CN113925884B - Use of metagen extract for promoting skin regeneration and anti-aging - Google Patents
Use of metagen extract for promoting skin regeneration and anti-aging Download PDFInfo
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- CN113925884B CN113925884B CN202010601261.4A CN202010601261A CN113925884B CN 113925884 B CN113925884 B CN 113925884B CN 202010601261 A CN202010601261 A CN 202010601261A CN 113925884 B CN113925884 B CN 113925884B
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Abstract
Use of an extract of metazoan for promoting skin regeneration and anti-aging, said metazoan being prepared by a process comprising the steps of: providing a first substance having a first isoelectric point in the range of pH 1 to pH 6 and a second substance having a second isoelectric point in the range of pH 4 to pH 8, wherein the second isoelectric point is higher than the first isoelectric point and they have a pH difference in the range of 0.5 to 3; mixing the first substance with a probiotic in water having a pH above the second isoelectric point to obtain a mixture; adding the second substance to the mixture, and then adjusting the pH of the mixture so that the pH of the mixture falls between the first isoelectric point and the second isoelectric point, thereby forming a precipitate; and taking the precipitate to perform cell wall separation and extraction treatment.
Description
Technical Field
The present invention relates to the use of a metazoan extract to promote skin regeneration (skin regeneration).
Background
Skin is the largest barrier protecting human individuals and has the function of combating pathogenic bacteria and various environmental damages. When the skin is exposed to various environmental factors (e.g., ultraviolet light, radiation, particulates), it may promote the skin's production of reactive oxygen species (reactive oxygen species, ROS) and free radicals (FREE RADICALS). When the amount of reactive oxygen species and free radicals exceeds the antioxidant capacity of the skin cells or tissues themselves, oxidative stress (oxidative stress) is developed to cause oxidative damage to the skin cells or tissues and even further induce inflammatory reactions.
Fibroblasts (fibroblasts) are mainly located in connective tissue (connective tissue), and play an important role in wound healing (wound-healing) and angiogenesis (angiogenesis), etc., for synthesizing cells of extracellular matrix (extracellular matrix, ECM) [ structural proteins (structural protein) including collagen (collagen) and elastin (elastin), hyaluronic acid (hyaluronic acid) and proteoglycan (proteoglycan) ]. In addition, it has been found that proteins such as fibroblast growth factor (fibroblast growth factor, FGF) and transforming growth factor-beta (transforming growth factor-beta, TGF-beta) have the effect of promoting fibroblast proliferation and collagen synthesis. When the secretion amount of these growth factors decreases with age, it causes gradual loss of collagen, elastin and hyaluronic acid in the skin (i.e., their synthesis rate is not the same as the degradation rate thereof), and the number of fibroblasts becomes smaller [ e.g., apoptosis (apoptosis) ], thereby resulting in skin atrophy (skin texture), deterioration of skin elasticity (SKIN ELASTICITY), roughening of skin texture (skin texture), deterioration of skin water retention capacity (SKIN WATER-holding capacity), and skin aging (SKIN AGING) such as wrinkles (wrinkle formation). Thus, there is currently a clinical use of fibroblasts to promote skin regeneration (skin regeneration) and to achieve the effects of skin repair (SKIN REPAIR) and anti-aging (antiaging).
Probiotics (probiotics) are a group of microorganisms that improve the intestinal flora (INTESTINAL MICROFLORA) and the intestinal immunity (INTESTINAL IMMUNITY). Probiotics have been widely used in foods and health products, and currently commonly used probiotics include: lactobacillus (Lactobacillus), bifidobacterium (Bifidobacterium), bacillus (Bacillus), lactococcus (Lactococcus), enterococcus (Enterococcus), saccharomyces (Saccharomyces), streptococcus (Streptococcus) and the like.
Although probiotics have the above benefits on the health of humans or animals, they can be susceptible to gastric acid damage after oral administration, rendering them ineffective in the gut. In addition, the probiotics is a living microorganism food component (live microbial food ingredient), so that the food and health care products prepared from the probiotics have the problem of difficult preservation.
Recent studies have found that inactivated probiotics (non-viable probiotics) and lysates (lysate), extracts or fractions of probiotics [ known as metazoans (postbiotics) ] have efficacy similar to probiotics and are more gastric acid tolerant and easier to preserve than probiotics. The effects of the metazoan are considered to be cell wall components derived from probiotics, and mainly include peptidoglycan (peptidoglycan), teichoic acid (teichoic acid), lipoteichoic acid (lipoteichoic acid), polysaccharides (polysacharide), proteins (proteins), and the like. There are many methods available for extracting metazoan from the cell walls of probiotics, but these methods generally suffer from poor extraction efficiency. Accordingly, there remains a need in the art to develop methods for efficiently extracting metazoan that meet the needs of the industry.
As a result of research, the applicant has unexpectedly found that: the metazoan extract prepared according to the method of the present invention is effective in promoting proliferation of human fibroblasts and secreting anti-inflammatory hormone, and is expected to be applied to promote skin regeneration and anti-aging (antiaging).
Disclosure of Invention
Accordingly, the present invention provides the use of a metazoan extract (postbiotics extract) for the preparation of a composition for promoting skin regeneration (skin regeneration), wherein the metazoan extract is prepared by a process comprising the steps of:
Providing a first substance and a second substance, wherein the first substance has a first isoelectric point (first isoelectric point) falling within a range of pH 1 to pH 6, the second substance has a second isoelectric point (second isoelectric point) falling within a range of pH4 to pH 8 and higher than the first isoelectric point, and the second isoelectric point has a pH difference of 0.5 to 3 from the first isoelectric point;
Mixing the first substance and a probiotic in water having a pH above the second isoelectric point to obtain a mixture;
adding the second substance to the mixture, and then adjusting the pH of the mixture so that the pH of the mixture falls between the first isoelectric point and the second isoelectric point, thereby forming a precipitate; and
The precipitate is subjected to a cell wall separation extraction process (cell wall isolation and extraction treatment), whereby the metazoan extract is obtained.
Preferably, the first substance is selected from the group consisting of: skim milk powder, casein, whey protein, soy protein, pea protein, egg protein, rice protein, hydrolyzed protein, corn protein, wheat protein, barley protein, branched chain amino acids, gelatin, collagen, amino acids, chitosan oligosaccharides, and combinations thereof.
Preferably, the second substance is selected from the group consisting of: sodium alginate, carrageenan, pectin, acacia, xanthan gum, locust bean gum, starch, trehalose, dextrin, syrup, guan Huadou gum, konjak fine powder, plant fiber, synthetic fiber, semisynthetic fiber, and combinations thereof.
Preferably, the probiotic is selected from the group consisting of: bacillus species, streptococcus species, lactococcus species, auxotroph species, balloon species, carnivorous species, enterococcus species, lactobacillus species, leuconostoc species, 9. Schwann species, micrococcus species, tetranecticoccus species, loiter species, weissella species, bifidus species, saccharomycetes species, kluyveromyces species, staphylococcus species, pediococcus species, propionibacterium species, and combinations thereof.
Preferably, the composition further comprises a health care substance selected from the group consisting of: hyaluronic acid, sodium hyaluronate, vitamin C, glucosamine, chondroitin, type one collagen, type two collagen, type three collagen, fish skin and scale collagen, non-denatured type two collagen, porcine collagen, bovine collagen, sialic acid, anthocyanin, polyphenol, flavonoid, eggshell membrane, and combinations thereof.
The invention has the beneficial effects that: the method of the invention can effectively extract metazoan from the cell wall of the probiotics, and the prepared metazoan extract contains more cell components containing protein and has excellent effects on promoting fibroblast proliferation and secreting anti-inflammatory hormone. Therefore, the metagen extract obtained by the method according to the present invention can be applied to promote skin regeneration and has a high potential to develop into a commodity for skin repair, promotion of skin wound healing and aging resistance.
Drawings
The above and other objects and features of the present invention will become more apparent by referring to the following description, appended claims and accompanying drawings in which:
FIG. 1 is a view of an electrophoresis film showing the results of protein electrophoresis analysis of the metazoan extract of the present invention and the existing metazoan extract.
Detailed Description
For the purposes of this specification, it will be clearly understood that: the word "comprising" means "including but not limited to", and the word "comprising" has a corresponding meaning.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. Of course, the invention is in no way limited to the methods and materials described.
In the present invention, the applicant found through experiments that: the method of the invention can effectively extract the metagen (postbiotics) from the cell wall of the probiotics, and the prepared metagen extract contains more cell components containing protein. In addition, the metazoan extract has the effect of promoting proliferation of human fibroblasts and secretion of transforming growth factor-beta (transforming growth factor-beta, TGF-beta), and thus is expected to be useful for promoting skin regeneration (skin regeneration) and anti-aging (antiaging).
Accordingly, the present invention provides the use of a metazoan extract for the preparation of a composition for promoting skin regeneration, wherein the metazoan extract is prepared by a process comprising the steps of:
Providing a first substance and a second substance, wherein the first substance has a first isoelectric point falling within a range from pH 1 to pH 6, the second substance has a second isoelectric point falling within a range from pH 4 to pH 8 and higher than the first isoelectric point, and the second isoelectric point and the first isoelectric point have a pH difference value falling between 0.5 and 3;
Mixing the first substance and a probiotic in water having a pH above the second isoelectric point to obtain a mixture;
adding the second substance to the mixture, and then adjusting the pH of the mixture so that the pH of the mixture falls between the first isoelectric point and the second isoelectric point, thereby forming a precipitate; and
The precipitate is subjected to a cell wall separation and extraction treatment, thereby obtaining the metazoan extract.
As used herein, the terms "skin regeneration (skin regeneration)", "skin regeneration (SKIN RENEWAL)" and "skin rejuvenation (skin rejuvenation)" may be used interchangeably.
According to the invention, the probiotic is selected from the group consisting of: bacillus species (Bacillus spp.), streptococcus species (Streptococcus spp.), lactococcus species (Lactococcus spp.), auxotroph species (Abiotrophia spp.), balloon species (Aerococcus spp.), carnivorous species (Carnobacterium spp.), enterococcus species (Enterococcus spp.), lactobacillus species (Lactobacillus spp.), leuconostoc species (Leuconostoc spp.), streptococcus species (Oenococcus spp.), pediococcus species (Pediococcus spp.), tetracoccus species (Tetragenococcus spp.), lozenges species (Vagococcus spp.), weissella species (Weissella spp), bifidobacterium species (Bifidobacterium spp.), saccharomyces species (Saccharomyces spp.), staphylococcus species (Staphylococcus spp.), staphylococcus spp), staphylococcus species (Staphylococcus spp.), staphylococcus spp.s..
Preferably, the probiotic is a lactobacillus species selected from the group consisting of: lactobacillus plantarum (Lactobacillus plantarum), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei (Lactobacillus casei), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus paracasei (Lactobacillus paracasei), and combinations thereof.
Preferably, the probiotic is a bifidobacterium species selected from the group consisting of: bifidobacterium bifidum (Bifidobacterium bifidum), bifidobacterium lactis (Bifidobacterium lactis), bifidobacterium longum (Bifidobacterium longum), bifidobacterium breve (Bifidobacterium breve), bifidobacterium animalis (Bifidobacterium animalis), and combinations thereof.
Preferably, the probiotic is a bacillus species selected from the group consisting of: bacillus coagulans (Bacillus coagulans), bacillus subtilis (Bacillus subtilis), bacillus clausii (Bacillus clausii), and combinations thereof.
According to the invention, the probiotic may be live or dead bacteria, concentrated (concentrated) or unconcentrated (non-concentrated), liquid (paste), semi-solid, or solid (solid) [ e.g. pellet, fine particle or powder ], and may be heat-inactivated (heat-inactivated), frozen (frozen), dried (dried), or freeze-dried (freeze-dried) [ e.g. in freeze-dried form or spray/fluid bed dried form ]. In a preferred embodiment of the invention, the probiotic is heat-inactivated and in the form of a spray-dried powder.
According to the invention, the heat inactivation of the probiotics may be performed by heating at 60-140 ℃ for 1 second to 30 minutes. In a preferred embodiment of the invention, the heat inactivation of the probiotic is carried out by heating at 73.+ -. 2 ℃ for 15 seconds.
According to the present invention, the first substance is selected from the group consisting of: skim milk powder (nonfat dry milk), casein (casein), whey protein (whey protein), soy protein (soy protein), pea protein (pea protein), egg protein (egg protein), rice protein (rice protein), hydrolyzed protein (hydrolyzed protein), corn protein (corn protein), wheat protein (wheat protein), barley protein (barley protein), branched chain amino acids (branched chain amino acid), gelatin (gelatin), collagen (collagen), amino acids (amino acid), chitosan (chitosan), chitin (chitosan), and combinations thereof. In a preferred embodiment of the invention, the first substance is whey protein.
According to the present invention, the second substance is selected from the group consisting of: sodium alginate (sodium alginate), agar (agar), carageenan (carageenan), pectin (pectin), acacia gum (arable gum), xanthan gum (locust bean gum) (locust bean gum), starch (starch) [ e.g. modified starch (modified starch) ], trehalose (fucose), dextrin (dextrin) [ e.g. resistant dextrin (RESISTANT MALTODEXTRIN) ], syrup (syrup), guan Huadou gum (guar gum), konjak flour (konjac powder), vegetable fiber (vegetable fiber), synthetic fiber (SYNTHETIC FIBER), semisynthetic fiber (semi-SYNTHETIC FIBER), and combinations thereof. In a preferred embodiment of the invention, the second substance is dextrin.
In a preferred embodiment of the invention, the second isoelectric point has a pH difference of 0.8 from the first isoelectric point.
According to the invention, the precipitate may be recovered by a solid-liquid separation (solid-liquid separation) treatment selected from the group consisting of: centrifugation, filtration, gravity sedimentation (GRAVITY SETTLING), and combinations thereof. In a preferred embodiment of the invention, the solid-liquid separation treatment is filtration.
As used herein, the terms "cell wall separation extract (cell wall isolation and extraction)" and "cell wall extract (cell wall extraction)" are used interchangeably and refer to the separation of a cell wall component (cell wall component) or a microbial metabolite (microbial metabolite) originally present on the cell wall component from the separated cell wall component.
The procedures and conditions for cell wall separation and extraction according to the present invention can be carried out using techniques well known and customary to those skilled in the art, such as the acid-alcohol method (acid alcohol method). In this respect, reference may be made, for example, to Pei-Jun Tian et al (2015), int.J. mol. Sci.,16 (8): 20033-20049.
According to the present invention, the composition may further comprise any skin friendly health care substance (HEALTHCARE MATERIAL), including, but not limited to: hyaluronic acid (sodium hyaluronate), vitamin C (vitamin C), glucosamine (glucosamine), chondroitin (chondroitin), type I collagen (type I collagen), type II collagen (type II collagen), type iii collagen (type III collagen), fish skin and scale collagen (FISH SKIN AND FISH SCALE collagen), non-denatured type II collagen (undenatured type II collagen), porcine collagen (porcine collagen), bovine collagen (bovine collagen), sialic acid (SIALIC ACID), anthocyanin (anthocyanin), polyphenol (polyphenol), flavonoid (flavonoid), eggshell membrane (egg shell membrane).
According to the present invention, the composition may be a pharmaceutical composition (pharmaceutical composition) or a food composition (food composition), and is not particularly limited in form, so that the composition can be used in the form of a preparation such as sterile powder (aseptic powder), lozenge (tablet), tablet (troche), buccal tablet (lozenge), pellet (capsule), capsule (caplet), dispersible powder (dispersible powder) or fine particles (granule), solution, suspension (emulsion), syrup (syrup), elixir (elixir), slurry (slurry), gel (jelly) or the like, which can be suitably prepared by publicly known methods.
According to the invention, when the composition is a pharmaceutical composition, the pharmaceutical composition may be manufactured into a dosage form (dosage form) suitable for parenteral administration (PARENTERAL ADMINISTRATION), oral administration (oral administration) or topical administration (topical administration) using techniques well known to those skilled in the art.
According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) 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 (solvents), buffers (buffers), emulsifiers (emulsifier), suspending agents (suspending agent), disintegrants (decomposer), disintegrants (DISINTEGRATING AGENT), dispersants (DISPERSING AGENT), binders (binding agents), excipients (excipients), stabilizers (stabilizing agent), chelating agents (CHELATING AGENT), diluents (diluent), gelling agents (GELLING AGENT), preservatives (PRESERVATIVE), fillers (filler), wetting agents (WETTING AGENT), lubricants (lubricant), absorption retarders (absorption DELAYING AGENT), liposomes (liposome), and the like. The choice and quantity of these reagents is within the skill and routine skill of those skilled in the art.
The pharmaceutical composition according to the present invention may also be formulated 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: emulsions (emulgation), gels (gel), ointments (ointment), creams (stream), patches (patch), wipes (liniment), powders (powder), aerosols (aerosol), sprays (spray), emulsions (condition), emulsions (serum), pastes (paste), foams (foam), drops (drop), suspensions (suspension), ointments (salve), and bandages (bandage).
According to the present invention, the external preparation is prepared by mixing the pharmaceutical composition of the present invention with a base (base) well known to those skilled in the art.
According to the invention, the substrate may comprise one or more additives (additives) selected from the group consisting of: water, alcohols, glycols, hydrocarbons (hydrocarbons) such as petroleum jelly (petroleum jelly) and white petrolatum (white petrolatum), waxes (wax) such as paraffin wax (Paraffin) and yellow wax (yellow wax), preservatives (PRESERVING AGENTS), antioxidants (antioxidants), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agents (GELLING AGENTS) such as941(/>941 Microcrystalline cellulose (microcrystalline cellulose), carboxymethyl cellulose (carboxymethylcellulose), active agent (ACTIVE AGENTS), humectant (humectants), odor absorbent (odor absorbers), perfume (fragrances), pH adjuster (pH adjusting agents), chelating agent (CHELATING AGENTS), emulsifier (emulsifiers), occlusive agent (occlusive agents), softener (emollients), thickener (thickeners), cosolvent (solubilizing agents), penetration enhancer (penetration enhancers), anti-irritant (anti-irritants), colorant (colorants), propellant (propellants), and the like. The choice and amounts of these additives are within the skill of the art of expertise and routine skill.
According to the present invention, when the composition is a food composition, the food composition may be added to an edible material (edible material) using a standard technique well known to those skilled in the art, for example, it may be directly added to the edible material, or it may be used to prepare an intermediate composition (INTERMEDIATE COMPOSITION) [ such as food additive (food additive) or premix (premix) ], which is then added to the edible material.
According to the present invention, the types of food products include, but are not limited to: fermented foods (FERMENTED FOOD), processed foods (processed foods), health foods (health foods), and dietary supplements (dietary supplements).
In accordance with the present invention, the food product may further comprise a food additive widely used in food manufacturing technology, including, but not limited to: starch (starch), dextrin (dextrin), lactose (lactose), corn flour (maize flour), rice flour (rice flour), tricalcium phosphate (tricalcium phosphate), silicon dioxide (silicon dioxide), magnesium stearate (magnesium stearate), calcium carbonate (calcium carbonate), glucose, sucrose (sucrose), fructose (fructose), sugar alcohol (sugar alcohol), oligosaccharides (oligosaccharides), sugar substitutes (sugar glucose), fruit juice flour (fruit juice powder), yeast flour (yeast powder), skim milk flour (nonfat dry milk), casein (casein), whey protein (whey protein), amino acids (amino acid), citric acid (CITRIC ACID), citrate (citrate), lactic acid (LACTIC ACID), lactate (lactate) and nucleotides (nucleotide).
The present invention also provides a method of promoting skin regeneration comprising administering to a subject in need thereof a metazoan extract as described above.
According to the present invention, the dose and the number of administrations of the metaextract may vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition and response of the individual to be treated. In general, the metaextract may be administered orally, parenterally or topically in a single dose or in divided doses.
The invention will be further illustrated with reference to the following examples, but it should be understood that the examples are for illustration only and should not be construed as limiting the practice of the invention.
< Example >
General experimental materials:
1. The probiotic strains used in the examples below were obtained from the microbiology laboratory of the food and applied bioscience system (DEPARTMENT OF FOOD SCIENCE AND Biotechnology at National Chung Hsing University) at the university of Zhongxing and have been integrated in table 1 below.
TABLE 1 respective probiotic strains
2. Source and culture of human foreskin fibroblast line (human foreskin fibroblast cell line) Hs 68:
The human foreskin fibroblast cell line Hs68 used in the following examples is a biological resource preservation and research center (Bioresource Collection AND RESEARCH CENTER, BCRC) available from Food Industry development institute (Food Industry RESEARCH AND Development Institute, FIRDI) of the national institute of health, taiwan.
Hs68 cells were cultured in 10cm dishes (PETRI DISH) containing Du Beike's Modified Eagle's Medium (DMEM) (Thermo FISHER SCIENTIFIC) [ 10% fetal bovine serum (fetal bovine serum, FBS) ] and cultured in an incubator set at 37℃under 5% CO 2. Thereafter, fresh medium was changed approximately every 2-3 days. When the cell density reached about 80-90% confluence (confluence), the subculture (subculture) was performed as follows: the medium was removed and the cells were washed with phosphate buffered saline (phosphate buffered saline, PBS) (pH 7.4), followed by the addition of trypsin-EDTA (trpsin-EDTA) to detach the cells from the bottom of the dish. Thereafter, fresh medium was added to neutralize trypsin activity and the medium was repeatedly aspirated with a quantitative pipette (pipette) to thoroughly break up the cells, and the resulting cell suspension was then dispensed into a new dish and cultured in an incubator set at 37 ℃ under 5% co 2.
General experimental method:
1. determination of the content of transforming growth factor-beta (transforming growth factor-beta, TGF-beta):
In the following examples, TGF-beta content was determined according to manufacturer's instructions using ELISA kits (BD Biosciences, cat. 559119) for enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA).
Example 1 preparation of metazoan extract (probiotics extract) of the invention
The experimental method comprises the following steps:
First, lactobacillus plantarum CB102, lactobacillus acidophilus JCM1132, lactobacillus casei JCM1134, bifidobacterium bifidum JCM1255, bifidobacterium lactis JCM10602 and Bifidobacterium longum CB108 in the above "general experimental materials" were inoculated into MRS medium (trade name BD Difco Lactobacilli MRS Broth, trade name DF 0881-17-5), respectively, and cultured at 37℃for 16 hours. Next, each culture was heat-inactivated using a high temperature short time sterilization method (high temperature short time, HTST) (73.+ -. 2 ℃ C., 15 seconds).
Next, each of the cultures after heat inactivation was centrifuged at 10,000rpm for 15 minutes at 25 ℃, followed by decanting the supernatant, and the obtained precipitate (pellets) was taken to be spray-dried, thereby obtaining dry powders of each strain.
The dry bacterial powders are respectively subjected to pretreatment according to the following steps: first, an appropriate amount of whey protein (whey protein) (trade name NZMP, trade name WPC 80) having an isoelectric point (isoelectric point) of pH 4.4 was dissolved in water to prepare a whey protein solution having a concentration of 10% (w/v, g/L), and then the pH of the solution was adjusted to 7.5 with food grade sodium carbonate. While stirring continuously, a suitable amount of dry bacterial powder was suspended in the solution to a final concentration of 5% (w/v, g/L), followed by slow addition of a suitable amount of dextrin (dextrin) (trade name ZHUCHENG DONGXIAO, cat. No. Maltodextrin DE-10) having an isoelectric point of pH 5.2 to a concentration of 6% (w/v, g/L), while slowly adding lactic acid to adjust the pH of the solution to about 4.8, at pH 4.8, whey protein and dextrin would reach charge neutralization (charge neutralization) to precipitate. Thereafter, stirring was continued until the precipitate no longer increased. Thereafter, filtration was performed using a filter paper having a pore size of 25 μm to separate and collect a precipitate, and then the resulting precipitate was taken to be spray-dried, thereby obtaining a pretreated bacterial powder.
The pretreated powders of the individual strains were each subjected to cell wall separation extraction (cell wall isolation and extraction), which was generally carried out with reference to the method described in Pei-Jun Tian et al (2015), int.j.mol.sci.,16 (8): 20033-20049, with minor modifications. Briefly, 50mg of the pretreated bacterial powder was weighed, followed by 1mL of 10% lactic acid (LACTIC ACID) and heated in a80℃water bath for 60 minutes. After that, centrifugation was performed at 10,000g for 15 minutes, then the supernatant was removed and 1mL of mixed solvent [ containing 4:10 (v/v) 0.5M citrate solution (ethanol, pH 4.6) followed by standing overnight. After that, centrifugation was performed at 10,000g for 20 minutes, and then the supernatant was removed and the formed precipitate was washed several times with 95% ethanol, followed by heating in a dry bath at 80 ℃ for about 40 minutes to completely remove ethanol, thereby obtaining the metaextract of the present invention.
In addition, the non-pretreated bacterial powders of the respective strains were also subjected to the same cell wall separation and extraction treatment to obtain metazoan extracts (hereinafter referred to as existing metazoan extracts).
EXAMPLE 2 analysis of the Components of the metazoan extract of the present invention
The existing metaextract obtained according to example 1 above and metaextract of the present invention were taken for the following extraction rate measurement (determination of extraction rate), protein content measurement (determination of protein content) and protein electrophoresis analysis (protein electrophoresis analysis).
The experimental method comprises the following steps:
A. Measurement of extraction yield:
The extraction yield was calculated by substituting the weight of the metagen extract obtained in example 1 above into the following formula (I):
Formula (I): a=b/50×100%
Wherein a=extraction yield (%)
B = weight of metazoan extract (mg)
B. determination of protein content:
The protein content was determined by dissolving the metaextract obtained in example 1 above in a phosphate buffer solution (containing 8g/L NaCl, 0.2g/L KCl, 1.44g/L Na 2HPO4 and 0.24g/L KH 2PO4, pH 6.2) and using Pierce TM BCA protein assay kit (Pierce TM BCA Protein Assay Kit) (trade name Thermo Scientific, product number 23225) according to the manufacturer's instructions.
C. Protein electrophoresis analysis:
The metachromatic extracts obtained in example 1 above were each taken 1g and dissolved in 20mL of water, followed by SDS-polyacrylamide gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) analysis using a Bio-Rad electrophoresis system and employing techniques known and customary to those skilled in the art.
Results:
A. Measurement of extraction yield:
table 2 below shows the extraction yield of the method with pretreatment according to the present invention and the extraction yield of the existing method without pretreatment.
TABLE 2 extraction yield of the process according to the invention and the prior art
As can be seen from Table 2, compared with the prior art, the method according to the present invention can obtain a higher extraction rate of the metazoan extract by separating and extracting the cell wall from the pretreated bacterial powder.
B. determination of protein content:
Table 3 below shows the protein content of the metaextract obtained by the process according to the invention and by the prior art processes.
TABLE 3 protein content of the metaextract obtained by the method according to the invention and the prior art methods
It can be seen from Table 3 that the protein content in the metaextract of the present invention is significantly higher than that in the existing metaextract.
C. Protein electrophoresis analysis:
FIG. 1 is a view of an electrophoresis film showing the results of protein electrophoresis analysis of the metazoan extract of the present invention and the existing metazoan extract. As can be seen from FIG. 1, the protein band (protein band) of the metaextract of the present invention is significantly more than that of the existing metaextract. The applicant believes that the metaextract obtained by the method according to the present invention contains a large amount of cell wall components such as peptidoglycan (peptidoglycan), lipoteichoic acid (lipoteichoic acid), teichoic acid (teichoic acid), glycoprotein (glycoprotein), proteoglycan (proteoglycan), and the like.
From a combination of the above experimental results, it can be found that the method of the present invention is capable of efficiently extracting the metazoan extract from the cell wall of the probiotics, and the metazoan extract thus obtained contains more cell wall proteins (cell wall protein).
Example 3 evaluation of the utility of the metaextract of the invention in promoting skin regeneration (skin regeneration)
In this example, the Lactobacillus plantarum and Bifidobacterium longum metazoan extracts of the present invention obtained according to example 1 above were taken to treat Hs68 cells, respectively, with existing Lactobacillus plantarum and Bifidobacterium longum metazoan extracts, and the effect of the inventive metazoan extracts in promoting skin regeneration was evaluated by measuring the TGF-beta secretion amount and the cell proliferation amount of the treated Hs68 cells. In addition, the applicant further evaluated the effect of the use of the metaextract of the present invention in combination with commercially available healthcare substances known to be applicable to skin care on this effect.
Experimental materials:
1. the healthcare materials used in this example were purchased from the company chuanbai co. (Chambio co., ltd.). The kinds of the health care substances and trade names thereof have been integrated in the following table 4.
TABLE 4 kinds of respective health substances and trade names thereof
The experimental method comprises the following steps:
A. effect of metagen extract on TGF- β secretion of Hs68 cells:
first, hs68 cells subcultured according to item 2 of the above "general test material" were divided into 25 groups including 1 control group, 12 comparison test groups (i.e., comparison test groups L-1 to L-6 and comparison test groups B-1 to B-6) and 12 test groups (i.e., test groups L-1 to L-6 and test groups B-1 to B-6). Hs68 cells of each group were cultured in each well of a 96-well plate containing 200. Mu.L of DMEM medium at a rate of 1X 10 4 cells/well, and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours. Next, the cell cultures of each group were replaced with fresh medium, respectively, and the post-metazoan extracts of lactobacillus plantarum and bifidobacterium longum according to the present invention obtained in example 1 above and the post-metazoan extracts of the existing lactobacillus plantarum and bifidobacterium longum were added to each group, respectively, according to the following table 5.
TABLE 5 final concentration of metaextract possessed by each group of cultures
After each group of cultures was cultured at 37℃for 24 hours, the TGF-beta content was determined according to the method described in item 1 of "measurement of TGF-beta content" of general Experimental method "above.
B. Effect of the combined use of metazoan extracts with other health care substances on TGF- β secretion of Hs68 cells:
First, hs68 cells subcultured according to item 2 of the above "general test material" were divided into 28 groups including 14 comparison test groups (i.e., 1X comparison test groups 1 to 7 and 2X comparison test groups 1 to 7) and 14 test groups (i.e., test groups 1-L to 7-L and test groups 1-B to 7-B). Hs68 cells of each group were cultured in each well of a 96-well plate containing 200. Mu.L of DMEM medium at a rate of 1X 10 4 cells/well, and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours. Next, the cell cultures of each group were replaced with fresh medium, respectively, and 7 kinds of health substances in item 1 of the above "experimental materials" and the metazoan extracts of lactobacillus plantarum and bifidobacterium longum of the present invention obtained according to example 1 above were added to each group, respectively, according to the following table 6.
TABLE 6 final concentration of metazoan extract or health care substances possessed by each group of cultures
After each group of cultures was cultured at 37℃for 24 hours, the TGF-beta content was determined according to the method described in item 1 of "measurement of TGF-beta content" of general Experimental method "above.
C. Effect of metazoan extract on cell proliferation of Hs68 cells (proliferation):
first, hs68 cells subcultured according to item 2 of the above "general test material" were divided into 3 groups including 1 control group and 2 test groups (i.e., test group L and test group B). Hs68 cells of each group were cultured in each well of a 96-well plate containing 200. Mu.L of DMEM medium at a rate of 1X 10 4 cells/well, and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours. Next, the cell cultures of each group were replaced with fresh medium, respectively, and the metagen extracts of lactobacillus plantarum and bifidobacterium longum of the present invention obtained according to the above example 1 were added to each group, respectively, according to the following table 7.
TABLE 7 final concentration of metaextract possessed by each group of cultures
After each group of cultures was incubated at 37 ℃ for 24 hours, the number of cells of Hs68 cells in each well was then counted.
D. effects of the combined use of metazoan extract with other health care substances on cell proliferation of Hs68 cells:
First, hs68 cells subcultured according to item 2 of the above "general test material" were divided into 28 groups including 14 comparison test groups (i.e., 1X comparison test groups 1 to 7 and 2X comparison test groups 1 to 7) and 14 test groups (i.e., test groups 1-L to 7-L and test groups 1-B to 7-B). Hs68 cells of each group were cultured in each well of a 96-well plate containing 200. Mu.L of DMEM medium at a rate of 1X 10 4 cells/well, and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours. Next, the cell cultures of each group were replaced with fresh medium, respectively, and 7 kinds of health substances in item 1 of the above "experimental materials" and the metazoan extracts of lactobacillus plantarum and bifidobacterium longum of the present invention obtained according to the above example 1 were added to each group, respectively, according to the above shown in table 6.
After each group of cultures was incubated at 37 ℃ for 24 hours, the number of cells of Hs68 cells in each well was counted and then the fold relative to that of the corresponding 1X comparative experimental group was calculated.
Results:
A. effect of metagen extract on TGF- β secretion of Hs68 cells:
table 8 below shows the TGF- β content measured for each group of cultures.
TABLE 8 TGF-beta content of cultures of each group
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From Table 8, it is seen that the effect of the Lactobacillus plantarum metazoatum extract of the present invention on promoting TGF-beta secretion of Hs68 cells is significantly superior to that of the existing Lactobacillus plantarum metazoatum extract. In addition, the metaextract of bifidobacterium longum of the present invention also exhibits similar excellent effects. The results of this experiment showed that: the metagen extract obtained by the method can effectively promote the Hs68 cells to secrete TGF-beta.
B. Effect of the combined use of metazoan extracts with other health care substances on TGF- β secretion of Hs68 cells:
Table 9 below shows the TGF- β content of each group of cultures.
TABLE 9 TGF-beta content of cultures of each group
As can be seen from Table 9, the TGF-beta content of each experimental group was significantly higher than that of the corresponding 1X comparison experimental group and 2X comparison experimental group. In particular, the amplitude of the increase of each experimental group compared to the corresponding 1X comparison experimental group is significantly higher than that of the 2X comparison experimental group compared to the corresponding 1X comparison experimental group, which means that: the use of the metaextract of the present invention to replace an equivalent amount of a healthcare substance can greatly enhance the utility of the healthcare substance in promoting the secretion of TGF-beta by Hs68 cells.
C. effect of metazoan extract on cell proliferation of Hs68 cells:
table 10 below shows the number of cells in each set of cultures.
TABLE 10 cell counts in each group of cultures
| Group of | Cell number (number) |
| Control group | 1.1×104 |
| Experiment group L | 1.7×104 |
| Experiment group B | 2.1×104 |
As can be seen from Table 10, the cell numbers of both experimental groups L and B were significantly higher than that of the control group. The results of this experiment showed that: the metagen extract of the invention can promote the cell proliferation of Hs68 cells.
D. effects of the combined use of metazoan extract with other health care substances on cell proliferation of Hs68 cells:
table 11 below shows the cell proliferation status in each group of cultures (fold relative to the corresponding 1X comparison experimental group).
TABLE 11 cell proliferation in cultures of each group
| Group of | Multiple x | Group of | Multiple x |
| Comparative experiment group 1X 1 | 1.0 | Comparative experiment group 1X 5 | 1.0 |
| 2X comparative experiment group 1 | 1.1 | 2X comparative experiment group 5 | 1.1 |
| Experimental group 1-L | 1.3 | Experimental group 5-L | 1.2 |
| Experimental group 1-B | 1.3 | Experimental group 5-B | 1.2 |
| Comparative experiment group 1X 2 | 1.0 | Comparative experiment group 6 1X | 1.0 |
| Comparative experiment group 2X 2 | 1.1 | 2X comparative experiment group 6 | 1.1 |
| Experimental group 2-L | 1.5 | Experimental group 6-L | 1.4 |
| Experimental group 2-B | 1.5 | Experimental group 6-B | 1.4 |
| Comparative experiment group 3 1X | 1.0 | Comparative experiment group 7 1X | 1.0 |
| 2X comparative experiment group 3 | 1.1 | 2X comparative experiment group 7 | 1.1 |
| Experimental group 3-L | 1.2 | Experimental group 7-L | 1.4 |
| Experimental group 3-B | 1.2 | Experimental group 7-B | 1.4 |
| Comparative experiment group 1X 4 | 1.0 | ||
| 2X comparative experiment group 4 | 1.1 | ||
| Experimental group 4-L | 1.3 | ||
| Experimental group 4-B | 1.3 |
* : The cell numbers of each group were compared against the corresponding 1X fold of those of the experimental group.
As can be seen from table 11, the cell proliferation status of each experimental group was significantly better than that of the corresponding 1X comparison experimental group and 2X comparison experimental group. In particular, the proliferation amplitude of each experimental group compared to the corresponding 1X comparison experimental group is significantly higher than that of the 2X comparison experimental group compared to the corresponding 1X comparison experimental group, which means that: the use of the metagen extract of the present invention to replace the equivalent amount of the health-care substance can greatly enhance the effect of the health-care substance in promoting the cell proliferation of the Hs68 cells.
According to the experimental results, the method can effectively extract the metazoan from the cell wall of the probiotics, and the prepared metazoan extract contains more cell components containing proteins and has excellent effects on promoting proliferation of fibroblasts and secreting anti-inflammatory hormone. Thus, the applicant believes that: the metagen extract obtained by the method according to the present invention can be applied to promote skin regeneration and has high potential to develop into a commodity for skin repair, promotion of skin wound healing and aging resistance.
All patents and documents cited in this specification are incorporated by reference herein in their entirety. In the event of conflict, the present specification, including definitions, will control.
Although the invention has been described with reference to the specific embodiments described above, it will be apparent that many modifications and variations may be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only as indicated by the claims appended hereto.
Claims (4)
1. Use of metazoan extract for the preparation of a composition for promoting skin regeneration, characterized in that: the metazoan extract is prepared by a process comprising the steps of:
Providing a first substance and a second substance, wherein the first substance has a first isoelectric point falling within a range from pH 1 to pH 6, the second substance has a second isoelectric point falling within a range from pH4 to pH 8 and higher than the first isoelectric point, and the second isoelectric point and the first isoelectric point have a pH difference value falling between 0.5 and 3;
Mixing the first substance and a probiotic in water having a pH above the second isoelectric point to obtain a mixture, wherein the probiotic is selected from the group consisting of: lactobacillus species, bifidobacterium species, and combinations thereof;
adding the second substance to the mixture, and then adjusting the pH of the mixture so that the pH of the mixture falls between the first isoelectric point and the second isoelectric point, thereby forming a precipitate; and
The precipitate is subjected to a cell wall separation and extraction treatment, thereby obtaining the metazoan extract.
2. Use of the metazoan extract supply according to claim 1 for the preparation of a composition for promoting skin regeneration, characterized in that: the first substance is selected from the group consisting of: skim milk powder, casein, whey protein, soy protein, pea protein, egg protein, rice protein, hydrolyzed protein, corn protein, wheat protein, barley protein, branched chain amino acids, gelatin, collagen, amino acids, chitosan oligosaccharides, and combinations thereof.
3. Use of the metazoan extract supply according to claim 1 for the preparation of a composition for promoting skin regeneration, characterized in that: the second substance is selected from the group consisting of: sodium alginate, carrageenan, pectin, acacia, xanthan gum, locust bean gum, starch, trehalose, dextrin, syrup, guan Huadou gum, konjak fine powder, plant fiber, synthetic fiber, semisynthetic fiber, and combinations thereof.
4. Use of the metazoan extract supply according to claim 1 for the preparation of a composition for promoting skin regeneration, characterized in that: the composition further comprises a health care substance selected from the group consisting of: hyaluronic acid, sodium hyaluronate, vitamin C, glucosamine, chondroitin, type one collagen, type two collagen, type three collagen, fish skin and scale collagen, non-denatured type two collagen, porcine collagen, bovine collagen, sialic acid, anthocyanin, polyphenol, flavonoid, eggshell membrane, and combinations thereof.
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| CN115569229B (en) * | 2022-08-30 | 2023-12-22 | 中南大学湘雅三医院 | Skin soft tissue protective material carrying multiple metaelements and preparation method thereof |
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| CN1997748A (en) * | 2004-04-20 | 2007-07-11 | 芝加哥大学 | Probiotic compounds from lactobacillus GG and uses therefor |
| CN101380289A (en) * | 2007-09-04 | 2009-03-11 | 欧莱雅 | Cosmetic use of a Bifidobacterium species lysate for treating dry skin |
| CN101724610A (en) * | 2009-12-29 | 2010-06-09 | 史知行 | Method for extracting SOD enzymes from animal blood (pig blood) by isoelectric equal charge method |
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| CN1997748A (en) * | 2004-04-20 | 2007-07-11 | 芝加哥大学 | Probiotic compounds from lactobacillus GG and uses therefor |
| CN101380289A (en) * | 2007-09-04 | 2009-03-11 | 欧莱雅 | Cosmetic use of a Bifidobacterium species lysate for treating dry skin |
| CN101724610A (en) * | 2009-12-29 | 2010-06-09 | 史知行 | Method for extracting SOD enzymes from animal blood (pig blood) by isoelectric equal charge method |
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| Lactobacillus plantarum protection by entrapment in whey protein isolate: k-carrageenan complex coacervates;L. Hernández-Rodríguez等;Food Hydrocolloids;摘要，第182页左栏第1段，2.4 复合物的形成，2.5 复合物产量，蛋白以及含水量，3.4复合物的浊度，表1 * |
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