Patent Application
20230118626
This non-provisional application claims priority under 35 U.S.C. § 119(a) to Patent Application No, 110138451 filed in Taiwan, R.O.C. on Oct. 15, 2021, the entire contents of which are hereby incorporated by reference,
The contents of the electronic sequence listing (P200860USI.xml; Size: 5 KB; and Date of Creation: Sep. 13, 2022) is herein incorporated by reference in its entirety.
The instant disclosure relates to an elderberry ferment, in particular to a magnetized elderberry ferment capable of antioxidation, preventing osteoporosis and improving immunity,
In modern society, as people get older, the function of osteoblasts declines, which makes the bone loss faster than regeneration, leading to fragile bones and easy fracture. About 200 million people in the world are affected by osteoporosis symptoms, which leads to 8.9 million cases of fragility fracture every year. About one-third of women and one-fifth of men over the age of 50 have experienced fragility fractures in their lifetime. In addition, after menopause, female hormones in the body are greatly reduced, which accelerates osteoporosis and bone density decline, and increases the risk of fractures.
Orthopedic physicians point out that basically osteoporosis is an irreversible symptom. Once the bone loss occurs, people can only slow down the speed of bone loss at best, but cannot recover to the previous bone density
For a long time, the commercially available calcium tablets have been calcium carbonate and calcium citrate, which have an absorption rate of only 25% to 35% for the human body despite of their high calcium content. Besides, after the calcium is absorbed into blood, part of the calcium is chelated with protein in the blood and cannot enter the bones, thus losing its proper bone protecting effect
In order to improve the above problems, it is urgent for those skilled m the art to develop functional foods that can improve the above problems, so as to benefit the vast population with such needs.
in view of this, the instant disclosure provides a magnetized elderberry ferment, which is obtained by fermenting elderberry (Sambucus nigra) with a plurality of strains in a magnetized environment and has good abilities of improving immunity, antioxidation and preventing osteoporosis.
In some embodiments, an use of a magnetized elderberry ferment in preparation of a composition for antioxidation is provided. The magnetized elderberry ferment is obtained by sequentially fermenting an elderberry extract with yeast, Streptococcus thermophilus and Acetobacier aceti in a fermenter under a magnetized environment
In some embodiments, a method for antioxidation, includes administering to a subject in need a composition containing a magnetized elderberry ferment, The magnetized elderberry ferment is obtained by sequentially fermenting, an elderberry extract with yeast, Streptococcus thermophilus, and Acetobacter aceti in a fermenter under a magnetized environment.
in some embodiments, the magnetized elderberry ferment is capable of increasing total antioxidant capacity (JAC), a concentration of erythrocyte glutathione S-transferase (GST-RBC) and a concentration of f-Thiols in blood of the subject and decreasing, a concentration of malondialdehyde (MDA) in the blood of the subject.
In some embodiments, an use of a magnetized elderberry ferment in preparation of a composition for preventing osteoporosis is provided. The magnetized elderberry ferment is obtained by sequentially fermenting an elderberry extract with yeast, Streptococcus thermphilius, and Acetobacter aceti in a fermenter under a magnetized environment.
In some embodiments, a method for preventing osteoporosis, includes administering to a subject in need a composition containing a magnetized elderberry ferment, The magnetized elderberry ferment is obtained by sequentially fermenting an elderberry extract with yeast, Streptococcus thermophilus, and Acelobacter aceti in a fermenter under a magnetized environment.
In some embodiments, the magnetized elderberry ferment promotes osteocyte differentiation.
in some embodiments, the magnetized elderberry ferment increases concentrations of osteocalcin and type 1 procollagen amino-terminal-propeptide (P 1 NP) in the blood of the subject to enhance bone formation.
In some embodiments, the magnetized elderberry ferment increases bone density of the subject.
In some embodiments, a method for improving immunity, includes administering to a subject in need a composition containing a magnetized elderberry ferment The magnetized elderberry ferment is obtained by sequentially fermenting an elderberry extract with yeast, Streptococcus thermophilus, and Acetobacter aceti in a fermenter under a magnetized environment.
In some embodiments, the magnetized elderberry ferment improves expression level of interleukin-18 gene in the subject and/or improves phagocytic activity of leukocytes in the subject.
In some embodiments, the magnetized elderberry ferment has a total polyphenol content of 2337.07 μg/mL.
In some embodiments, the elderberry extract is obtained by mixing an elderberry raw material with water to obtain a mixture and heating the mixture at 80° C.-100° C. for 0.5 hour-1 hour.
In some embodiments, the elderberry raw material with the water are mixed in a ratio of 1:10
In some embodiments, the elderberry extract is obtained by mixing an elderberry raw material with water and heating the mixture at 95° C. for 1 hour,
In some embodiments, the magnetized environment is formed by a plurality of magnets arranged outside a body of the fermenter.
In some embodiments, the magnets are N35 strong neodymium-iron-boron magnets.
In some embodiments, the magnet has a length of 100 mm, a width of 20 mm and a height of 5 mm.
In some embodiments, the composition is used in an amount of 7.5 mL/day the magnetized elderberry ferment in a liquid form.
In some embodiments, the composition is used in an amount of 0.75g/day of the magnetized elderberry ferment in a solid form,
Based on the above, the magnetized elderberry ferment in any of the embodiments is obtained by mixing the elderberry and water, heating the mixture at a specific temperature for some time and sequentially fermenting the mixture with the yeast, Streptococcus thermophilus, and Acetobacter aceti in the fermenter under the magnetized environment, and can be used in the preparation of the composition for antioxidation, preventing osteoporosis and/or improving immunity, In some embodiments, the maimetized elderberry ferment has the total polyphenol content of 2337.07 μg/mL. In some embodiments, the magnetized elderberry ferment is capable of increasing the total antioxidant capacity (TAC), the concentration of erythrocyte glutathione S-transferase (GST-RBC) and the concentration of f-Thiols in the blood of the subject and decreasing the concentration of malondialdehyde (MDA) in the blood of the subject, and thus has the ability of antioxidation,
In some embodiments, the magnetized elderberry ferment increases the concentrations of osteocalcin and type 1 procollagen amino-terminal-propeptide (P1NP) in the blood of the subject to enhance the bone formation. In some embodiments, the magnetized elderberry ferment increases the bone density.of the subject, thereby preventing osteoporosis.
Some specific implementations of the instant disclosure are described below. Without departing from the spirit of the instant disclosure, the instant disclosure may still be practiced in many different forms, and a scope of protection should not he limited to the conditions specifically stated in the specification.
In this instant disclosure, statistical analysis is performed using software Excel. Data are presented as mean ± standard deviation (SD), and differences between groups are analyzed by student's t-test. In the figures, “*” represents a p-value less than 0.05, “**” represents a p-value less than 0.01, and “***” represents a p-value less than 0.001. The more “*”, the more statistically significant the difference.
Some numerical values used in this specification are approximations, and all experimental data are expressed in the range between plus and minus 10%, preferably in the range of phis or minus 5%.
In this specification, “wt %” refers to weight percent, and “vol %” refers to volume percent.
Sambucus nigra is a plant of the genus Sambucus in the family Adartweae mainly grown in Europe. Its fruit is a dark purple (bright black) berry with a diameter of about 3 mm-S mm, which is often used as food for wild animals or used to make wine and medicines. The fruit of Sambucus nigra is also known as elderberry and European elderberry, and the maturity of fruit takes place in 7-8 months. Sambucus nigro is native mostly to Italy, such as Merano, a hot spring town in Italy.
First, after a elderberry raw material and an extraction solvent are mixed in a certain ratio to obtain a mixture, the mixture is heated at a specific temperature for a specific time to obtain an elderberry extract, The elderberry raw material may be the raw or processed fruit of Sambucus nigra (i.e., elderberry), or elderberry juice made from elderberry, The extrEtction solvent may be water. In some embodiments, the specific temperature may be 80° C.-100° C., and the specific time may be 0.5 hour-1 hour. In some embodiments, the elderberry raw material and the extraction solvent are mixed in a ratio of 1-3:5-29 to obtain a mixture. Herein, the specific ratio of the extraction solvent to the substance to be extracted (such as crushed fruit or whole fruit juice) or the specific extraction time can significantly improve the extraction efficiency The specific extraction time can avoid the possible degradation of active ingredients in the extract caused by too long extraction time.
In some embodiments, the aforementioned “processing” refers to drying, or other physical methods, such as chopping, dicing, milling, grinding or other methods that can affect the size and physical integrity of the raw material. In some embodiments, the aforementioned “elderberry juice” is a juice extracted from elderberry For example, the elderberry juice is obtained by crushing and squeezing elderberry, removing the pomace and fine suspended matters and then concentrating the mice to a certain sugar content (degrees Brix, ° Bx).
In some embodiments, the elderberry extract may be obtained by mixing whole fruit juice of elderberry and water in a weight ratio of 1-3:5-29 and heating the mixture at 80° C.-100° C. for 0.5 hour-1 hour. In an exemplary example, the elderberry extract is obtained by mixing whole fruit juice of elderberry and water in a weight ratio of 1:10 and heating the mixture at 95° C. for 1 hour. In another exemplary example, the elderberry extract is obtained by mixing whole fruit juice of elderberry and water in a weight ratio of 1:5 and heating the mixture at 95° C. for 1 hour.
In some embodiments, the elderberry extract has a sugar content of 9° Bx-10° Bx. Herein, sufficient sugar content can ensure the smooth progress of subsequent fermentation steps and the strains to have sufficient nutrients during the fomentation process,
Next, the elderberry extract is added to a fermenter under a magnetized environment, then a plurality of strains are sequentially added to the elderberry extract, and fermentation is performed for 5 days-10 days to obtain a magnetized elderberry ferment The plurality of strains include yeast, lactic acid bacteria and Acetobacter aceti. For example, the yeast is added in an amount of 0.01 wt %-0.5 wt %, the lactic acid bacteria are added in an amount of 0.01 wt %-0.25 wt %, and Acetobacter aceti are added in an amount of 3 wt %-10 wt %. In some embodiments, when the elderberry extract is made from elderberry (not pure juice), the elderberry extract is directly fermented with the strains without filtering out the solids inside (i.e., elderberry fruit fragnients), and such that active ingredients in the solids are further extracted by using the strains.
In some embodiments, the magnetic environment is formed by a plurality of magnets arranged outside a body of the fermenter. The magnets used are strong magnets, and the strong magnets may be, but not limited to, N35 strong neodymium-iron-boron magnets. The outside of the tank body refers to four peripheral sides of the fermenter that are connected continuously, ha some embodiments, the magnet has a length of 100 mm, a width of 20 mm and a height of 5 mm. In an exemplary example, one 35 strong neodymiurn-iron-boron magnet is attached to each of the four peripheral sides of the body of the fermenter, and the 35 strong neodymium-iron-boron magnet has a size of 100 trim (length×20 mm (width)×5 mm (height). In some embodiments, the magnetic environment has a magnetic field strength (magnetic flux density) of 120 milliteslas-300 milliteslas. Based on this, when the fermentation is performed in the magnetic environment, microbes cultured in this environment may be converted by enzymes into energy by utilizing the potential difference formed by the distribution of protons inside and outside the mitochondrial. membrane, The S pole of the magnet may attract protons through the magnetic lines of force to promote the distribution of charges and accelerate the formation of potential difference, thereby increasing the production rate of microbial cells, in other words, the magnetic environment can increase the glucose utilization and growth rate of the microbes, so as to greatly improve the fermentation efficiency.
In some embodiments, the fermentation with the plurality of strains used is performed according to the following sequence: after fermentation with. the yeast is performed for 1 day-2.5 days, fermentation with the lactic acid bacteria is performed for 1 day-3 days, and femientation with Acelobacier acell is performed for 3 days-10 days.
First, 0.1 wt %-0.5 wt % of yeast is added to the elderberry extract, and fermentation is performed at 28° C.-37° C. for 1 day-2.5 day to obtain a first primary ferment liquid. The yeast may be commercially available Saccharomyces cerevisiae or Saccharomyres cerevisiae with the deposit number BCRC20271 (purchased from Bioresource Collection and Research Center (BCRC) of Food industry Research and Development Institute). For example, the first primary ferment liquid is obtained by adding 0.1% of Sacchommyces cerevisiae to the elderberry extract and performing fermentation at 30° C. for 1 day. Here, by adding the yeast first, the elderberry extract may be fermented to produce ethanol, which helps to extract active ingredients from the elderberry extract. In some embodiments, the first primary ferment liquid has a pH of less than 4 and a sugar content of about 9° Bx.
Next, 0.01 wt %-0.25wt % of lactic acid bacteria is added to the first primary ferment liquid, tend fermentation is performed at 28° C.37° C. for 1 day-3 days to obtain a second primary ferment liquid, The lactic acid bacteria may be commercially available Lactobacillus plomarum, Lactobacillus plantarumwith the deposit number BCRC910760 (purchased from BCRC), commercially available Streptococcus thermophilus or Streptococcus thermophilus TCI633 with the deposit number BCRC910636 (international deposit number DSM28121) (purchased from BCRC). For example, the second primary ferment liquid is obtained by adding 0.05% of Streptococcus thermophilus TCI633 to the first primary ferment liquid and performing fermentation at 30° C. for 1 day, Here, by adding the lactic acid bacteria, glucose in the first primary ferment liquid may be further consumed, such that the sugar content is reduced and lactic acid is produced, thereby reducing the pH of the first primary ferment liquid. Here, reducing the pH of the first primary ferment liquid helps to further extract other different active ingredients of elderberry. In some embodiments, the second primary ferment liquid has a pH of less than 3.5 and a sugar content of about 6° Bx.
Then, 5% of Acetobacter aceti (purchased from BCRC, with the deposit number BCRC11688 and the international deposit number ATCC15973) is added to the second primary ferment liquid, and fermentation is performed at 28° C.-37° C. for 3 days-10 days to obtain a primary ferment liquid, For example, the primary ferment liquid is obtained by adding 5% of Acetobacter aceti to the second primary ferment liquid and performing fermentation at 30° C. for 5 days. Here, by adding Acetobacter aceti, the ethanol in the second primary ferment liquid can be consumed, thereby further reducing the glucose content. In some embodiments, the primary ferment liquid has a pH of less than 3.5 and a sugar content of about 3° Bx.
Next, the primary ferment liquid is subjected to a plurality of processing procedures to obtain the magnetized elderberry ferment. Specifically. the processing procedure may be, but not limited to, one or more processing steps such as concentration under reduced pressure, filtration, adjustment of sugar content and sterilization.
In some embodiments, the primary ferment liquid is concentrated under reduced pressure at 55° C.-65° C. and filtered through a screen with a specific mesh to obtain a raw fermentation solution, and the sugar content of the raw fermentation solution is adjusted to obtain the magnetized elderberry ferment. In other embodiments, the filtration and the concentration under reduced pressure may be performed in a reverse older, that is, the primary ferment: liquid is first filtered through a screen with a specific mesh and then concentrated under reduced pressure to obtain the raw fermentation solution. For example, the aforementioned concentration may be concentration under reduced pressure at 60° C., and the aforementioned filtration may be performed using a 200-mesh screen.
In some embodiments, the raw fermentation solution obtained by the concentration reduced pressure and the filtration (which may be performed in a reverse order) has a sonar content of about 2° Bx and a pH of less than 3.5.
In some embodiments, the sugar content of the raw fermentation solution is adjusted to obtain the magnetized elderberry ferment. For example, the sugar content of the raw fermentation solution may be adjusted to 39° Bx by adding an oligosaccharide to the raw fermentation solution to form the elderberry ferment, The oligosaccharide added may be fructoolig,osaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide.
etc. In some embodiments, the oligosaccharide added may be an oligosaccharide solution containing 40 wt %-70 wt % isomaltooligosaccharide.
In some embodiments, the magnetized elderberry ferment in a liquid form may be spray-dried to remove the solvent, thereby obtaining the magnetized elderberry ferment in a solid form (for example, magnetized elderberry ferment powder).
In some embodiments, the magnetized elderberry ferment has a total polyphenol content of 2337.07 μg/mL. Based on this, the magnetized elderberry ferment has the ability of antioxidation and is capable of scavenging free radicals in the subject.
In some embodiments, the magnetized elderberry ferment has a flavone content of 12703 μg/mL.
In some embodiments, the magnetized elderberry ferment has an amioxidation function, and can be used in preparation of a composition for antioxidation. For example, the magnetized elderberry ferment is capable of increasing total antioxidant capacity (TAC), a concentration of erythrocyte glutathione S-transferase (GST-RBC) and a concentration of f-Thiols in blood of a subject and decreasing a concentration of malondialdehyde (MDA) in the blood of the subject. Based on this, after the subject takes the magnetized elderberry ferinent, the antioxidation ability of the subject can be improved.
In some embodiments, the magnetized elderberry ferment has the ability of preventing osteoporosis, and can be used in preparation of a composition for preventing osteoporosis. For example, the magnetized elderberry ferment promotes osteocyte differentiation. In some embodiments, the magnetized elderberry ferment increases the concentrations of osteocalcin and type 1 procollagen amino-terminal-propeptide (P INP) in the blood of the subject to enhance the bone formation, After the subject takes the magnetized elderberry ferment, the magnetized elderberry ferment can promote osteocyte differentiation, enhance the bone formation, increase the bone density and prevent osteoporosis.
In some embodiments, the magnetized elderberry ferment has the ability of improvinu, immunity, and can be used in preparation of a composition for improving immunity. For example, the magnetized elderberry ferment improves expression level of interleukin-18 gene of the subject andior improves phagocytic activity of leukocytes (such as macrophages). After the subject takes the magnetized elderberry ferment, the magnetized elderberry ferment can stimulate the innate immunity of the subject and improve the phagocytic activity of macrophages in the subject.
In some embodiments, the subject is a human.
In some embodiments, any of the aforementioned compositions may be a pharmaceutical. In other words, the pharmaceutical includes an effective content of tlae magnetized elderberry ferment.
In some embodiments, the aforementioned pharmaceutical can be manufactured into dosage forms suitable for being enterally, parenterally, orally or topically administered using techniques well known to those skilled in the art.
In some embodiments, the enterally or orally administered dosage forms may be, but not limited to, tablets, troches, lozenges, pills, capsules, dispersible powder or granules, solutions, suspensions, emulsions, syrup, elixir, slurry or the like. In some embodiments, the parenterally or topically administered dosage forms may be, but not limited to, injections, sterile powder, external preparations or the like, In some embodiments, the injections may be administered by subcutaneous injection, intraepidermal injection, intradermal injection or intralesional injection.
In some embodiments, the aforementioned pharmaceutical may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques, In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following carriers: a solvent, a buffer, an emulsifier, a suspending agent, a disintegrating agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling, agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome and the like, The type and quantity of carrier to be used are within the professional quality and routine skill of those skilled in the art. In some embodiments, the solvent used as a pharmaceutically acceptable carrier may be water, normal saline, phosphate buffered saline (PBS), or an aqueous solution containing alcohol.
In some embodiments, the aforementioned pharmaceutical may be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including an emulsion, a gel, an ointment, a cream, a patch, as liniment, powder, an aerosol, a spray, a lotion, a serum, a paste, foam, drops, a suspension, a salve and bandage.
In some embodiments, the aforementioned external preparation is prepared by mixing the aforementioned pharmaceutical and a base well known to those skilled in the art. For example, the substrate may include one or more additives selected from water, alcohols, glycol, hydrocarbons (such as petroleum jelly and white petrolatum), wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers, stabilizing agents, gelling agents (such as carbopol®974P, microcrystalline cellulose and carboxymethylcerllulose), active agents humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants, etc. The selection and quituttity of these additives are within the professional quality and routine skill of those skilled in the art.
In some embodiments, an of the aforementioned compositions may be a skincare product in other words, the skin care product includes a specific content of the magnetized elderberry ferment. Moreover, the skincare product may further include an acceptable adjuvant widely used in skincare product manufacturing techniques. For example, the acceptable adjuvant may include one or more agents selected from a solvent, a gelling auent, an active auent, a preservative, au antioxidant, a screening agent, a chelating anent, a surfactant, a coloring agent, a thickening agent, a filler, a fragrance and an odor absorber. The selection and quantity of these agents are within the professional quality and routine skill of those skilled in the art.
In some embodiments, the aforementioned skincare product can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art, including, but not limited to, an aqueous solution, an aqueous-alcohol solution or oily solution, an oil-in-water type, water-in-oil type or composite emulsion, a gel, an ointment, a cream, a mask, a patch, a pack, a liniment, powder, an aerosol, a spray, a lotion, a. serum, a paste, foam, a dispersion, drops, a mousse, sunblock, tonic water, a foundation, makeup remover products, soap and other body cleansing products, etc.
In some embodiments, the aforementioned skincare product may also be used in combination with one or more external use agents with known activities selected from whitening agents (such as tretinoin, catechin, kojic acid, arbutin and vitamin C), humectants, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts (such as aloe extract), skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, antisehorrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these external use agents are within the professional quality and routine skill of those skilled in the art.
In some embodiments, any of the aforementioned compositions may be an edible product. In other words, the edible product includes a specific content of the magnetized elderberry ferment. In some embodiments, the edible product may be a general food, a health food or, a dietary supplement.
In some embodiments, the aforementioned edible product may be manufactured into dosage forms suitable for being orally adrninistered using techniques well known to those skilled in the art. In some embodiments, the aforementioned general food may be the edible product itself. In some embodiments, the general food may be, but not limited to, beverages, fermented foods, bakery products or seasonings,
In some embodiments, the obtained magnetized elderberry ferment may be further used as a food additive to prepare a food composition containing the plant ferment liquid-magnetized elderberry ferment. Here, the edible product (i.e. food composition) for ingestion by humans and non-human animals can be prepared from any edible material by adding the magnetized elderberry ferment of any example during the preparation of raw materials or by adding the magnetized elderberry ferment of any example during the production of food by conventional methods.
In some embodiments, dosage forms of the aforementioned composition may be liquids or solids (for example, powder packets or tablets).
In some embodiments, the aforementioned composition is used in an amount of 0.75 mL/day of the magnetized elderberry ferment in a liquid form (i.e., magnetized elderberry ferment liquid).
In some embodiments, the aforementioned composition is used in an amount of 0.75g/day of the magnetized elderberry ferment in a solid form.
Firstly, elderberry juice (origin: Merano,ltaly) and water were mixed in a weight ratio of 1:10 to obtain an elderberry solution, Elderberry juice was purchased from a supplier in ItalY (Carriére G), with the product name Elderberry Juice.
Next, the elderberry solution was heated to 95° C., and held for 1 hour after the heating environment reaches 95° C. to obtain an elderberry extract. Then, glucose was added in an amount of 10 wt. % of the total weight of the elderberry solution. Afier the temperature of the elderberry extract dropped to room temperature, the elderberry extract may be used as an elderberry culture. solution for the subsequent fermentation process,
In the subsequent fermentation, an ordinary fermenter and a magnetic fermenter were used. Four sides outside a body of the magnetic fermenter were respectively provided with one magnet (four magnets in total). The magnets used were N35 strong neodymium-iron-boron magnets. Each magnet had a length of 100 mm, a width of 20 mm and a height of 5 mm.
Two aliquots of elderberry culture solution were respectively transferred to the ordinary fermenter and the magnetic fermenter, 0.1 wt % of Saccharomyces cerevisiae (purchased from BCRC, with the deposit number BCRC20271) was added to the elderberry culture solution, and static culture was performed at 30° C. for 1 day to form a first primary ferment liquid. Here, the first primary ferment. liquid had a pH of less than 4 and a sugar content of about 9° Bx.
Next, 0.05 wt % of Streptococcus thermophilus (purchased from BCRC, with the deposit number BCRC 910535) was added to the first primary ferment liquid, and static culture was performed at 30° C. for 1 day to form a second primary ferment liquid, Here, the second primary ferment liquid had a pH of less than 3.5 and a sugar content of about 6° Bx.
5 wt % of Acetobacter aceti (purchased from BCRC, with the deposit number BCRC11688) was added to the second primary ferment liquid, and static. culture was performed at 30° C. for 5 days to form a primary ferment liquid. Herein, the second primary ferment liquid had a pH of less than 3.5 and a sugar content of about 3° Bx.
Next, the primary ferment liquid was concentrated under reduced pressure at 60° C., and filtered through a 200-mesh screen to obtain a raw fermentation solution. Herein, the raw fermentation solution had a sugar content of about 2° Bx and a pH of less than 3,5. Then, isomaltooligosaccharide and the raw fermentation solution were mixed in a ratio of 60 wt %:40 wt % to adjust the sugar content of the raw fermentation solution to 39° Bx, thereby obtaining an elderberry fermentation product. Herein the elderberry fermentation product obtained by the fermentation in the ordinary cementer was an “elderberry ferment”, and the elderberry fermentation product obtained by the fermentation in the magnetic fermenter was an “magnetized elderberry ferment”.
Besides, the sugar content of the aforementioned elderberry extract was adjusted with glucose to 9° Bx (pH 6,3) to obtain an “elderberry water extract”.
10.0 mg of gallic acid was added into a 10 ml volumetric flask, and then made up to 10 mL with water (H2O) to obtain a stock solution of gallic acid. The stock solution of gallic acid was diluted 10-fold, i.e., 900 μL of water was added to 100 μL of stock solution of gallic acid to obtain a 100 μg/mL initial solution of gallic acid (containing 1000 ppm of gallic acid). Then, 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL standard solutions of gallic acid were prepared according to the following Table 1. and 100 μg/mL of the standard solution of each concentration was taken and added to a glass test tube. 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck) was added to each glass test tube and uniformly mixed with the standard solution. After the mixture was allowed to stand for 3 minutes, 400 μL of 7.5% sodium carbonate was added, and the resulting mixture was uniformly mixed and allowed to react for 30 minutes to obtain a standard reaction solution. 200 μL of the standard reaction solution was added to a 96-well plate, and the absorbance at 750 rim was measured to obtain a standard curve.
The magnetized elderberry ferment, the elderberry ferm..nt and the elderberry water extract prepared in Example 1 were respectively taken as test samples for the experimental group (magnetized elderberry ferment), the contrast group (elderberry ferment) and the control group (elderberry water extract).
The test sample in each group was diluted 25-fold with water, and then 100 mL of the diluted solution was taken. and added to a centrifuge tube. Next, 500 μL of Folin-Ciocaltens phenol reagent virus added to the glass test tube and uniformly mixed with the sample. After the mixture was allowed to stand for 3 minutes, 400 μL of 7.5% sodium carbonate was added, and the resulting mixture was uniformly mixed and allowed to react for 30 minutes to obtain a reaction solution to be tested. The glass test tube filled with the reaction solution to be tested was shaken until no bubbles. 200 μL of the reaction solution to be tested was added to a 96-well plate, and the absorbance at 750 nm of the reaction solution to be tested was measured.
Next, the total polyphenol content was calculated based on the .absorbance of the reaction solution to be tested by using the standard curve and interpolation, as shown in
Therefore, after administrating the subject, the magnetized elderberry ferment significantly improve the antioxidant activity in the subject as compared with the elderberry water extract or the elderberry ferment.
Here, the ability of the magnetized elderberry ferment to promote osteogenesis was confirmed by Alizarin Red S (purchased from Sigma-Aldrich) staining. The Alizarin Red S was a water-soluble orange-yellow crystal that may bind to a calcium salt to form an orange-red precipitate. The Alizarin Red S can be used to detect calcium deposits in cells or detect calcified substances.
The cell differentiation medium used was DMEM (brand: Gibco) with 10 vol % FBS (brand: Gibco). 1 vol. % penicillin-streptomycin (purchased from Gibco), 0.1 μM. dexamethasone (DEXA; brand: Sigma), 0.5 μM ascorbic acid (brand: Sigma, and 10 mIM β-glycerol (brand: Sigma). The cell culture medium was Minimum Essential Medium Alpha (MEMα, brand: Gibco) with 20 vol % FBS and 1 vol % penicillin-streptomycin.
At a cell density of 2×104 cells per well, mouse bone marrow stromal cell line OP9 (purchased from BCRC, No, 6566) was inoculated into wells of a 24-well plate containing 500 μL of cell differentiation medium, and cultured at 37° C. for 7 days. During the 7 days of culture, the cell differentiation medium was replaced every 3 days.
The O P9 cells were divided into four groups, namely blank group, control group, contrast group and experimental group. After 7 days of culture, the cell differentiation media of the four groups were replaced with experimental media, and culture was performed at 37° C. for 7 days. During the 7 days of culture, the experimental medium was replaced every 3 days. Here, the experimental medium used by the experimental group was a cell culture medium containing 0.0625 vol % magnetized elderberry ferment, the experimental medium used by the contrast group was a cell culture medium containing 0.0625 vol % elderberry ferment, the experimental medium used by the control group was a cell culture medium containing 0.0625 vol % elderberry water extract, and the experimental medium used by the blank group was an ordinary cell culture medium (with no magnetized elderberry ferment, elderberry ferment or elderberry water extract added).
Next, after 7 days of culture, the differentiation of OP9 cells into osteocytes the four groups were confirmed by Alizarin Red S (brand: Sigma) staining, Herein, the cells in each group included OP9 cells and osteocytes obtained after differentiation, so the treatment objectives in the groups will be described as “cells” below
Alizarin Red S staining: First, the experimental medium in each group was removed, and the cells .in each group were washed with PBS (brand: Gibco). Then, the PBS was removed, 4% formaldehyde was added, and the reaction was performed for 10 minutes to fix the cells in each group. Next, the cells in the two groups were washed with water (ddH2O), 200 μL of Alizarin Red S solution was added, and the reaction was performed for 1 minutes. Here, the Alizarin Red S solution was obtained by dissolving 2 g of Alizarin Red S powder in 100 mL of water (ddH2O) and filtering the mixture through a 0.22 μm syringe filter.
The Alizarin Red S solution in each group was pipetted out, and the stain in the cells in each group was destained with water (ddH2O). Next, the calcium ion content in the stained cells was observed with a microscope (brand: ZEISS), Herein, when the stained cells appeared red, it indicated the calcium ion content in the cells and may represent the amount of osteocytes differentiated from OP9 cells.
10% CPC buffer was added to the destained cells in each group, and the reaction was performed for 1 hour with gentle shaking. The CPC buffer was prepared by mixing cetylpyridinium chloride (CPC) with water.
100 μL of the cell-containing reaction solution in each group was taken and added to a 96-well plate, and the absorbance (OD550) of each well was read using an ELISA reader (brand: Bidlek). The amount of osteocytes differentiated from OP9 cells was calculated based on the absorbances measured. Regarding, the amount of osteocytes in the blank group as 100%, the relative amount of osteocytes (also known as osteogenesis rate) in the other three groups was calculated, as shown in
Referring to
At a cell density of 5×105 cells per well, human monocytic cell line THP-1 was inoculated into a 6-well plate (purchased from GeneDireX) containing cell culture medium. 1-lere, the cell culture medium used was an RPMI medium containing 10% FBS, Next, 500-nanornolar differentiation reagent (phorbol 12-myristate 13-acetate (PMA), purchased from Sigma) was added to each well, and culture was performed for 24 hours such that the monoeytic cell line THP-1 was differentiated into macrophages.
The macrophages were divided into four groups, namely blank group, control group, contrast group and experimental group. The cell culture medium containing the differentiation reagent in the four groups was respectively replaced with an experimental medium, and culture was performed in an environment of 5% CO2 and 37 QC for 24 hours. The experimental medium used by the experimental group was 200 μL of magnetized elderberry ferment, the experimental medium used by the contrast group was 200 μL of elderberry ferment, the experimental medium used by the control group was 200 μL of elderberry water extract, and the experimental medium used by the blank group was 200 μL of RPMI medium without FBS.
After 24 hours of culture, the expression level of interleukin-18 (IL-18, Gene ID: 3606) gene after treatment with the macrophages in each group was determined using RNA extraction kits, a reverse transcriptase, KAPA SYBR® FAST qPCR kits and a quantitative PCR instrument. Here, the interleukin-18 mays stimulate the innate immunity and promote the aggregation and phagocytosis of the macrophages, and thus, may be used as a marker of phagocytic activity of macrophages.
R is REVERSE, and. is FORWARD,
Referring to
At a cell density of 5×105 cells per well, human monocytic cell line THP-1 was inoculated into a 6-well plate (purchased from GeneDireX) containing cell culture medium. Here, the cell culture medium used was an RPMI medium containing 10% FBS. Next, 500-nanoinolar differentiation reagent (phorbol 12-myristate 13-acetate (PMA), purchased from Sigma) was added to each well, and culture was performed for 24 hours such that the monocytic cell line THP-1 was differentiated into macrophages.
The macrophages were divided into four groups, namely blank group, control group, contrast group and experimental group. The cell culture medium containing the differentiation reagent in the four groups was respectively replaced with an experimental medium, and culture was performed in an environment of 5% CO2 and 37° C. for 24 hours. The experimental medium used by the experimental group was 200 μL of magnetized elderberry ferment, the experimental medium used by the contrast group was 200 μL of elderberry ferment, the experimental medium used by the control group was 200 μL of elderberry water extract, and the experimental medium used by the blank group was 200 μL of RPMI medium without FBS.
0,5% fluorescent microbeads (1.7 to 2,2 microns itt size, purchased from Spheroteth) were added to the macrophages in each group, and the Culture was Continued for 4 hours. Next, the experimental medium in each group was removed, and the macrophages were washed with 1X PBS 3 times. Each well in each group was divided into 5 areas. From 2 fields of view randomly selected in each area, pictures were taken with a fluorescence microscope (purchased from ZEISS). Further, the number of fluorescent microbeads in the macrophages in the pictures was calculated. The results are shown in
Referring to
In order to further confirm the effects of the magnetized elderberry ferment on the human body, relevant human experiments were also performed as follows,
6-1, Sample preparation
The magnetized elderberry ferment prepared. in Example 1 was made into a test sample containing 7.5 ml of magnetized elderberry ferment per bottle.
6-2. Subjects: 6 menopausal, female subjects aged 45 to 65.
6-3. Test mode: 6 subjects were given one bottle a day for 8 consecutive weeks. Before taking (0th week) and 8 weeks after taking (8th week), blood of the subjects was collected. LEZEN Reference Lab was commissioned to perform blood tests, Cathay Pacific Health Management was commissioned to perform bone density tests using a dual-energy X-ray absorptiometry (DXA).
6-4. Blood test items: antioxidant markers and bone formation markers. The antioxidant markers included: total antioxidant capacity (TAC), malondialdehyde (MAD) as an oxidative stress marker, glutathione S-transferase (GST-RBC) as an antioxidant marker, and f-Thiols. The bone formation markers included: concentrations of osteocalcin and concentration of type 1 procollagen am ino-term inal-propepti de (P1NP).
Referring to
Referring to
Based on this, after being taken by the subjects, the magnetized elderberry ferment improves the overall antioxidant capacity of the subjects, and may also help the subjects to protect against the injury caused by five radicals by improvthg the in vivo antioxidant capacity of the subjects.
Referring to
Referring to
As a result, administrating the subjects with the magnetized elderberry ferment effectively increase the in vivo antioxidant marker and improve the body's ability of protecting against free radicals, thereby reducing the oxidative stress injury caused by free radicals.
Referring to
Referring to
6-5. Bone density test items: presented as “T score” by instrument measurement table. “T score” is used as an indicator for determining bone density. If the “T score” value is less than −1, it means osteopenia. If the “T score” value is less than or equal to −2.5, it means osteoporosis.
Referring to
As a result, after the subjects tookn the magnetized elderberry ferment for 8 weeks, the magnetized elderberry ferment may reduce the oxidative stress injury in the 6 subjects, improve the antioxidant capacity of the subjects and increase the bone formation marker concentrations in the blood and the bone density value of the subjects. This indicated that the magnetized elderberry ferment had the potential of antioxidation and preventing osteoporosis,
Based on the above, the magnetized elderberry ferment according to any example of the disclosure can improve the antioxidation ability of the subject and prevent osteoporosis of the subject. The magnetized elderberry ferment is obtained by sequentially fermenting the elderberty extract with the yeast, Streptococcus thermophilus, and Acetobacter aceti in the fermenter under a magnetic environment. In some embodiments, the magnetized elderberry ferment is capable of increasing total antioxidant capacity (TAC), the concentration of erythrocyte glutathione S-transferase (GST-RBC) and the concentration of f-Thiols in the blood of the subject and decreasing the concentration of malondialdehyde (MDA) in the blood of the subject. In some embodiments, the magnetized elderberry ferment has the abilities of promoting osteocyte differentiation, increasing the concentrations of osteocalcin and P1NP in the subject and increasing the bone density of the subject. In some embodiments, the magnetized elderberry ferment improves the expression level of interleukin-18 gene in the subject andfor improves phagocytic activity of leukocytes to achieve the effects of immunity improvement and health care. Thereby, the magnetized elderberry ferment according to any example has the potential of improving the immunity, antioxidation and preventing osteoporosis, and can achieve the effects of improving the immunity, enhancing systemic antioxidant ctipacity and protecting bones.
Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, the disclosure is not for limiting the scope of the invention, Persons having ordinary skill in the art may make various modifications and changes without departirm from the scope and spnil of the invention. Theretfore, the scope of the appended claims should not be limited to the description of the preferred embodiments described above.
| Number | Date | Country | Kind |
|---|---|---|---|
| 110138451 | Oct 2021 | TW | national |
