TWI737086B - Fermentation method for increasing content of effective components in plants - Google Patents

Abstract

The present disclosure provides a fermentation method for increasing content of effective components in plants.

Description

Fermentation method for increasing the content of plant neutral components

The present invention relates to a fermentation method for increasing the content of plant neutral components.

Fat is an essential component in the human body, but excessive fat content can cause damage to the human body. In countries where the national economy is taking off, the obesity problem is only increasing. Therefore, from the perspective of the Asian region, China will be the next wave of countries where the increase in obesity problems will increase sharply.

In addition, Taiwan has now become "the fattest country in Asia." Among the people over 18 years old, the proportion of men who are overweight is 51.1%, and the proportion of women who are overweight is 35.8%, and the proportion is still rising, even being overweight. The age group is also declining year by year, and the proportion of overweight students in elementary and middle schools has risen to more than 25%, making it the eighth-highest region in the world. Therefore, obesity is a problem that needs to be solved urgently.

In addition, glycation is related to the aging of the skin and the body, and it is also related to diabetes, which is very harmful to the health of Chinese people. The essence of saccharification reaction is Maillard reaction or carbonyl-amine browning, which is the combination of aldehyde (ketone) group of sugar and amine-containing substances (such as protein, peptide, amine group). A nonenzymatic glycosylation (glycation) reaction between the amine groups of acids, phospholipids, nucleic acids, or derivatives of the above. Under the symptoms of hyperglycemia for a long time, it will cause glucose auto-oxidation and protein glycation to form advanced glycation end products (AGEs). And AGEs can cause a variety of aging diseases, such as skin wrinkles, cataracts, atherosclerosis, kidney failure and so on.

Skin tissue is composed of epidermis, dermis and subcutaneous tissue. The dermis contains a lot of collagen and hyaluronic acid, which are closely related to the water retention and elasticity of the skin. Human skin will experience oxidation, aging, rough skin or wrinkles with age, physiological factors or environmental factors, such as Normal young people’s skin has a certain degree of elasticity and tension. When the expression muscles relax, the skin will quickly recover and the wrinkles will disappear; but after entering middle age, the skin begins to age significantly, the skin becomes thinner, harder, dry, and tensioned. Decrease; dermal collagen decreases, elastic fibers degeneration, and breakage, which reduces the tension and elasticity of the skin. Therefore, when the expression muscles are relaxed, the skin cannot recover quickly, and wrinkles are formed over time; and with age, the skin becomes weaker The subcutaneous tissue is more relaxed, coupled with the atrophy or lack of facial support tissues, and the softness of muscles, the skin will fall under the action of gravity, forming deeper wrinkles. Rough skin is caused by externally important factors such as dryness, ultraviolet rays, cleansers, chemical substances and other irritating substances, or internal important factors such as hormonal balance disorder, and is accompanied by a decline in the barrier function of the stratum corneum. Phenomenon such as the decrease of water content, the increase of epidermal metabolism, the production of scales and the roughening of keratin. Therefore, if the cells on the skin lose their elasticity and moisturizing function, it will cause skin wrinkles, dryness, and loss of luster.

For a long time, the manufacture of fermented food has relied on microorganisms. Microorganisms are integrated into the raw materials. During their growth, they will produce the required enzymes. The process of decomposing organic matter into beneficial substances in human life is called fermentation; therefore, this metabolic activity is actually catalyzed. The performer is the enzyme produced by the microorganism. The range of products obtained by fermentation is extremely wide, ranging from extremely high unit price pharmaceutical-grade products, such as lipid-lowering drugs or antibiotics, to industrial enzymes, such as starch decomposition, fiber decomposition, and proteolytic enzymes; there are also beneficial food products. Microorganisms, such as lactic acid bacteria, red yeast bacteria, natto bacteria, edible and medicinal mushrooms, as well as seasonings of monosodium glutamate, agricultural biological insecticides, such as suli bacteria, or antagonism such as Bacillus subtilis, actinomycetes, and Trichoderma Fungus microorganisms; even, from animal feed medicines or microorganisms directly added to feed, to waste recycling, various types of pollution treatment and cleaning microorganisms, and other special enzymes. In particular, in recent years, some researchers have begun to ferment plants (such as fruits and vegetables), and the obtained plant fermented products can be used to improve human health. Therefore, people's demand for fermented food has increased day by day.

However, the traditional fermentation process used to prepare fermented products often takes a lot of time for the growth of microorganisms, and requires industrial-grade fermentation equipment, such as a fermentation tank, which is extremely costly. Therefore, if a novel method for fermenting plants can be developed, and the method can be used to increase the growth rate of brewer's yeast and the content of the effective components of the plant, this will bring a major breakthrough to the technology in the field and benefit those in need The vast ethnic group.

In view of this, the object of the present invention is to provide a method for fermenting plants, comprising the following steps: (a) extracting a plant with water to obtain a plant extract; and (b) combining the plant extract with a beer Saccharomyces cerevisiae , Lactobacillus plantarum , and Acetobacter aceti sequentially undergo a fermentation process to obtain a plant fermentation product, wherein the fermentation process includes the plant extract, the beer yeast The bacteria, the germ lactic acid bacteria, and the acetic acid bacteria irradiate a red light source with a wavelength between 620 nm and 750 nm.

In an embodiment of the present invention, the plant is rambutan ( Nephelium lappaceum ).

In an embodiment of the present invention, before the extraction, glucose with a concentration of 10% (w/w) is added according to the total weight.

In an embodiment of the present invention, the fermentation time of the beer yeast is 1 to 2.5 days, the fermentation time of the germ lactic acid bacteria is 1 to 3 days, and the fermentation time of the acetic acid bacteria is 3 to 10 days.

In an embodiment of the present invention, the concentration of the saccharomyces cerevisiae is between 0.01 and 0.5% (v/v), the concentration of the germ lactic acid bacteria is between 0.01 and 0.25% (v/v), and the concentration of the acetic acid bacteria Between 3~10%(v/v).

In an embodiment of the present invention, the weight ratio of the plant to water is 1:5-10.

In an embodiment of the present invention, in step (a), the temperature of the extraction is between 50 and 100° C., and the time of the extraction is between 0.5 and 1.5 hours.

Another object of the present invention is to provide an application of the method as described above for increasing the growth rate of Saccharomyces cerevisiae and the content of effective components in plants.

In an embodiment of the present invention, the plant is rambutan ( Nephelium lappaceum ), and the active ingredient is polyphenol.

Another object of the present invention is to provide a plant fermented product, which is prepared by a method as described above.

Another object of the present invention is to provide a use of the aforementioned plant fermented product for preparing a skin care and fat-reducing composition.

In an embodiment of the present invention, the skin care includes enhancing the antioxidant capacity of the skin, enhancing the anti-glycation activity of the skin, and promoting the proliferation of elastin.

In an embodiment of the present invention, the fat reduction includes promoting lipolysis and increasing leptin secretion.

In an embodiment of the present invention, the composition is in the form of a medicine, a skin care product or a food product.

Another object of the present invention is to provide a method for increasing the growth rate of microorganisms, comprising the following steps: (a) extracting a plant with water to obtain a plant extract; and (b) combining the plant extract with a plant extract The microorganism undergoes a fermentation process to obtain a plant fermented product, wherein the microorganism is a Saccharomyces cerevisiae or Acetobacter aceti . The fermentation process includes irradiating the plant extract and the microorganism with a wavelength medium. Red light source at 620~750nm.

In an embodiment of the present invention, the plant is rambutan ( Nephelium lappaceum ).

In an embodiment of the present invention, before the extraction, glucose with a concentration of 10% (w/w) is added according to the total weight.

In an embodiment of the present invention, the fermentation time of the microorganism is 1 to 3 days.

In an embodiment of the present invention, the concentration of the beer yeast is between 0.01 and 0.5% (v/v), and the concentration of the acetic acid bacteria is between 5% (v/v).

In an embodiment of the present invention, the growth rate of the beer yeast is increased by at least 47%, and the growth rate of the acetic acid bacteria is increased by at least 22%.

In summary, the method of the present invention for fermenting plants and the effect of the plant fermented product prepared by the method are: it can increase the growth rate of microorganisms (such as brewer's yeast and acetic acid bacteria) and the content of effective components in plants , And by enhancing the skin's antioxidant capacity, enhancing the skin's anti-glycation activity and promoting the proliferation of elastin to achieve the effect of skin care and beautiful skin. On the other hand, the plant fermented product can achieve the effect of reducing fat by promoting lipolysis and increasing the secretion of leptin.

The following will further explain the implementation of the present invention. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with the art will not depart from the spirit and scope of the present invention. Some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 is a data chart of the microbial growth rate detection.

Figure 2 is a data diagram of the analysis of the content of the effective components in the plant, where *** means compared with the control group, p <0.001.

Fig. 3 is a data chart of the effectiveness of the plant fermented product prepared by the method of the present invention in enhancing the antioxidant capacity of the skin, where * represents the comparison with the control group, and p <0.05.

Fig. 4 is a data diagram of the efficacy of the plant fermented product prepared by the method of the present invention in enhancing the anti-glycation activity of the skin, where * represents the comparison with the control group, p <0.05.

Figure 5 is a data diagram of the efficacy of plant fermented products prepared by the method of the present invention in promoting the proliferation of elastin.

Figures 6A and 6B are cell staining diagrams showing the effect of plant fermented product prepared by the method of the present invention on promoting the proliferation of elastin, where blue represents the nucleus and green represents elastin.

Fig. 7 is a data chart of the effect of plant fermented product prepared by the method of the present invention on promoting lipolysis, wherein * represents the comparison with the control group, and p <0.05.

Fig. 8 is a data chart showing the effect of plant fermented product prepared by the method of the present invention on increasing the secretion of leptin.

Fig. 9 is a data diagram showing the efficacy of the method for increasing the growth rate of microorganisms of the present invention in increasing the growth rate of Saccharomyces cerevisiae.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Use Excel software for statistical analysis. Data mean ± standard deviation (SD) represented by the difference between the two herein by Student t test (student's t -test) analysis.

According to the present invention, rambutan ( Nephelium lappaceum ) is a large tropical fruit tree belonging to the genus Nephelium in the Sapindaceae family. Ripe rambutan fruits are not all red, but there are also yellow fruits. The core size of some rambutans is similar to that of sesame. The taste of rambutan is similar to that of lychee. The flesh (aril) is white, the flesh is transparent and juicy, sweet and sour, and contains 1 seed, which is flat oval.

As used in this article, the terms " Saccharomyces cerevisiae ", " Lactobacillus plantarum " and " Acetobacter aceti " are intended to cover beer that can be easily obtained by those skilled in the art. Yeast, germ lactic acid bacteria and acetic acid bacteria (for example, can be purchased from domestic or foreign depository institutions), or beer yeast and germ isolated and purified from natural sources using the usual microbial isolation method in this art Lactic acid bacteria and acetic acid bacteria strains.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally, orally or topically by using techniques well known to those skilled in the art, including: But not limited to: injection [for example, sterile aqueous solution or dispersion], sterile powder, tablet, troche, oral Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir (elixir), thick slurry (slurry), external preparation (external preparation) and the like.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvent, buffer, emulsifier, suspending 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 Things. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), Aqueous solution containing alcohol and combinations thereof.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intradermal injection Injection (intraepidermal injection), intradermal injection (intradermal injection), intramuscular injection (intramuscular injection), intravenous injection (intravenous injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serum, paste, foam, drop, suspension, salve and bandage.

According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and White petrolatum], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers ( absorption enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents (occlusive agents), emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants (propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the skin care product can further include an acceptable adjuvant that is widely used in skin care product manufacturing technology. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the skin care product can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution, water -Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel Glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, makeup remover products, soap, and other body cleansing products.

According to the present invention, skin care products can also be used in combination with one or more external use agents with known activities selected from the following: whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [ Such as aloe extract, skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, and anti-dermatitis agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, corticosteroids Corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

Example 1. Microbial growth rate detection

First, the rambutan (Nephelium lappaceum ) juice from Rayong, southern Thailand is mixed with water in a weight ratio of 1:5-10, and glucose with a concentration of 10% (w/w) is added according to the total weight. Then, extract at 50°C-100°C for 0.5-1.5 hours to obtain a plant extract.

Then, after the plant extract culture medium is colonized with Saccharomyces cerevisiae BCRC 20271 and the fermentation begins, 1 mL of the bacterial liquid is taken out every 24 hours, and after serial dilution, the bacterial liquid is applied to Yeast peptone dextrose, YPD) plate, wherein the whole fermentation process is irradiated with a red LED light source with a wavelength of 620~750nm (preferably 635nm) to obtain the plant fermented product irradiated with the red LED light source. After that, the plant fermented product irradiated with the red LED light source was used as the experimental group. The preparation method of the plant fermented product of the comparison group was roughly the same as that of the experimental group, except that the red LED was replaced by blue light with a wavelength of 400~450nm Light source: As for the preparation method of the plant fermented product of the control group, the preparation method is roughly the same as that of the experimental group, except that the red LED light source is replaced by natural light. Next, place the YPD plate coated with bacterial solution in an incubator at 30°C for 48 hours, and then count the colonies to evaluate the growth rate of the brewer's yeast. The experimental results are shown in Figure 1.

Figure 1 is a data chart of the microbial growth rate detection. It can be seen from Figure 1 that compared with the control group and the comparison group, the growth rate of S. cerevisiae in the experimental group has been significantly improved. In particular, during the fermentation process, irradiating a red LED light source with a wavelength of 620 to 750 nm throughout the fermentation process can increase the growth rate of beer yeast by 47% (compared to the control group), whereas irradiating natural light and blue light has no effect on the growth rate. The results of this embodiment show that irradiating a red LED light source during the fermentation of plants can effectively increase the growth rate of brewer's yeast.

Example 2. Analysis of the content of plant neutral components

First, prepare a standard solution, dissolve 10 g of gallic acid in water and add a volume of 10 mL to a measuring flask. Next, prepare 0 μL/mL, 20 μL/mL, 40 μL/mL, 60 μL/mL, 80 μL/mL, and 100 μL/mL standard solutions, and then take 100 μL of each standard solution to a centrifuge tube having a volume of 10 mL. After that, add 500 μL of Folin-Ciocalteu's phenol reagent, mix and stand upright for 3 minutes, then add 400 μL of 7.5% sodium carbonate (sodi μM carbonate), mix and stand upright for 30 minutes. Then, 200 μL of each reaction solution was transferred to a 96-well plate, and the absorbance was measured at 750 nm.

In addition, the plant fermented product irradiated with the red LED light source described in Example 1 was used as the experimental group. The preparation method of the plant fermented product of the control group was roughly the same as that of the experimental group, except that natural light was used instead of the red LED light source. . The experimental group and the control group were respectively diluted five times with water and a volume of 100 mL was transferred to a microcentrifuge tube. After that, add 500 μL of phoschorol reagent, mix and stand upright for 3 minutes, and then add 400 μL of 7.5% sodium carbonate, mix and stand upright for 30 minutes. Then, 200 μL of the reaction solutions of each group were transferred to a 96-well plate, and the absorbance was measured at 750 nm. The results of the total polyphenol content are shown in Figure 2.

Figure 2 is a data diagram of the analysis of the content of effective components in plants. It can be seen from Figure 2 that compared with the control group, the total polyphenol content of the experimental group has a significant increase (up 60%). The results of this embodiment show that irradiating a red LED light source during the fermentation of plants can effectively increase the content of neutral components (that is, total polyphenols) in plants (that is, rambutans).

Example 3. The manufacturing process of the method for fermenting plants of the present invention

Based on the results of Example 1 and Example 2, the present invention decided to use a red LED light source to prepare plant fermented products. First, the rambutan (Nephelium lappaceum ) juice from Rayong, southern Thailand is mixed with water in a weight ratio of 1:5-10, and glucose with a concentration of 10% (w/w) is added according to the total weight. Then, extract at 50°C-100°C for 0.5-1.5 hours to obtain a plant extract. The plant extract is cooled to room temperature for subsequent three-stage fermentation. The three-stage fermentation system is to inoculate plant extracts in sequence with 0.01~0.5% (v/v) Saccharomyces cerevisiae (Saccharomyces cerevisiae) BCRC 20271 for 1~2.5 days; inoculate 0.01~0.25% (v/v) germ lactic acid bacteria ( Lactobacillus plantarum ) TCI028 (deposited at the Biological Resources Preservation and Research Center of the Food Industry Development Institute on December 13, 2006, deposit number BCRC910805) Fermentation for 1~3 days; inoculation of 3~10% (v/v) acetic acid bacteria ( Acetobacter aceti ) BCRC 11688 (the above strains are all purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan) fermentation 3 ~10 days, and irradiate a red LED light source with a wavelength of 620~750nm (preferably 635nm) throughout the fermentation process. After that, it was concentrated under reduced pressure at 45°C to 70°C, and then filtered with a mesh of 200 to 400 mesh. Next, add 40 to 70% isomalto-oligosaccharides and sterilize to obtain a plant fermented product.

Example 4. Evaluation of the effectiveness of the plant fermented product prepared by the method for fermenting plants of the present invention in enhancing the antioxidant capacity of the skin

According to reports, after the age of 35, the body's antioxidant capacity drops drastically, free radicals accumulate, and skin ages rapidly. Therefore, this example explores whether the plant fermented product prepared according to the method of Example 3 can improve the antioxidant capacity of the skin. Use Ferric reducing antioxidant power (FRAP) colorimetry to determine the antioxidant capacity of the test substance; among them, the test substance with reducing activity can be used to reduce iron ions (Fe ³⁺ ) It becomes ferrous ion (Fe ²⁺ ), and the color of the solution turns from reddish brown to green. The intensity of the green compound is proportional to the reducing power, so it can be used to quantify the reducing activity of the test substance.

First, a standard curve is prepared using ascorbic acid, which is known to have reducing activity. Accurately weigh 10mL of L-Ascorbic acid (L-Ascorbic acid, Vit C, Vitamin C, purchased from Sigma, USA, No. A5960-25g), place it in a 10mL volumetric flask, add ddH ₂ O to quantify to 10mL, and prepare It is 1mg/mL of L-ascorbic acid. Next, take 1mL of the prepared L-ascorbic acid and place it in a 10mL volumetric flask, then add ddH ₂ O to quantify to 10mL, prepare 1μg/mL L-ascorbic acid, and dilute the 100μg/mL L-ascorbic acid serially Make 0, 10, 20, 40, 60, 80, and 100μL/mL L-ascorbic acid (as shown in Table 1), and respectively take 250μL of each concentration standard solution into the test tube, and then add 250μL of phosphate to each Buffer solution (prepared by mixing anhydrous sodium dihydrogen phosphate (NaH ₂ PO ₄ , purchased from JTBaker, code 3828-01) and disodium hydrogen phosphate (Na ₂ HPO ₄ , purchased from Sigma, code 04270) in a ratio of 1:1 After mixing with a vibrator (Vortex), add 250μL of 1% red blood salt (Potassium ferricyanide, K ₃ Fe (CN), purchased from Sigma, No. 244023) and mix with a vibrator at 50°C. Heat in a water bath for 20 minutes, then add 250 μL of 10% trichloroacetic acid (TCA, CCl ₃ COOH, purchased from JT Baker, No. 0414-01), mix well with a shaker, and centrifuge at 300 g for 10 minutes. And be careful not to shake when taking it out, then take out 300μL of the supernatant of each tube reaction solution, then add 300μL of ddH ₂ O and 120μL of 1% ferric chloride (FeCl ₃ , purchased from Alfa Aesar, number A16231) to shake After mixing the instrument evenly, let it react for 10 minutes, and measure its absorbance at 700nm to draw the regression curve formula of the standard solution.

Next, the plant fermented product prepared in Example 3 was used as the experimental group. The preparation method of the plant fermented product of the comparative group was roughly the same as the method for fermenting plants described in Example 3, except that: Replace the red LED light source with natural light. In addition, the plant extract of Example 3 was used as a control group. After the samples of each group were diluted 20 times, follow-up experiments were carried out. After that, take 250μL of each group of samples into a 10mL glass test tube, then add 250μL of phosphate buffer solution and mix well with a shaker, add 250μL of 1% red blood salt and mix well with a shaker, and then mix well with a shaker. Heat in a water bath at ℃ for 20 minutes, then add 250μL of 10% trichloroacetic acid to mix well with a shaker, centrifuge at 300g for 10 minutes, and take care not to shake when taking it out, then take out 300μL of the supernatant of each tube reaction solution , Then add 300 μL of ddH ₂ O and 120 μL of 1% ferric chloride, mix well with a shaker, and react for 10 minutes, and measure its absorbance at 700 nm, and then use the regression curve formula of the above standard solution to use the inner difference method After calculating the concentration, multiply the dilution factor to obtain the reduction ability of each group of samples. The results of this example are shown in FIG. 3.

Fig. 3 is a data diagram showing the effectiveness of the plant fermented product prepared by the method of the present invention in enhancing the antioxidant capacity of the skin. It can be seen from Figure 3 that compared with the control group and the comparison group, the reduction ability of the experimental group has been significantly improved. In particular, compared with the control group, the reduction ability of the experimental group increased by 57%. The results of this example show that the plant fermented product prepared by the method for fermenting plants of the present invention can effectively enhance the antioxidant capacity of the skin.

Example 5. Evaluation of the effectiveness of the plant fermented product prepared by the method for fermenting plants of the present invention in enhancing the anti-glycation activity of the skin

This example is to test the anti-saccharification activity of the plant fermented product prepared by the method for fermenting plants of the present invention. Therefore, the efficiency of saccharification of collagen (Collagen) produced by inhibiting D-fructose is used to perform saccharification. Quantification of activity. Sugar molecules and proteins are cross-linked to form advanced glycation end-products (AGEs), which trigger oxidative stress in cells and cause skin aging and damage. First, the plant fermented product prepared in Example 3 was used as the experimental group. The preparation method of the plant fermented product of Comparative Group 1 was roughly the same as the method for fermenting plants described in Example 3, except that : Substituting natural light for the red LED light source. The preparation method of Comparative Group 2 is different from the method for fermenting plants described in Example 3 in that the three-stage fermentation and red LED light source are not carried out after the plant extract is obtained by water extraction. Irradiate. As for the control method, there is no light and no plant fermentation or extracts are added. The samples of each group were diluted 10 times and then the follow-up experiment was carried out. After that, add 0.2mL of 60mg/mL collagen solution (prepared with 200mM sodium phosphate buffer, pH 7.4) containing 0.06% of NaN _{3, and add 0.2mL 1.5M D-fructose (with 200mM sodium phosphate)} Buffer preparation, pH 7.4) The solution is uniformly mixed, 0.1 mL of the mixed solution is taken as the origin product, and the remaining mixed solution is reacted at 50°C for 24 hours, and 0.1 mL is taken as the end product, and then the origin product and the The end product is the fluorescence value of excitation wavelength 360nm and emission wavelength 460nm. Among them, Aminoguanidine (AG) is known to have the effect of inhibiting glycation. Finally, the following formula is used to calculate the efficiency of the ability to eliminate AGEs to represent its anti-glycation activity. The less highly glycated end products produced, the higher the anti-glycation activity.

The results of this example are shown in FIG. 4. Figure 4 is a data diagram of the efficacy of plant fermented products prepared by the method of the present invention in enhancing the anti-glycation activity of the skin. It can be seen from Figure 4 that compared with the control group, the comparison group 1 and the comparison group 2, the glycation ratio percentage of the experimental group has a significant decrease. Among them, compared with the control group, the percentage of saccharification in the experimental group was reduced by about 55%. The results of this example show that the plant fermented product prepared by the method for fermenting plants of the present invention can effectively improve its ability to remove highly saccharification end products and has excellent anti-saccharification activity of the skin.

Example 6. Evaluation of the utility of the plant fermented product prepared by the method for fermenting plants of the present invention in promoting the proliferation of elastin

In the skin, elastin and collagen cooperate with each other to help support the structure of the skin, enhance the elasticity of the skin, strengthen the skin's firmness, and reduce the production of wrinkles. Therefore, this example explores whether the plant fermented product prepared according to the method of Example 3 can promote the proliferation of elastin. First, the plant fermented product (concentration of 0.0625%) prepared in Example 3 was used as the experimental group. The preparation method of the plant fermented product of the comparative group was roughly the same as the method for fermenting plants described in Example 3. , The difference is: replace the red LED light source with natural light. As for the control method, there is no light and no plant fermentation or extracts are added.

Next, perform elastin content analysis. The process is as follows: Use Fastin ^{TM Elastin Assay kit to inoculate 1×10 5} cells/well with 2 mL of medium in a 6-well plate. The cell line used is Human skin fibroblasts CCD-966sk (ATCC ^® number is CRL-1881), the medium is the minimum essential medium (MEM) (supplemented with 10% FBS, 1% penicillin/streptomycin (Penicillin/streptomycin) and 1mM sodium pyruvate (Gibco)) and incubated at 37°C for 24 hours. After that, the samples of each group were treated with 2 mL of medium, and then acted for 48 hours. Then, the medium was removed and washed once with 1X PBS, and then the cells were treated with trypsin for 3 minutes. Stop trypsin activity with culture medium, then transfer to 1.5 mL tube. Next, the cells were collected with trypsin, then transferred to a 1.5 mL microcentrifuge tube, centrifuged at 300 xg for 5 minutes, and then washed with PBS (Gibco). After that, centrifuge at 300 xg for 5 minutes, and then keep the cell pellet in about 300 μL of PBS. Next, 100 μL of 1.0 M oxalic acid was added to 300 μL of cell suspension, and the mixture was reacted in a heating block at 100° C. for 1 hour. Add an equal volume of Elastin Precipitating Reagent to each test tube, cover the test tube and vortex briefly to mix, and let it stand for 15 minutes to complete the precipitation. After that, centrifuge at 10,000 xg for 10 minutes to drain the liquid content of the test tube, and then gently tap the inverted test tube onto a paper towel to remove most of the remaining fluid in the test tube. Next, add 1 mL of dye reagent, mix the contents by inverting the test tube, and then place the test tube on the mechanical shaker for 90 minutes. After emptying the unbound dye tube, the elastin-dye complex can be observed as a reddish brown deposit in the bottom and inner lower wall of the test tube. Add 250μL of Dye Dissociation Reagent to each test tube, cover the test tube, and release the dye into the solution by vortexing the mixer to ensure that all the bound dye has entered the solution. After that, the contents of each test tube were transferred to a 96-well plate, and then the plate was placed in an ELISA reader (BioTek), and the absorbance was measured at 513nm.

Then, perform elastin immunofluorescence staining. The procedure is as follows: 5×10 ³ human skin fibroblasts CCD-966sk (BCRC number 60153) are inoculated on each well of the chamber slide, and the medium is the minimum essential medium (MEM). ) (Eagle) (prepared in Earle's Balanced Salt Solution (Earle's BSS), GIBCO, No. 41500-034, USA) (added with 0.1mM non-essential amino acid, 1.5g/L sodium bicarbonate (sodium bicarbonate), 1mM sodium pyruvate (90%), and 10% fetal bovine serum (FBS) (Gibco)), and incubate overnight. Afterwards, fix the cells with 4% paraformaldehyde at room temperature for 10 minutes, then wash 3 times with 1X PBS, and then add 0.2% Triton X-100 (in 1X PBS (Gibco)) to penetrate the cells , And act for 10 minutes at room temperature. Then, add 1% BSA (in 1X PBS), block at 37°C for 1 hour, and then wash 3 times with 1X PBS. After that, an antibody (elastin antibody (Merck; MAB2503) was added, diluted with 1% BSA, and reacted at 37°C for 1 hour), and washed with 1X PBS for 3 times. Then, the secondary antibody (anti-mouse-alexa 488 antibody (Thermo), diluted with 1% BSA, and reacted at 37° C. for 1 hour) was added, and washed with 1X PBS 3 times. After that, add Hoechst 33342 (Thermo) and incubate at room temperature for 3~5 minutes, then wash 3 times with 1X PBS. Then, the glass slide was fixed with a mounting gel and observed under a fluorescent microscope. The results of this example are shown in Figs. 5 and 6A and 6B.

Figure 5 is a data diagram of the efficacy of plant fermented products prepared by the method of the present invention in promoting the proliferation of elastin. Fig. 6A and Fig. 6B are cell staining diagrams showing the effect of the plant fermented product prepared by the method of the present invention on promoting the proliferation of elastin. It can be seen from Figure 5 that compared with the control group and the comparison group, the elastin content of the experimental group has a significant increase. Among them, compared with the control group, the elastin content of the experimental group increased by 13%. It can be seen from Figure 6A and Figure 6B that the cell staining diagram of the plant fermented product prepared by the method of the present invention in promoting the proliferation of elastin, where blue represents the nucleus and green represents elastin. Fig. 6A is the control group of Fig. 5, and Fig. 6B is the experimental group of Fig. 5. Observation results under a fluorescent microscope show that there is more elastin under the action of plant fermentation products, and the elastin content of Fig. 5 is increased by 13% after data conversion. The results of this example show that the plant fermented product prepared by the method for fermenting plants of the present invention can effectively promote the proliferation of elastin.

Example 7. Evaluation of the effectiveness of the plant fermented product prepared by the method for fermenting plants of the present invention in promoting lipolysis

In this example, mouse bone marrow stromal cells were used to test the efficacy of the plant fermented product prepared by the method for fermenting plants of the present invention in promoting lipolysis. The mouse bone marrow stromal cell OP9 line was purchased from the American Type Culture Collection (ATCC ^® ), numbered CRL-2749 ^TM . The cells are cultured in pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) before differentiation, which contains 90% of the minimum essential medium Alpha medium (minimum essential medium alpha medium, purchased from Gibco, USA, 12100-046) , 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA), and 1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA); and use differentiation medium (Differentiation Medium) Differentiate mouse bone marrow stromal cells, which contains 90% minimum essential medium Alpha medium, 20% fetal bovine serum, and 1% penicillin/streptomycin.

To demonstrate the present invention for the fermentation of plant obtained fermented plant having the effect of promoting lipolysis, first mouse bone marrow stromal cells to differentiate into adipocytes (adipocyte), practices 8 × 10 ⁴ to mouse bone marrow stromal Cell OP9 and 500 μL of pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) were seeded in a 24-well culture dish and cultured at 37°C for 7 days, with fresh differentiation medium replaced every 3 days. Next, use a microscope (ZEISS) to observe the formation of lipid droplets to ensure that the cells have fully differentiated.

After that, OP9 cells were divided into 3 groups, including 1 control group, 1 comparison group and 1 experimental group. The plant fermented product obtained according to the method described in Example 3 was added to the cells of the experimental group at a concentration of 0.0625%. The preparation method of the substances added to the cells of the comparison group is roughly the same as the method for fermenting plants described in Example 3, except that the red LED light source is replaced by natural light. As for the cells in the control group, no treatment was done. Then, the cells of each group were cultured for 7-10 days, and the medium was changed every 3 days. After that, refer to the user manual of Cayman's Glycerol cell-based assay kit to perform glycerol assay on each group of cells.

Glycerin is the final product of lipolysis, and the rate of lipolysis is tested by detecting the amount of glycerol produced. Collect the cell culture supernatant from each well of the culture plate, and then take 25 μL of the cell culture supernatant and standard into a new 96-well culture plate. After that, 100 μL of modified Free Glycerol Assay Reagent was added to each well, and then reacted at room temperature for 15 minutes. _{Then, the OD 540nm} absorbance value of each group was read with an ELISA reader (BioTek).

The results of this example are shown in FIG. 7. Fig. 7 is a data chart of the effect of plant fermented product prepared by the method of the present invention on promoting lipolysis. It can be seen from Figure 7 that compared with the control group and the comparison group Bottom, the oil droplet content of the experimental group was significantly reduced. The results of this example show that the plant fermented product prepared by the method for fermenting plants of the present invention has the effect of promoting fat decomposition.

Example 8. Evaluation of the utility of the plant fermented product prepared by the method for fermenting plants of the present invention in increasing the secretion of leptin

Leptin is an important factor for the body to regulate body weight. It can suppress appetite and fat hyperplasia, increase the basal metabolic rate, and control body fat within an appropriate range. Many long-term obese people have the problem of insufficient leptin secretion, which makes it difficult to lose weight and easy to gain weight. Therefore, this example explores whether the plant fermented product prepared according to the method of Example 3 can increase the secretion of leptin. First, inoculate 1×10 ⁴ mouse adipocyte cell ( Mus musculus , mouse adipocyte cell) 3T3-L1 (ATCC ^® coded CL- 173 ^TM ), which contains 90% of Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, purchased from Gibco), 10% of bovine serum (Bovine Serum, purchased from Gibco), and 1% of penicillin /Streptomycin (Penicillin-streptomycin, purchased from Gibco), and then cultured at 37°C for 48 hours. After 48 hours, remove the medium and add the differentiation medium for 4 days (change the fresh differentiation medium every two days), which contains 90% Dubek's modified Eagle's medium, 10% FBS, 1% penicillin/streptomycin (Penicillin-streptomycin), 1.0μM/mL Dexamethasone (DEXA) (Sigma), 0.5mM/mL Methylisobutylxanthine (IBMX) (Sigma) and 1.0μg/mL insulin (insulin) (Sigma). After 4 days, change the differentiation medium to Adipocyte Maintenance Medium (fresh maintenance medium every two days), which contains 90% Dubek’s modified Eagle’s medium, 10% FBS, 1% penicillin/streptomycin (Penicillin-streptomycin), and 1.0μg/mL insulin. Seven to 10 days after the start of differentiation induction, the cells were fully differentiated, and then the formation of lipid droplets was observed under a microscope. After that, samples of each group (concentration of 0.0625%) were added, and the medium was changed every 48 hours after acting for another 12 days. Next, the culture medium was collected, and the leptin content was determined using the Mouse LEP (Leptin) ELISA Kit (Elabscience). The results of this example are shown in FIG. 8.

Fig. 8 is a data chart showing the effect of plant fermented product prepared by the method of the present invention on increasing the secretion of leptin. It can be seen from Figure 8 that compared with the control group and the comparison group, the leptin content of the experimental group has a significant increase. In particular, compared with the control group, the leptin content of the experimental group increased by 30%. This implementation The results of the example show that the plant fermented product prepared by the method for fermenting plants of the present invention can effectively increase the secretion of leptin.

Example 9. The manufacturing process of the method of the present invention for increasing the growth rate of microorganisms

First, the rambutan (Nephelium lappaceum ) juice from Rayong, southern Thailand is mixed with water in a weight ratio of 1:5-10, and glucose with a concentration of 10% (w/w) is added according to the total weight. Then, extract at 50°C-100°C for 0.5-1.5 hours to obtain a plant extract.

Next, the plant extract culture medium is planted with 0.01~0.5% (v/v) Saccharomyces cerevisiae BCRC 20271. After 1~3 days of fermentation, 1mL of bacterial solution is taken out every 24 hours, and after serial dilution, The bacterial liquid is coated on a Yeast peptone dextrose (YPD) plate, and the whole fermentation process is irradiated with a red LED light source with a wavelength of 620-750nm (preferably 635nm) to obtain a plant fermented product that irradiates the red LED light source. After that, the plant fermented product irradiated with the red LED light source was used as the experimental group. The preparation method of the plant fermented product of the comparison group was roughly the same as that of the experimental group, except that the red LED was replaced by blue light with a wavelength of 400~450nm Light source: As for the preparation method of the plant fermented product of the control group, the preparation method is roughly the same as that of the experimental group, except that the red LED light source is replaced by natural light. Next, place the YPD plate coated with bacterial solution in an incubator at 30°C for 48 hours, and then count the colonies to evaluate the growth rate of the brewer's yeast. The experimental results are shown in Figure 9.

Fig. 9 is a data diagram showing the efficacy of the method for increasing the growth rate of microorganisms of the present invention in increasing the growth rate of Saccharomyces cerevisiae. It can be seen from Fig. 9 that compared with the control group and the comparison group, the growth rate of S. cerevisiae in the experimental group has been significantly improved. In particular, during the fermentation process, irradiating a red LED light source with a wavelength of 620~750nm (the experimental group) throughout the fermentation process can increase the growth rate of beer yeast by 47% (compared to the control group). On the contrary, irradiating natural light and blue light does not increase the growth rate. Effect. The results of this example show that the method for increasing the growth rate of microorganisms of the present invention can effectively increase the growth rate of Saccharomyces cerevisiae.

Next, in the plant extract medium (AAB medium supplemented with 0.5% yeast extract, 0.3% peptone and 2.5% mannitol), colonize 5% (v/v) Acetobacter aceti 1~3 days after BCRC 11688 starts to ferment, take out 1mL of bacterial solution every 24 hours, after serial dilution, apply the bacterial solution to Yeast peptone dextrose (YPD) plate, and the whole fermentation process is irradiated with a wavelength of 620 ~750nm (preferably 635nm) red LED light source to obtain plant fermented products irradiating the red LED light source. After that, the plant fermented product irradiated with the red LED light source was used as the experimental group. The preparation method of the plant fermented product of the comparison group was roughly the same as that of the experimental group, except that the red LED was replaced by blue light with a wavelength of 400~450nm Light source: As for the preparation method of the plant fermented product of the control group, the preparation method is roughly the same as that of the experimental group, except that the red LED light source is replaced by natural light. Next, place the YPD plate coated with bacterial solution in an incubator at 30°C for 48 hours, and then count the colonies to evaluate the growth rate of acetic acid bacteria. The experimental results are shown in Table 2.

It can be seen from Table 2 that compared with the control group and the comparison group, the growth rate of acetic acid bacteria in the experimental group has been significantly improved. In particular, during the fermentation process, irradiating a red LED light source with a wavelength of 620~750nm (the experimental group) can increase the growth rate of acetic acid bacteria by 22% (compared with the control group), while irradiating natural light and blue light has no effect on the growth rate. . The results of this example show that the method for increasing the growth rate of microorganisms of the present invention can effectively increase the growth rate of acetic acid bacteria.

In summary, the method for fermenting plants of the present invention and the plant fermented product prepared by the method can increase the growth rate of microorganisms (such as saccharomyces cerevisiae and acetic acid bacteria) and the content of plant neutral components, and by Enhance the skin's antioxidant capacity, enhance the skin's anti-glycation activity, and promote the proliferation of elastin to achieve the effect of skin care and beautiful skin. On the other hand, the plant fermented product can achieve the effect of reducing fat by promoting lipolysis and increasing the secretion of leptin.

The above descriptions are merely illustrative and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application.

【Biological Material Deposit】

Food Industry Development Research Institute (Taiwan); December 13, 2006; No. BCRC910805.

Claims (18)

A method for fermenting plants includes the following steps: (a) extracting a plant with water to obtain a plant extract, wherein the plant is rambutan (Nephelium lappaceum); and (b) combining the plant extract with A brewer’s yeast ( Saccharomyces cerevisiae ), a germ lactic acid bacteria ( Lactobacillus plantarum ) and an acetic acid bacteria ( Acetobacter aceti ) undergo a fermentation process in sequence to obtain a plant ferment, wherein the fermentation process includes the plant extract, the The brewer's yeast, the germ lactic acid bacteria and the acetic acid bacteria irradiate a red light source with a wavelength between 620 nm and 750 nm. According to the method described in item 1 of the scope of patent application, before the extraction, glucose with a concentration of 10% (w/w) is added according to the total weight. The method according to item 1 of the patent application, wherein the fermentation time of the beer yeast is 1 to 2.5 days, the fermentation time of the germ lactic acid bacteria is 1 to 3 days, and the fermentation time of the acetic acid bacteria is 3 to 10 days . The method described in item 1 of the scope of patent application, wherein the concentration of the brewer's yeast is between 0.01 and 0.5% (v/v), the concentration of the germ lactic acid bacteria is between 0.01 and 0.25% (v/v), and the The concentration of acetic acid bacteria is between 3-10% (v/v). The method described in item 1 of the scope of patent application, wherein the weight ratio of the plant to water is 1:5-10. The method according to claim 1, wherein in step (a), the temperature of the extraction is between 50 and 100° C., and the time of the extraction is between 0.5 and 1.5 hours. An application of the method as described in any one of items 1 to 6 of the scope of the patent application for improving the growth rate of brewer's yeast and the content of effective components in plants. The use described in item 7 of the scope of patent application, wherein the effective ingredient is polyphenol. A plant fermented product is prepared by the method described in any one of items 1 to 6 of the scope of the patent application. A use of the plant fermented product as described in item 9 of the scope of patent application for preparing a skin care and fat-reducing composition. The use as described in item 10 of the scope of patent application, wherein the skin care includes enhancing the skin's antioxidant capacity, enhancing the skin's anti-glycation activity and promoting the proliferation of elastin. The use described in item 10 of the scope of patent application, wherein the fat reduction includes promoting lipolysis and increasing the secretion of leptin. The use as described in item 10 of the scope of patent application, wherein the composition is in the form of a medicine, a skin care product or a food product. A method for increasing the growth rate of microorganisms includes the following steps: (a) extracting a plant with water to obtain a plant extract, wherein the plant is rambutan ( Nephelium lappaceum ); and (b) the plant The extract and a microorganism undergo a fermentation process to obtain a plant fermented product, wherein the microorganism is a Saccharomyces cerevisiae or an acetic acid bacteria ( Acetobacter aceti ), and the fermentation process includes the plant extract and the microorganism Irradiate a red light source with a wavelength between 620 and 750 nm. According to the method described in item 14 of the scope of patent application, before the extraction, glucose with a concentration of 10% (w/w) is added based on the total weight. The method described in item 14 of the scope of patent application, wherein the fermentation time of the microorganism is 1 to 3 days. According to the method described in item 14 of the scope of patent application, the concentration of the beer yeast is between 0.01 and 0.5% (v/v), and the concentration of the acetic acid bacteria is between 5% (v/v). According to the method described in item 14 of the scope of patent application, the growth rate of the beer yeast is increased by at least 47%, and the growth rate of the acetic acid bacteria is increased by at least 22%.

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