to form the guarana fermentation broth. The method for preparing guarana fermentation broth according to claim 1, wherein the total polyphenol content of the guarana fermentation broth is more than 1600 μg/ml. The use of a guarana fermentation broth prepared by a method for preparing a guarana fermentation broth for preparing a composition that reduces the formation of a receptor, wherein the guarana fermentation broth is used to increase a lipolytic enzyme ( ATGL) gene expression to reduce fat formation. Use of a guarana fermentation broth prepared by a method for preparing guarana fermentation broth for preparing a composition for reducing the formation of a receptor, wherein the guarana fermentation broth is used to reduce the formation of lipid oil droplets To reduce fat formation. The use of a guarana fermentation broth prepared by a method for preparing a guarana fermentation broth for preparing a composition for reducing the formation of a receptor, wherein the guarana fermentation broth is used to reduce amylolytic enzymes and alpha -Glucosidase activity and reduce the heat absorption after starch is broken down into sugars to reduce fat formation.

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