CN115969033A - Preparation method and application of elderberry magnetic field fermentation product - Google Patents
Preparation method and application of elderberry magnetic field fermentation product Download PDFInfo
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- CN115969033A CN115969033A CN202111203264.3A CN202111203264A CN115969033A CN 115969033 A CN115969033 A CN 115969033A CN 202111203264 A CN202111203264 A CN 202111203264A CN 115969033 A CN115969033 A CN 115969033A
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Abstract
The invention provides a preparation method of an elderberry magnetic field leavening and application of the elderberry magnetic field leavening in preparing a composition for promoting hard bone differentiation, enhancing immunity or resisting oxidation, wherein the preparation method is to provide a culture solution, the culture solution comprises an elderberry solution formed by the weight ratio of the elderberry to water of 1-3:5-29 and 10 wt% of glucose in the total weight of the elderberry solution; fermenting the culture solution and a plurality of strains in a magnetic field for 4-15.5 days to obtain a fermentation stock solution, wherein the plurality of strains comprise 0.01-0.5 wt% of yeast, 0.01-0.25 wt% of lactic acid bacteria and 3-10 wt% of acetic acid bacteria; and adjusting the sugar degree of the fermentation stock solution to form the elderberry magnetic field fermentation product.
Description
Technical Field
The present application relates to an application of elderberry (Sambucus nigra) fermentation product, and more particularly to an application of elderberry fermentation product in preparation of a composition for promoting hard bone differentiation, enhancing immunity or resisting oxidation.
Background
With the change of environment, the ultraviolet index and air pollution are increasingly serious, the ultraviolet radiation in sunlight is the main cause of skin damage, aging and suntan, and the skin can even generate melanin to form spots and is difficult to fade after being exposed to the sun for a long time. In addition, ultraviolet radiation in sunlight can promote the production of Reactive Oxygen Species (ROS), which are also known as free radicals, in skin cells, leading to skin aging, loss of luster, dryness, and even reddening.
In addition, skin cells age gradually, skin texture becomes rough gradually and wrinkles are generated, the recovery speed of the skin gradually becomes slow due to slow metabolism speed of the skin cells, and the accumulated large amount of active oxygen substances, melanin and the like cannot be completely metabolized in time due to long-term external environmental stimulation such as air pollution and the like, so that the skin condition cannot be improved.
Conventional cosmetics, health care products and health foods are mostly made of chemical components, and long-term use of the cosmetics, the health care products and the health care foods not only causes harm to human health, but also causes irritation to sensitive skin and aggravates the condition of skin redness, and in addition, the manufacturing process is not environment-friendly and ecological, causes environmental pollution, and even enters into an ecological circle through a drainage system.
In order to solve the above problems, there is a need in the art to develop a skin-conditioning composition having anti-uv, anti-oxidant, melanin formation-inhibiting, skin red spot-reducing, redness-relieving and skin moisturizing-improving effects, so as to benefit the broad group in need thereof.
Disclosure of Invention
In view of the above, the present invention provides a preparation method of elderberry (Sambucus nigra) fermented product, an elderberry fermented product prepared by the preparation method, and an application of the elderberry fermented product in preparation of a composition for promoting hard bone differentiation, enhancing immunity or resisting oxidation.
In one embodiment, a method for preparing an elderberry ferment comprises:
providing a culture solution, wherein the culture solution comprises an elderberry solution formed by the elderberry and water according to the weight ratio of 1-3:5-29 and glucose accounting for 10 weight percent of the total weight of the elderberry solution;
in one embodiment, the culture solution and a plurality of strains are fermented in a magnetic field for 4-15.5 days to obtain a fermentation stock solution, wherein the plurality of strains comprise 0.01-0.5 wt% of yeast, 0.01-0.25 wt% of lactic acid bacteria and 3-10 wt% of acetic acid bacteria; and
adjusting the sugar degree of the fermentation stock solution to form the elderberry ferment.
In one embodiment, the step of fermenting the culture fluid and the plurality of bacterial species in the magnetic field comprises:
fermenting the microzyme in the magnetic field for 1 to 2.5 days to form a first primary fermentation liquid;
adding the lactic acid bacteria into the first primary fermentation liquid and fermenting in the magnetic field for 1 to 3 days to form a second primary fermentation liquid;
adding the acetic acid bacteria into the second primary fermentation liquid and fermenting in the magnetic field for 3-10 days to form the primary fermentation liquid; and
filtering the primary fermentation broth to obtain the fermentation stock.
In one embodiment, the magnetic field has a magnetic flux density of 120 to 300 milli-tesla.
In one embodiment, the total polyphenol content in the elderberry fermentation broth is 1134.6 μ g/ml.
In one embodiment, the yeast is Saccharomyces cerevisiae with accession number ATCC26602, the lactic acid bacteria is Lactobacillus plantarum with accession number DSM33108, and the acetic acid bacteria is Acetobacter aceti with accession number ATCC15973.
The invention also provides application of the elderberry fermentation liquor in preparing a composition for promoting hard bone differentiation, enhancing immunity or resisting oxidation.
In one embodiment, the elderberry fermentation broth increases the concentration of Osteocalcin (ostocelcin) and/or type i procollagen nitrogen-terminal pro-peptide (P1 NP) in the blood.
In one embodiment, the elderberry fermentation broth increases interleukin 18 and/or leukocyte phagocytosis.
In one embodiment, the elderberry broth increases the concentration of glutathione S transferase (GST-RBC), sulfur-containing compounds (f-Thiols), malondialdehyde (MDA), or a combination thereof, in red blood cells in blood.
In one embodiment, the weight ratio of elderberry to water in the elderberry fermentation liquid is 1-3:5-29, and the fermentation is performed in a magnetic field for 4-15.5 days by a plurality of strains, wherein the plurality of strains comprises 0.01-0.5 wt% of yeast, 0.01-0.25 wt% of lactic acid bacteria and 3-10 wt% of acetic acid bacteria.
In one embodiment, the elderberry fermentation liquid is further prepared into a cosmetic composition, a health food composition, a cosmetic composition or a skin external agent.
In summary, the manufacturing method of any embodiment doubles the total polyphenol content of the elderberry fermentation product compared with the conventional process, and greatly improves multiple effects of the elderberry fermentation product. In addition, the elderberry fermentation product of any embodiment of the present invention can be used for preparing a composition for promoting differentiation of hard bone, enhancing immunity or resisting oxidation, wherein the composition is used for promoting differentiation of hard bone, increasing calcium ion content in bone cells, preventing osteoporosis, strengthening bone density and/or strengthening bone, enhancing immunity by increasing phagocytosis of interleukin 18 and/or white blood cells, achieving health care effects, increasing glutathione S transferase (GST-RBC), sulfur-containing compounds (f-Thiols), and Malondialdehyde (MDA) in red blood cells, increasing reducing substances in blood, eliminating oxidizing substances in body, reducing oxidation pressure, preventing damage to protein molecules, enzymes in body and interfering with their effects, and preventing unsaturated fatty acids from being over-oxidized, by increasing the concentration of Osteocalcin (ostealcin) and/or the nitrogen-terminal pro-peptide chain of type i procollagen (P1 NP) in blood.
The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.
Drawings
FIG. 1 is a flow chart of a method for preparing a fermentation broth according to an embodiment of the present invention.
Fig. 2 is a flowchart of the details of step S02 in fig. 1.
FIG. 3 shows the total polyphenol content of elderberry fermentation product prepared by the method of the present invention.
FIG. 4 is a graph comparing the results of the control group, control group 1, control group 2 and experimental group in terms of the number of sclerosteous cells. * P value <0.01, compared to control group.
FIG. 5 is a graph showing comparison of the results of the expression levels of interleukin 18 in the control group, control group 1, control group 2 and experimental group. * p value <0.05, compared to control.
FIG. 6 is a graph showing the comparison of the results of the phagocytic activity of leukocytes in the control group, control group 1, control group 2 and experimental group. * P value <0.001, compared to control group.
Fig. 7 is a graph comparing the results of subjects continuously ingesting beverages containing the experimental group for the sum of antioxidant capacity at week 0 and week 8.
Fig. 8 is a graph comparing the results of subjects continuously ingesting the beverage containing the experimental group in the erythrocytes at week 0 and week 8 with the glutathione S transferase.
Fig. 9 is a graph comparing the results of subjects continuously ingesting beverages containing the experimental group at weeks 0 and 8 for sulfur-containing compounds.
Fig. 10 is a graph comparing the results of subjects continuously taking drinks containing the experimental group at week 0 and week 8 of malondialdehyde.
Fig. 11 is a graph comparing results of subjects who continuously ingested drinks containing the experimental group at weeks 0 and 8 for osteocalcin.
FIG. 12 is a graph comparing the results of the nitrogen-terminal pro-peptide chain of procollagen type I at weeks 0 and 8 after the subjects continuously ingest the drinks containing the experimental group.
Fig. 13 is a graph comparing the results of T-score (T-score) of bone density at week 0 and week 8 of subjects continuously ingesting beverages containing the experimental group.
Detailed Description
Some embodiments of the present disclosure will be described below. The present disclosure may be embodied in many different forms without departing from the spirit thereof, and the scope of protection should not be limited to the details set forth in the specification.
Statistical analysis was performed using Excel software. Data are expressed as mean ± Standard Deviation (SD) and differences between groups were analyzed by student's t-test (student's t-test).
As used herein, the numerical values are approximate and all experimental data are shown to be within a range of plus or minus 10%, and more preferably within a range of plus or minus 5%.
As used herein, the term "juice extraction" refers to the product produced by the action of extraction. The extract may be presented as a solution dissolved in a solvent, or the extract may be presented as a concentrate or serum free or substantially free of solvent.
In accordance with the present invention, the pharmaceutical may be manufactured in a dosage form suitable for parenteral (parenteral), oral (oral) or topical (topically) administration using techniques well known to those skilled in the art, including, but not limited to: injections (injections) [ for example, sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion) ], sterile powder (sterile powder), troche (tablet), tablet (troche), buccal tablet (dosage), pill (pill), capsule (capsule), dispersible powder (dispersible powder) or granule (granule), solution, suspension (suspension), emulsion (emulsion), syrup (syrup), elixir (elixir), syrup (syrup), external preparation (external preparation), and the like.
According to the present invention, the pharmaceutical may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents (solvent), buffers (buffer), emulsifiers (emulsifying), suspending agents (suspending agent), disintegrating agents (disintegrant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizers (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving), wetting agents (wetting agent), lubricants (lubricating), absorption delaying agents (absorption delaying agent), liposomes (liposome) and the like. The selection and amounts of such agents are within the skill and routine skill of those skilled in the art.
According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline (normal saline), phosphate Buffered Saline (PBS), aqueous alcohol-containing solutions (aqueous solution linking alcohol), and combinations thereof.
According to the invention, the medicament may be administered by a parenteral route (parenteral routes) selected from the group consisting of: intraperitoneal injection (intraepithelial injection), subcutaneous injection (subcutaneous injection), intraepidermal injection (intraepithelial injection), intradermal injection (intraradial injection), intramuscular injection (intramyocardial injection), intravenous injection (intravenous injection), and intralesional injection (intrafocal injection).
According to the present invention, pharmaceuticals can be manufactured into an external preparation (external preparation) suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: creams (lotions), liniments (liniments), powders (powders), aerosols (aerogels), sprays (sprays), emulsions (positions), serums (serums), pastes (pastes), foams (foams), drops (drops), suspensions (suspensions), ointments (salves), and bandages (bandages).
According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base (base) as well known to those skilled in the art.
According to the present inventionThe substrate may comprise one or more additives (additives) selected from the group consisting of: water, alcohols (alcohols), glycols (glycols), hydrocarbons (hydrocarbons) [ such as petroleum jelly (jelly), white petrolatum (white jelly)]Waxes (wax) [ such as paraffin (paraffin) and yellow wax (yellow wax)]Preservatives (preserving agents), antioxidants (antioxidant), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilisers (stabilizing agents), gelling agents (gelling agents) [ such as
974P(/>
974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethyl cellulose)]Active agents (actives), moisturizers (humectants), odor absorbers (odor absorbers), perfumes (fragrans), pH adjusting agents (pH adjusting agents), chelating agents (chelating agents), emulsifiers (emulsifiers), occlusive agents (occlusive agents), softeners (emulsifiers), thickeners (thickeners), solubilizing agents (solubilizing agents), penetration enhancers (penetration enhancers), anti-irritants (anti-irritants), colorants (colorants), and propellants (propellants). The selection and amounts of such additives are within the skill and routine skill of those skilled in the art.
According to the present invention, the care product may further comprise an acceptable adjuvant (acceptable adjuvant) which is widely used in the art of care product manufacture. For example, the acceptable adjuvant may comprise one or more agents selected from the group consisting of: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, colouring agents, thickening agents, fillers, fragrances and odour absorbers. The selection and amounts of such agents are within the skill and routine skill of those skilled in the art.
In accordance with the present invention, the cosmetic may be manufactured in a form suitable for skin care (skincare) or makeup (makeup) using techniques well known to those skilled in the art, including, but not limited to: aqueous solutions (aqueous solutions), aqueous-alcoholic solutions (aqueous-alcoholic solutions) or oily solutions (oily solutions), emulsions in the form of oil-in-water type, water-in-oil type or complex type, gels, ointments, creams, masks (masks), patches, liniments, powders, aerosols, sprays, emulsions, slurries, pastes, foams, dispersions, drops, mousses (mousses), sun blocks (sunblocks), lotions (toilet waters), foundations (foundations), make-up removal products (make-up removal products), soaps (soaps) and other body cleansing products (body cleansing products) and the like.
In accordance with the present invention, the cosmetic may also be used in combination with one or more known active topical agents (external use agents) selected from the following: whitening agents (whitening agents) [ such as retinoids (tretinoin), catechins (catechin), kojic acid, arbutin and vitamin C ], moisturizing agents, anti-inflammatory agents (anti-inflammatory agents), bactericides (bacteriodes), ultraviolet absorbers (ultraviet absorbers), plant extracts (plant extracts) [ such as aloe extracts (aloe extracts) ], skin nutrients (skin nutrients), anesthetics (anesthesics), antiacne (anti-acne agents), antipruritics (antipruritics), analgesics (analgesics), anti-dermatitis agents (anti-dermal agents), anti-hyperkeratosis agents (anti-hyperkeratotic agents), anti-dry skin agents (anti-dry skin agents), anti-perspiration agents (anti-inflammatory agents), anti-aging agents (anti-aging agents), anti-obesity agents (anti-obesity agents), anti-seborrheic agents (anti-dermatitis agents), anti-aging agents (anti-seborrheic agents), anti-dermatitis agents (anti-dermatitis agents), and anti-wrinkle agents (anti-dermatitis agents). The selection and amounts of these external agents are within the skill and routine skill of those skilled in the art.
According to the present invention, the food product may be used as a food additive (food additive) to be added during the preparation of the raw material or during the preparation of the food by conventional methods, and formulated with any edible material into a food product for ingestion by humans and non-human animals.
According to the present invention, the types of food products include, but are not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) and dietary supplements (dietary supplements).
In the description of the following embodiments, the "%" symbol refers to weight percent unless otherwise indicated.
Elderberry (known under the name Sambucus nigra, also known as cichorus nigra) is a shrub fruit of the genus elderberry of the family pentaforaceae, and is grown primarily in europe, for example, milano (Merano), the term of fruit ripening is 7-8 months. The fruit is bright black and the diameter is 3-5 mm. In one embodiment, the elderberry is grown in Milano of Italian by irrigation with snow melt in Alps and hot springs in the pervasive environment, so that the elderberry has abundant bioactive substances such as minerals.
Refer to fig. 1. In one embodiment, the preparation method of the elderberry ferment comprises the following steps: providing a culture solution (step S01), fermenting the culture solution and a plurality of strains in a magnetic field for 7 days to obtain a fermentation stock solution (step S02), and adjusting the sugar degree (Degrees Brix, ° Bx) of the fermentation stock solution to complete the elderberry fermentation product (step S03).
In step S01, the culture solution includes an elderberry solution formed by the elderberry and water in a weight ratio of 1-3:5-29 and glucose which is 10% of the total weight of the elderberry solution. In some embodiments, "elderberry" generally refers to a fruit of a plant, wherein the fruit may comprise the fruit that was originally, dried, or otherwise physically processed to facilitate handling, which may further comprise the fruit that was intact, chopped, diced, milled, ground, or otherwise processed to affect the size and physical integrity of the raw material.
In some embodiments, the elderberry in the "elderberry ferment" can be juice extracted from the fruit of the elderberry. For example, in some embodiments, the elderberry fruit is crushed, squeezed, and concentrated to a sugar content after the pomace and finer suspended matter are removed. In other embodiments, the elderberry ferment may be subjected to other procedures during the manufacturing process that do not substantially affect the efficacy of the elderberry ferment described herein, such as fermentation, to add flavor or other uses.
In some embodiments, elderberry in the "elderberry ferment" may also be an extract obtained by extracting the fruit of elderberry with a suitable extraction solvent. For example, the elderberry fruit extract can be obtained by soaking the elderberry in water, extracting for a proper time at normal temperature, and filtering to remove solid impurities.
In one embodiment, step S01 may include: mixing elderberry and glucose according to the proportion to form a mixed solution; and extracting the mixture at 50-100 deg.C for 0.5-1.5 hr to obtain culture solution. In this way, the extraction process is performed with glucose and elderberry solution, which helps to dissolve glucose and avoid contamination.
In another embodiment, step S01 includes: extracting herba Sambuci Williamsii with water at 50-100 deg.C for 0.5-1.5 hr to obtain water extract; and adding glucose to the aqueous extract to obtain a culture solution. In this way, the elderberry is separately extracted to facilitate the effective components in the elderberry to be released really.
In some embodiments, extraction refers to maintaining the mixture at 50-100 ℃ and standing for 0.5-1.5 hours. In one embodiment, extraction refers to maintaining the mixture at 95 ℃ and standing for 1 hour.
In one embodiment, the sugar degree of the culture medium is 9 ° Bx to 10 ° Bx. In this way, the sugar degree is enough to ensure the smooth proceeding of the subsequent fermentation, and the strains can have enough nutrients.
Subsequently, the culture solution and the plurality of bacterial seeds are fermented in a magnetic field for 4 to 15.5 days to obtain a fermentation stock solution (step S02). Wherein the plurality of strains comprise 0.01-0.5% of yeast, 0.01-0.25% of lactic acid bacteria and 3-10% of acetic acid bacteria (Acetobacter aceti). In one embodiment, the culture solution is directly added with the strain without filtering out the solid matter (the extracted elderberry) therein for fermentation, so as to further extract the active ingredients in the solid matter by using the strain.
In one embodiment, the yeast may be a commercially available brewer's yeast (Saccharomyces cerevisiae). For example, the lager brewing yeast strain deposited at the institute for food development with accession number BCRC20271 (International deposit ATCC 26602) was purchased.
In one embodiment, the lactic acid bacteria can be commercially available Lactobacillus plantarum (Lactobacillus plantarum), streptococcus thermophilus (Streptococcus thermophilus) or Lactobacillus plantarum. For example, lactobacillus plantarum (available from institute for food development, accession number BCRC 910760) was used. As a further example, streptococcus thermophilus TCI633 (International deposit for Streptococcus thermophilus TCI 633: german Collection of microorganisms, 12.12.12.2013, deposit number DSM28121, deposit number Germany) with the deposit number BCRC910636 strain is used.
In one example, acetobacter aceti strain having the accession number BCRC11688 (International deposit ATCC 15973) was purchased from the American Type Culture Collection.
In one embodiment, "brewers yeast (Saccharomyces cerevisiae)", "Lactobacillus plantarum (Lactobacillus plantarum)", and "acetic acid bacterium (Acetobacter aceti)" are respectively intended to encompass brewers yeast, lactobacillus plantarum, and acetic acid bacterium, respectively, that are readily available to those skilled in the art (e.g., available from domestic or foreign depositories), or that are isolated and purified from natural sources using microbial isolation methods customary in the art.
Refer to fig. 2. In one embodiment, step S02 may include: fermenting yeast in a magnetic field for 1-2.5 days to form a first primary fermentation liquid (step S21); adding lactic acid bacteria into the first primary fermentation liquid and fermenting in a magnetic field for 1-3 days to form a second primary fermentation liquid (step S22); adding acetic acid bacteria into the second primary fermentation liquid and fermenting in a magnetic field for 3-10 days to form primary fermentation liquid (step S23); and filtering the primary fermentation liquid to obtain a fermentation raw liquid (step S24).
In step S21, the culture solution may be fermented to produce alcohol by first adding yeast; therefore, the alcohol is beneficial to extracting different effective components of the human body participating in the medlar. In some embodiments, the first primary fermentation broth has a PH of less than 4 and a brix of about 9 ° Bx.
In step S22, the PH of the first primary fermentation broth is lowered by adding lactic acid bacteria such that glucose in the first primary fermentation broth is further consumed to lower the sugar degree and produce lactic acid; therefore, the pH value of the first primary fermentation liquid is reduced, so that other different effective components in the ginseng and the Chinese wolfberry can be further extracted. In some embodiments, the PH of the second primary fermentation broth is less than 3.5 and its brix is about 6 ° Bx.
In step S23, the content of glucose can be further reduced by adding acetic acid bacteria to consume the alcohol in the second primary fermentation broth. In some embodiments, the PH of the primary fermentation broth is less than 3.5 and its sugar content is about 3 ° Bx.
In one embodiment of step S24, the primary fermentation broth may be filtered and concentrated to obtain a fermentation stock. In one example, the concentration may be performed by vacuum concentration at 60 ℃, and the filtration may be performed by 200mesh filtration. In some embodiments, the sugar content of the fermentation liquor is about 2 ° Bx. In some embodiments, the PH of the fermentation broth is less than 3.5.
In one embodiment of step S03, oligosaccharides may be added to the fermentation stock to make the sugar degree of the fermentation stock reach 40 ° Bx to form an elderberry ferment. Herein, the oligosaccharide refers to an oligosaccharide obtained by polymerizing 3 to 10 monosaccharide molecules. Wherein the oligosaccharide can be fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, etc. In one embodiment, the oligosaccharides added may be an oligosaccharide solution containing 40% to 70% isomalto-oligosaccharides.
In one embodiment, the total polyphenol content of the elderberry fermentation product obtained in step S03 is 436.33 μ g/ml. In one embodiment, the elderberry ferment has a flavone content of 12703 μ g/ml.
[ example 1]: preparation method of fermentation product of elderberry
Mixing the elderberry of Milano purchased from Italian university, water and glucose to form a mixed solution. In the mixed solution, the weight ratio of elderberry to water is 1:5 to form an elderberry solution (i.e., one time the weight of elderberry and five times the weight of water), and the glucose is 10% of the total weight of the elderberry solution formed by elderberry and water. Subsequently, the mixture was extracted at 95 ℃ for 1 hour to obtain a culture solution. The magnet arranged on the periphery of the fermentation barrel can form a magnetic field in the fermentation barrel, and the culture solution is placed into the fermentation barrel with the magnet arranged on the periphery after being cooled, so that the culture solution is fermented in the magnetic field. Saccharomyces cerevisiae of BCRC20271 strain was added to the culture broth in a fermenter in an amount of 0.01% relative to the culture broth, and then fermented at 30 ℃ for 1 day to form a first primary fermentation broth. 0.05% of Streptococcus thermophilus (Streptococcus thermophilus TCI633, deposited number BCRC910636; streptococcus thermophilus TCI633 International depositary, german Collection of microorganisms, deposited number DSM28121, 12.12.2013) is added to the first primary fermentation liquid in the fermentation vat, and then fermentation is performed for 1 day to form a second primary fermentation liquid. And finally adding 5 percent of acetic acid bacteria of the BCRC11688 strain relative to the culture solution into the second primary fermentation liquid in the fermentation barrel, and fermenting for 5 days to obtain a fermentation stock solution. After the fermentation stock solution was obtained, the primary fermentation liquid was concentrated under reduced pressure at 60 ℃ and filtered with 200mesh to obtain a fermentation stock solution. Then, 60% of isomalto-oligosaccharide and 40% of fermentation stock solution are mixed to make the sugar degree of the fermentation stock solution reach 40 ° Bx, so as to form the elderberry magnetic field fermentation product.
Preparing a solid elderberry magnetic field fermentation product:
in order to obtain the solid-state elderberry magnetic field fermentation product, the solvent of the elderberry magnetic field fermentation product subjected to reduced pressure concentration can be removed in a spray drying manner, so that elderberry magnetic field fermentation product powder is obtained.
Example 2 preparation of fermented product of elderberry (general fermentation)
The same raw material ratio and fermentation procedure as the elderberry fermented product of example 1 were adopted, but no magnet was disposed on the periphery of the fermentation barrel to obtain the elderberry fermented product.
Example 3 preparation of aqueous extract of elderberry (no fermentation)
Mixing Williams Elder Raspberry of Milano of Italian university with water at a ratio of 1:5, adding glucose solution to adjust sugar degree to 9 to obtain Williams Elder water extract with pH of 6.3.
Example 4 Total Polyphenol content test
The fermented product of elderberry obtained in example 1 and the fermented product of elderberry obtained in example 2 were used the elderberry water extract is used as an experimental group and a control group. Each sample was diluted 10-fold with water and 100mL of the diluted sample was placed in a centrifuge tube. Then, 500. Mu.L of Folin-Ciocalteu phenol reagent is added into a centrifuge tube to be mixed with the diluted sample and kept stand for 3 minutes, and then 400. Mu.L of 7.5% sodium carbonate is added to be mixed and kept stand for 30 minutes to obtain a reaction solution to be detected. After the secondary standing, 200 mu L of reaction solution to be detected is taken to a 96-well plate, and the light absorption value of the reaction solution to be detected under 750nm is measured.
Then, a calibration curve was prepared using Gallic acid (Gallic acid) as a standard. Here, 0. Mu.L/mL, 20. Mu.L/mL, 40. Mu.L/mL, 60. Mu.L/mL, 80. Mu.L/mL, and 100. Mu.L/mL of a gallic acid standard solution were prepared, and 100. Mu.L of each concentration of the standard solution was taken out into a 10mL centrifuge tube. Add 500. Mu.L Folin-Ciocalteu phenol reagent into the centrifuge tube, mix with the standard solution and stand for 3 minutes, add 400. Mu.L 7.5% sodium carbonate, mix well and stand for 30 minutes to obtain the standard reaction solution. A standard curve was obtained by taking 200. Mu.L of a standard reaction solution in a 96-well plate and measuring the absorbance at 750 nm.
And then, converting the light absorption value of the reaction solution to be detected into the total polyphenol content by using a standard curve. Thus, the total polyphenol content of the elderberry magnetic field fermentation product of the experimental group is 1134.6 mug/ml and the total polyphenol content of 557.6 mug/ml in the elderberry fermentation product of the control group can be obtained, as shown in fig. 3.
The experimental result shows that the total polyphenol content of the elderberry magnetic field fermentation product is increased by 203.5 percent compared with the total polyphenol content of the elderberry fermentation product; therefore, the fermentation in the magnetic field is more beneficial to the extraction of the effective components.
Example 5 Osteogenesis (Osteogenesis) promotion test
To confirm that the elderberry magnetic field fermentate of the present invention has the ability to promote the formation of hard bone, the present invention was confirmed using Alizarin Red S (Sigma-Aldrich) staining. Alizarin Red S is a yellow-orange crystal, soluble in water, and capable of binding with calcium salt to form an orange-Red precipitate for detection of calcium deposition or calcified material in cells.
The invention uses the elderberry magnetic field fermentation product to treat OP9 new mouse bone marrow interstitial cell strain (obtained by ATCC number CRL-2749), uses untreated OP9 cell strain as control group, uses the elderberry water extract of example 3 as control group 1, uses the elderberry fermentation product of example 2 as control group 2, and observes the situation that the elderberry magnetic field fermentation product of example 1 differentiates OP9 cell strain into hard bone cells. The results show that alizarin red staining analysis shows that the OP9 cells treated by the elderberry magnetic field fermentation product can differentiate into hard bone cells, thereby increasing the accumulation of calcium ions. And the analyzed data result is shown in fig. 4, compared with the control group which is not treated by the elderberry magnetic field fermentation product and has 100% of differentiation into hard bone cells, the control group 1 has 72.05% of differentiation into hard bone cells of the OP9 cell strain treated by the elderberry water extract, the control group 2 has 74.38% of differentiation into hard bone cells, and the experimental group has 129.45% of differentiation into hard bone cells of the OP9 cell strain treated by the elderberry magnetic field fermentation product, so that the invention has the advantage that the invention can effectively improve the cartilage differentiation capacity by 1.29 times, and the invention has proved that the invention has the capability of effectively promoting the hard bone cell growth and hard bone formation.
[ example 6] detection of Gene involved in macrophage phagocytic Activity
A6-well plate (purchased from GeneDireX) was prepared, and the human monocytic cell lines THP-1 (containing RPMI medium and 10% FBS) of the control, control 1, control 2 and experimental groups of example 6 were injected into each well at 5X10 5 The mononuclear cell strain THP-1 is cultured in each wellIn 6-well plates, recipients were cultured for 24 hours by adding a differentiation agent (phorbol 12-myristate 13-acetate (PMA), purchased from Sigma) at a concentration of 500 Nemomol per well to differentiate THP-1, a monocyte cell strain, into macrophages. Then, the medium was removed from each well and the cells were divided into 4 groups, control 1, control 2 and experimental. For the cells of the control group, only 200 μ L of FBS-free medium was added; adding only 200 μ L of aqueous extract of elderberry to the cells of control group 1; for the cells of the control group 2, 200 μ L of elderberry fermentation product was added; for the cells of the experimental group, 200. Mu.L of elderberry magnetic field fermentation product was added. Each group of cells was incubated in 5% CO 2 And cultured at 37 ℃ for 24 hours.
Using RNA extraction kit, reverse transcriptase, KAPA
The FAST qPCR reagent set is combined with a quantitative PCR instrument, and the gene expression quantity change of interleukin 18 (IL-18, gene ID 3606) in cells after macrophages are treated by a elderberry magnetic field fermentation product is measured, and the interleukin 18 can stimulate the innate immunity and promote the aggregation and phagocytosis of the macrophages.
Table 1:
* R is REVERSE and F is FORWARD.
Please refer to fig. 5. Regarding the IL-18 gene expression level of the control group as 1 (i.e., 100%), the IL-18 gene expression level of the control group 2 was increased to 2.04, the IL-18 gene expression level of the experimental group was increased to 2.48, and the IL-18 gene expression level of the representative experimental group was 2.48 times higher than that of the control group, compared to the IL-18 gene expression level of the control group 1 increased to 1.74. Therefore, after mouse macrophages are treated by the magnetic enzyme containing elderberry, the expression level of the IL-18 gene is increased, which shows that the magnetic enzyme containing elderberry has the effects of stimulating the innate immunity and improving the phagocytic activity of the macrophages.
[ example 7] macrophage phagocytic Activity assay
0.5% fluorescent beads (1.7-2.2 μm in size, commercially available from Spheroeth) were added to macrophages in the control, control 1, control 2 and experimental groups of example 6 and incubation was continued for 4 hours. The recipient, the culture medium was removed, 1 XPBS was added for 3 washes, each well was divided into 5 zones, 2 fields were randomly selected for each zone and photographed with a fluorescence microscope (purchased from ZEISS), and the number of fluorescent beads in macrophages in the photograph was further counted and the statistics of the results are shown in FIG. 6.
As shown in FIG. 6, the number of fluorescent beads in the macrophages of control group was 100%, the number of fluorescent beads in the macrophages of control group 1 was 106.03%, the number of fluorescent beads in the macrophages of control group 2 was 113.79%, and the number of fluorescent beads in the macrophages of experimental group 1 was 145.69%. Therefore, compared with the control group, macrophages co-cultured with elderberry magnetic ferment can phagocytose more fluorescent micro-beads, which shows that the elderberry magnetic ferment can significantly improve the phagocytosis ability of macrophages.
In order to confirm the efficacy of the elderberry magnetic enzyme of the present invention on human body, the following human body experiments were also performed.
[ example 8]: human body experiment-determination of antioxidant capacity
This experiment was conducted on 6 women with a age of 45-65 who had stopped taking a drink containing 7.5ml of elderberry magnetic ferment prepared in example 1 for 8 consecutive weeks. Blood was drawn at weeks 0 and 8 to detect the amount of total antioxidant activity (TAC), glutathione S transferase (GST-RBC), sulfur compounds (f-Thiols), and Malondialdehyde (MDA) in the blood before and after the drink containing the present elderberry magnetoferment was taken.
The Total Antioxidant Capacity (TAC), glutathione S transferase (GST-RBC), sulfur compounds (f-Thiols), and Malondialdehyde (MDA) in the blood of the subjects of this example were determined by the agency of the King-independent laboratory (Taiwan, china).
FIG. 7 shows the variation of the Total Antioxidant Capacity of the subjects at week 0 and week 8 after taking the beverage containing the elderberry magnetometers of the invention, the Total Antioxidant Capacity (TAC) value of the subject at week 0 is about 0.56mmol/L, the Total Antioxidant Capacity of the subject at week 8 is increased to about 0.64 mmol/L, which is increased by 14.3%, and the Total Antioxidant Capacity (TAC) measurement measures the Total Antioxidant Capacity of the non-enzymatic Antioxidant population in plasma, which is more responsive to the Total Antioxidant Capacity of the whole body than the measurement of one Antioxidant alone. Higher TAC values measured indicate higher antioxidant capacity in vivo. The elderberry magnetic ferment disclosed by the invention can strengthen the in-vivo antioxidant capacity and help resist the injury induced by free radicals.
Fig. 8 shows the amount of change of glutathione S transferase in the subjects at week 0 and week 8 after taking the beverage containing elderberry magnetic ferment of the present invention, the glutathione S transferase (GST-RBC) value of the subject at week 0 is about 5.17U/g-Hb, the glutathione S transferase of the subject at week 8 is increased to about 6.19U/g-Hb by 19.7%, the increase of the glutathione S transferase enhances the efficacy of oxidation resistance and immunity, reduces hydrogen peroxide in vivo to water and oxygen, and reduces lipid peroxide to harmless products, reducing oxidation pressure in vivo.
Fig. 9 shows the variation of sulfur-containing compounds in the subjects at week 0 and week 8 after taking the drink containing elderberry magnetomase of the invention, the concentration of sulfur-containing compounds in the subjects at week 0 was 311.00 μ g/mL, and the concentration of sulfur-containing compounds in the subjects at week 8 rose to about 348.50 μ g/mL, which increased by 12%. The elderberry magnetic ferment can increase the content of sulfur-containing compounds, and the sulfur-containing compounds are good antioxidant substances, are quite sensitive to free radicals, can alleviate the damage of Reactive Oxygen Species (ROS), and have the functions of detoxifying and strengthening liver functions.
Fig. 10 shows the variation of malondialdehyde at week 0 and week 8 of the beverage containing the elderberry magnetic ferment of the present invention, the malondialdehyde concentration at week 0 of the subject is about 1.36 nmol/mL, the malondialdehyde concentration at week 8 of the subject is reduced to about 1.30nmol/mL, which is reduced by 4.4%, malondialdehyde (MDA) is formed by lipid peroxidation between free radicals in the body and phospholipids (phospholipids) on the cell membrane, the faster the MDA formation indicates poor body antioxidant capacity, the more the MDA accumulation indicates high injury probability. The elderberry magnetic ferment can reduce the content of MDA, avoid skin from generating black spots and freckles caused by MDA, accelerate the aging process, damage cholesterol to cardiovascular diseases such as arterial blood vessels and coronary arteriosclerosis, and cancer caused by DNA damage. Lipid peroxidation occurs while lowering phospholipids (phospholipids) on cell membranes.
[ example 9]: human body experiment-determination of capability of promoting bone formation
The experiment was conducted on the subjects of example 8 at weeks 0 and 8 to detect the changes in the concentrations of osteocalcin and type i procollagen nitrogen-terminal pro-peptide chain (P1 NP) before and after taking the beverage containing the present invention.
The concentrations of osteocalcin and nitrogen-terminal pro-peptide of type I procollagen in the blood of the subjects of this example were determined by the test division of the same laboratory as Wei, taiwan, china.
Fig. 11 shows the change of osteocalcin concentration of the subject at week 0 and week 8 after taking the beverage containing the elderberry magnetoferment of the invention, and the results are shown in fig. 11, wherein the osteocalcin concentration of the subject at week 0 is about 17.70ng/mL, and the osteocalcin concentration of the subject at week 8 is increased to about 18.02ng/mL, which is increased by 1.8%, so that the effects of promoting the bone formation rate and slowing down osteoporosis are achieved.
Fig. 12 shows the change of the concentration of the nitrogen-terminal front chain of the type i precollagen at week 0 and week 8 after the subjects take the beverage containing the elderberry magnetoferment of the present invention, and the results are shown in fig. 11, the concentration of the nitrogen-terminal front chain of the type i precollagen at week 0 is 60.66ng/mL, and the concentration of the nitrogen-terminal front chain of the type i precollagen at week 8 of the subjects is increased to about 66.65ng/mL, which is increased by 9.9%, and the effects of promoting the bone formation rate and slowing down osteoporosis are achieved.
[ example 10]: human body experiment-bone density determination
The subject of example 8 was evaluated for bone density by measuring the T-score using a dual-energy X-ray absorption instrument (dual-energy X-ray absorption) at week 0 and week 8.
Fig. 12 shows the change of T scores of the subjects at week 0 and week 8 after taking the beverage containing elderberry magnetometers of the invention, and the results are shown in fig. 13, wherein the T score at week 0 is about-0.92, and the T score of the subjects at week 8 is increased to-0.85, which is 7.6% higher.
In summary, the manufacturing method of any embodiment doubles the total polyphenol content of the elderberry fermentation product compared with the conventional process, and greatly improves multiple effects of the elderberry fermentation product. In addition, the fermented elderberry of any embodiment may be used for preparing a composition for promoting differentiation of hard bone, enhancing immunity or resisting oxidation, wherein the composition is used for promoting differentiation of hard bone, increasing calcium ion content in bone cells, preventing osteoporosis, strengthening bone density and/or strengthening bone by increasing the concentration of Osteocalcin (osteopecalin) and/or nitrogen-terminated pre-peptide (P1 NP) of procollagen type i in blood, enhancing immunity by increasing phagocytic power of interleukin 18 and/or white blood cells, achieving health care effects, reducing oxidation by increasing glutathione S transferase (GST-RBC), sulfur-containing compounds (f-Thiols), and Malondialdehyde (MDA) in blood, increasing reducing substances in blood, eliminating oxidizing substances in body, reducing oxidation pressure, avoiding damage to protein molecules, enzymes in body, interfering with their effects, and avoiding peroxidation of unsaturated fatty acids.
The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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Claims (11)
1. A preparation method of a elderberry magnetic field fermentation product is characterized by comprising the following steps: the elderberry magnetic field fermentation product comprises:
providing a culture solution, wherein the culture solution comprises an elderberry solution formed by the weight ratio of the elderberry to water of 1-3:5-29 and 10 wt% of glucose of the total weight of the elderberry solution;
fermenting the culture solution and a plurality of strains in a magnetic field for 4-15.5 days to obtain a fermentation stock solution, wherein the plurality of strains comprise 0.01-0.5 wt% of yeast, 0.01-0.25 wt% of lactic acid bacteria and 3-10 wt% of acetic acid bacteria; and
adjusting the sugar degree of the fermentation stock solution to form the elderberry magnetic field fermentation product.
2. The method of claim 1, wherein: the step of fermenting the culture solution and the plurality of strains in the magnetic field comprises:
fermenting the microzyme in the magnetic field for 1 to 2.5 days to form a first primary fermentation liquid;
adding the lactic acid bacteria into the first primary fermentation liquid, and fermenting in the magnetic field for 1-3 days to form a second primary fermentation liquid;
adding the acetic acid bacteria into the second primary fermentation liquid and fermenting in the magnetic field for 3-10 days to form primary fermentation liquid; and
filtering the primary fermentation liquid to obtain the fermentation stock solution.
3. The method of claim 1, wherein: a magnetic flux density of the magnetic field is 120 to 300 millitesla.
4. The method of claim 1, wherein: the total polyphenol content in the elderberry fermentation liquor is 1134.6 mug/ml.
5. The method of claim 1, wherein: the yeast is Saccharomyces cerevisiae with the preservation number of ATCC26602, the lactic acid bacteria is Lactobacillus plantarum with the preservation number of DSM33108, and the acetic acid bacteria is Acetobacter aceti with the preservation number of ATCC15973.
6. Use of fermentation product of magnetic field of herba Sambuci Williamsii in preparing composition for promoting hard bone differentiation, enhancing immunity or resisting oxidation is provided.
7. Use according to claim 6, characterized in that: the fermentation liquor of elderberry can increase the concentration of Osteocalcin (Osteocalcin) and/or the nitrogen-terminal pro-peptide chain (P1 NP) of the type I procollagen in blood.
8. Use according to claim 6, characterized in that: the elderberry fermentation liquor increases the phagocytic capacity of interleukin 18 and/or white blood cells.
9. Use according to claim 6, characterized in that: the elderberry fermentation broth increases the concentration of glutathione S-transferase (GST-RBC), sulfur-containing compounds (f-Thiols), malondialdehyde (MDA), or combinations thereof, in red blood cells in blood.
10. Use according to claim 6, characterized in that: the weight ratio of elderberry to water in the elderberry fermentation liquid is 1-3:5-29, and the elderberry fermentation liquid is fermented in a magnetic field for 4-15.5 days by a plurality of strains, wherein the plurality of strains comprise 0.01-0.5 wt% of yeast, 0.01-0.25 wt% of lactic acid bacteria and 3-10 wt% of acetic acid bacteria.
11. Use according to claim 6, characterized in that: the elderberry fermentation liquor is further prepared into a maintenance composition, a health food composition, a cosmetic composition or a skin external agent.
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| CN112106909A (en) * | 2019-06-21 | 2020-12-22 | 百岳特生物技术（上海）有限公司 | Ginseng and medlar fermentation liquor and preparation method thereof |
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| TWI797780B (en) * | 2021-10-15 | 2023-04-01 | 大江生醫股份有限公司 | Manufacturing method of sambucus nigra magnetic ferments and use thereof for promoting hard bone formation, enhancing immunity, or anti-oxidation |
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| CN108077925A (en) * | 2017-12-15 | 2018-05-29 | 石家庄君乐宝乳业有限公司 | The preparation method of synthetism rasp berry extract and the probiotic powder of tool enhancing immune function |
| CN112106909A (en) * | 2019-06-21 | 2020-12-22 | 百岳特生物技术（上海）有限公司 | Ginseng and medlar fermentation liquor and preparation method thereof |
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