CN114072128A - Method for preparing skin probiotic block composition with skin moisturizing and skin regenerating functions - Google Patents
Method for preparing skin probiotic block composition with skin moisturizing and skin regenerating functions Download PDFInfo
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Abstract
In the present invention, a spherical skin probiotic block composition in which inactive probiotics are captured using hyaluronic acid may be formed by performing a dropping reaction of hyaluronic acid of different pH and chitosan inactive probiotics produced by fermenting lactic acid bacteria in a medium of known composition including hyaluronic acid or chitosan. When the skin probiotic block is applied to the skin, the expression of aquaporin 3(AQP-3) and the expression of hyaluronan synthase 2(HAS-2) of skin cells can be increased, thereby exhibiting a function of improving moisturizing ability and a skin regeneration function of promoting the growth of skin cells, and particularly, exhibiting a greater function in dermal cells. Since the skin probiotics having the block formed as described above can exhibit healthy skin moisturizing and skin regenerating effects, they can be effectively applied to bio-cosmetics such as cosmetic compositions, patches for skin, and masks, which use them as functional ingredients.
Description
Technical Field
The present invention relates to a skin probiotic block composition which maximizes the skin moisturizing and skin regenerating effects of dermal cells by capturing a lactic acid bacteria fermented product having skin moisturizing and skin regenerating functions using hyaluronic acid.
Specifically, the present invention relates to a method for manufacturing a skin probiotic bar composition in which skin probiotics, which are functional lactic acid bacteria for skin moisturizing and skin regeneration, are fermented using a medium of known ingredients including hyaluronic acid or chitosan, and then heated and filtered, thereby inducing the synthesis of spherical bar particles through a mixing reaction of different pH of the fermented products and thereby capturing the lactic acid bacteria fermented products using hyaluronic acid. The composition can capture the lactobacillus leavening with skin moisturizing and skin regeneration functions by utilizing hyaluronic acid with high skin affinity, thereby effectively transferring the lactobacillus leavening to skin dermal cells and maximizing the effects of skin moisturizing and skin regeneration.
Background
The skin has a three-layer structure including a stratum corneum, an epidermis and a dermis. The outermost stratum corneum in the skin structure is composed of very thin layers of about 0.5mm in thickness. The stratum corneum, which is composed of 25 to 30 apoptotic skin cells, can function to protect human skin, and the skin cutin that has performed the function will fall off by itself and become cutin again after the skin neogenetic cells grow into new skin, and by repeatedly performing the above-described configuration, human skin can be configured and protected. The epidermis layer is located just below the stratum corneum layer of the skin and may serve functions such as a barrier to permeation, innate immunity, uv protection, wound healing, and vitamin D synthesis. The dermis layer is a layer for supplying nutrients to the epidermis and responsible for skin regeneration, and may function to inhibit pathogen invasion, wound repair, and skin structure and elasticity maintenance.
Probiotic bacteria (Probiotics) are in latin meaning "for life" (for live), mainly live cells, and also microorganisms that can help the health of the host when ingested in appropriate amounts (WHO/FAO, 2002). Microorganisms such as Lactobacillus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), and Streptococcus (Streptococcus sp.) are the most representative of probiotic bacteria, and can be isolated from the intestinal tract. The lactic acid bacteria as described above have high affinity with intestinal mucosa in the intestinal tract, and thus can enhance immunity of each organ of the human body by interacting with immune cells existing in the intestinal mucosa after attachment, thereby preventing diseases. Recently, by confirming the function of probiotics, there have been studies indicating that probiotics not only are beneficial to intestinal health but also have skin moisturizing effects. Therefore, it can be applied to the field of cosmetics by fermenting Streptococcus, Lactobacillus, and lactococcus, and then filtering or extracting.
Since the beginning of 2014, a revision has been made in the academic field for a portion of the probiotic concept proposed in 2002. That is, unlike the conceptual definition of probiotics by WHO/FAO in 2002, a completely new concept of inactive probiotics (Taverniti & Guglielmetti, 2011; ant a & Knorr, 2009) appeared, accompanied by systematization of the results of the study that not only viable bacteria would exhibit probiotic effects.
Although studies on the formulation of the non-active probiotics as described above are actively being conducted, when a bio-cosmetic is manufactured using lactic acid bacteria as a raw material based on the prior art, if lactic acid bacteria growing under anaerobic conditions are directly used, there occurs a problem that the lactic acid bacteria easily undergo apoptosis due to exposure to the air on the surface of the skin and are detached from the skin together with the skin glial cells. In addition, recently, many functional ingredients have excellent efficacy, but the expected efficacy cannot be achieved because the functional ingredients are hardly absorbed into the skin. Therefore, research activities for promoting skin permeation of functional ingredients are actively being conducted. As described above, although the development of lactic acid bacteria fermented products is very important, formulation technology for maximizing the efficacy of raw materials is also indispensable.
Hyaluronate and hyaluronic acid are negatively charged glycosaminoglycan polysaccharides, and are present in various parts of the body, particularly in large amounts on the skin. Hyaluronic acid is present as a part of a cell matrix including collagen and other proteins surrounding cells such as keratinocytes of the epidermis and fibroblasts of the dermis, and its content in both the dermis and the epidermis is very high. Hyaluronic acid plays a very important role in the skin, and can improve the moisturizing ability of the skin, maintain the elasticity of the skin, reduce the damage to the underlying part of the skin when the skin is damaged, and play a role similar to lubricating oil in ensuring smooth movement of collagen, which is a main constituent of the skin, between cells. In particular, the natural moisturizer can function as a top moisturizing factor on the skin surface because it has excellent hydrophilicity. However, it has been known that hyaluronic acid of 400kDa or more cannot penetrate the skin barrier, and even if hyaluronic acid can penetrate the skin barrier, the penetration ratio is very small, and thus its efficacy cannot be exhibited in the dermal layer.
The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a skin probiotic block composition, which is a lactic acid bacteria fermented product having skin moisturizing and skin regenerating functions of dermal cells, by capturing a lactic acid bacteria fermented product with hyaluronic acid to produce a spherical block, so that it is possible to transplant a different type of lactic acid bacteria having skin moisturizing and skin regenerating functions, which are physiologically and ecologically different from a skin resident flora, without rejection.
Documents of the prior art
Patent document
(patent document 1) Korean registered patent No. 10-1967809 (name of invention: cosmetic composition for skin wrinkle improvement or skin moisture-keeping containing nano kimchi lactic acid bacteria, Applicant: 1 person out of BIOGENICS KOREA, registration date: 2019, 04.04.04.4.)
(patent document 2) Korean registered patent No. 10-1764412 (title of the invention: functional hydrated hyaluronic acid and method for producing coated lactic acid bacteria having excellent intestinal mucosal adhesive ability and selective antagonistic action using the hydrated hyaluronic acid, applicant: ILDONG BIOSCIENCE, registration date: 2017, month 07, day 27)
(patent document 3) Korean registered patent No. 10-1280232 (name of the invention: method for producing a quadruple-coated lactic acid bacterium and a quadruple-coated lactic acid bacterium produced by the method, Applicant: ILDONG pharmaceutical Co., Ltd., registration date: 2013, 06 months and 25 days)
(patent document 4) Korean registered patent No. 10-1742542 (title of invention: composition for moisturizing skin or improving wrinkles, containing killed bacteria of intermittent sterilized lactic acid bacteria as active ingredient, applicant: Korean institute of medicine, registration date: 26/05/2017)
(patent document 5) Korean registered patent No. 10-1106077 (name of invention: humectant, applicant: House Wellness Foods Corporation, registration date: 09 month 01 2012)
(patent document 6) Korea publication No. 10-2018-0108209 (name of invention, composition for treating skin injury comprising Lactobacillus rhamnosus JC1225 strain, Applicant: Myeon-Yeog Biolab Ex 1, published: 2018, 10 months, 04 days)
(patent document 7) Japanese laid-open patent No. 2006-199608 (name of invention: skin external preparation, Applicant: ACE BIO PRODUCT KK, registration date: 2005, 01 month 19)
Non-patent document
(non-patent document 1) Hyaluronic Acid Coated Nanoparticles Reduced the immunological sensitivity of the Formed Protein Corona, Abdulaziz Almalik, SCIENTIFIC REPORTS,7:10542, DOI:10.1038/s41598-017-10836-7(www.nature.com)
(non-patent document 2) The immunological properties of microorganisms and The biological properties of microorganisms and The clinical laboratory 2011, Genes Nutr.6:261-
(non-patent document 3) compatibility of inactivation pathway of bacteria genus ATCC 53103by flow cytometry analysis, E.Ananta, D.Knorr, sciences direct, Volume 26, Issue 5, August 2009, Pages 542-
Disclosure of Invention
The purpose of the present invention is to provide a skin probiotic block composition that maximizes the skin moisturizing and skin regenerating effects of dermal cells by capturing a lactic acid bacteria fermentation product having skin moisturizing and skin regenerating functions with hyaluronic acid. Specifically, the present invention relates to a method for producing a skin probiotic cake composition in which skin functional lactic acid bacteria fermented product is captured by hyaluronic acid by heating each fermented product produced by fermenting skin probiotics, which are skin moisturizing and skin regenerating functional lactic acid bacteria, with a medium of known ingredients including hyaluronic acid or chitosan, filtering the heated product, and inducing the synthesis of spherical masses through a mixed reaction of the fermented products at different pH. The composition can capture the lactobacillus leavening with skin moisturizing and skin regeneration functions by utilizing hyaluronic acid with high skin affinity, thereby effectively transferring the lactobacillus leavening to skin dermal cells and maximizing the effects of skin moisturizing and skin regeneration. The skin probiotic mass composition may be provided as a raw material in the form of, for example, various cosmetic compositions, patches for skin, masks, and the like.
In order to solve the above problems, the method for producing a skin microbial cake composition of the present invention includes the following steps.
Preferably, the method comprises the following steps: a step of preparing a first medium of known composition containing hyaluronic acid and glucose and a second medium of known composition containing chitosan and glucose;
(second step) a step of preparing a fermentation product of hyaluronic acid bacteria by inoculating lactic acid bacteria to the first known component medium and fermenting the same, and preparing a fermentation product of chitosan lactic acid bacteria by inoculating lactic acid bacteria to the second known component medium and fermenting the same;
(third step) a step of producing inactive probiotics by heating the fermented product of hyaluronic acid bacteria and the fermented product of chitosan lactic acid bacteria, respectively; and the number of the first and second groups,
(fourth step) a step of manufacturing a skin probiotic bar composition by drop-mixing the hyaluronic acid non-viable probiotic and the chitosan non-viable probiotic.
The invention is characterized in that: in the first step, the first known component medium is an aqueous solution containing 0.1 to 1.0 (w/v)% of medium molecular weight hyaluronic acid (1,500 to 2,000kDa) and 0.1 to 2.0 (w/v)% of glucose, and the second known component medium is an aqueous solution containing 0.1 to 1.0 (w/v)% of chitosan and 0.1 to 2.0 (w/v)% of glucose. The medium of each known composition is preferably used after being sterilized by a usual medium production method. Specifically, sterilization is preferably performed at 110 to 125 ℃ and 1.1 to 1.3 atmospheres for 10 to 60 minutes. In the case of using the medium of known composition as described above, the fermentation odor inevitably generated when culturing lactic acid bacteria can be significantly reduced.
In the case where the molecular weight of hyaluronic acid in the first known composition medium is less than 1,500kDa or exceeds 2,000kDa, problems of poor culture of lactic acid bacteria or poor formation of skin probiotic clumps may result. Furthermore, in case the molecular weight of chitosan in the second known composition medium exceeds 100kDa, it may cause a problem of poor formation of skin probiotic lumps. In addition, when the content of hyaluronic acid and glucose in the first known component culture medium is beyond the concentration range or the content of chitosan and glucose in the second known component culture medium is beyond the concentration range, the problems of poor culture of lactic acid bacteria and poor formation of skin probiotic bacteria block may be caused.
In the second step, when the first known component medium is inoculated with lactic acid bacteria and fermented, a fermented product of hyaluronic acid bacteria is produced at a pH of 3.0 to 4.5 (preferably pH 4.0), and when the second known component medium is inoculated with lactic acid bacteria and fermented, a fermented product of chitosan lactic acid bacteria is produced at a pH of 5.0 to 6.5 (preferably pH 5.5).
The skin-functional lactic acid bacteria in the second step may be selected from the group consisting of Lactobacillus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), Streptococcus (Streptococcus sp.), Lactococcus (Lactococcus sp.), Enterococcus (Enterococcus sp.), Pediococcus (Pediococcus sp.), Leuconostoc (Leuconostoc sp.), and Weissella (Weissella sp.), but is not limited thereto.
The Lactobacillus includes, for example, Lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus curvatus (Lactobacillus cubatus), Lactobacillus sake (Lactobacillus sakei), Lactobacillus plantarum (Lactobacillus plantarum), and Lactobacillus gasseri (Lactobacillus gasseri), but is not limited thereto.
The lactobacillus of the genus Bifidobacterium includes, for example, Bifidobacterium longum (Bifidobacterium longum) and Bifidobacterium bifidum (Bifidobacterium bifidum), but is not limited thereto.
The Streptococcus genus lactic acid bacteria include, for example, Streptococcus thermophilus (Streptococcus thermophilus) and Streptococcus visceral (Streptococcus entericus), but are not limited thereto.
The lactic acid bacteria belonging to the genus lactococcus include, for example, lactococcus lactis (lactococcus lactis) and lactococcus garvieae (lactococcus garvieae), but are not limited thereto.
The Enterococcus genus lactic acid bacteria include, for example, Enterococcus faecium (Enterococcus faecium) and Enterococcus faecalis (Enterococcus faecalis), but are not limited thereto.
The genus Pediococcus includes, for example, Pediococcus pentosaceus (Pediococcus acidilactici), Pediococcus damascenus (Pediococcus damnosus), and Pediococcus cellularis (Pediococcus cellularia), but is not limited thereto.
The Leuconostoc lactic acid bacteria include, for example, Leuconostoc lactis (Leuconostoc lactis), but are not limited thereto.
The Weissella lactic acid bacteria include, but are not limited to, Weissella fusca (Weissella convusa) and Weissella mesenteroides (Weissella paramenteroides).
In the third step, the heating treatment of the lactic acid bacteria fermented product for producing each of the inactive probiotics is preferably performed at 90 to 100 ℃ for 5 to 15 minutes (more preferably at 100 ℃ for 10 minutes). At this time, in the case where the heat treatment conditions are less than 90 ℃ or more than 100 ℃ and the heating time is less than 5 minutes or more than 15 minutes, there may be caused a problem that the formation of skin probiotic lumps in the subsequent process is not good.
In the fourth step, when mixing the hyaluronic acid non-viable probiotic and the chitosan non-viable probiotic, it may be performed by dropping the chitosan non-viable probiotic while stirring the hyaluronic acid non-viable probiotic. Alternatively, it may be performed by dropping the hyaluronic acid-inactive probiotic while stirring the chitosan-inactive probiotic. In this case, if the non-viable probiotics are directly mixed without using the dropping method, there is a problem that the formation yield of the skin probiotic cake is significantly reduced.
In this case, when the hyaluronic acid-inactive probiotic and the chitosan-inactive probiotic are mixed in the process of producing the skin probiotic mass, the hyaluronic acid-inactive probiotic having a pH of 4.0 is preferably added dropwise while stirring the chitosan-inactive probiotic having a pH of 5.5.
The present invention may also provide a cosmetic composition containing the skin probiotic pellet manufactured by the method as described above. The cosmetic composition is particularly excellent in skin moisturizing and skin regenerating functions, and more preferably can improve the skin moisturizing and skin regenerating functions of dermal cells of the skin. In the cosmetic composition, all ingredients generally used may be contained. For example, it may contain common auxiliary components such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and perfumes. More specifically, in the case where the cosmetic composition of the present invention is in the form of a paste, cream or gel, as the carrier ingredient, there may be used, for example, animal fibers, plant fibers, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc or zinc oxide. In the case where the cosmetic composition of the present invention is in the form of powder or spray, for example, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and particularly in the case of spray, a propellant such as chlorofluorocarbon, propane-butane or dimethyl ether may be further contained. In the case where the cosmetic composition of the present invention is in the form of a solution or emulsion, as a carrier component, a solvent, a solvating agent or an emulsifying agent may be used, and for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butyl glycol oil, glycerin fatty ester, polyethylene glycol or fatty acid ester of sorbitan may be used.
When the cosmetic composition of the present invention is in the form of a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester or polyoxyethylene sorbitol ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth can be used as a carrier component. In the case where the formulation of the cosmetic composition of the present invention is a surfactant-containing detergent, examples of the carrier component include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazoline derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative, and ethoxylated glycerin fatty acid ester. The cosmetic composition of the present invention may further comprise an excipient such as a fluorescent substance, a fungicide, a hydration-inducing substance, a humectant, an aromatic substance, an aromatic carrier, a protein, a solubilizer, a sugar derivative, a solar barrier, a vitamin, a plant extract, and the like. The addition amount of the ingredient may be selected within a range that does not impair the inherent effects of the cosmetic composition, depending on the formulation form or the purpose of use. For example, the amount of the component added may be 0.1 to 10% by weight, preferably 0.1 to 6% by weight, based on the total weight of the composition, but is not limited thereto.
Further, the type of the formulation of the cosmetic composition is not particularly limited, and examples thereof include skin care cosmetics such as lotions, milky lotions, gels, creams, essences, masks, ampoules, skin creams, lotions, soaps, body scrubs, soaps, and essential oils, and makeup cosmetics such as lipsticks, foundations, and hair care cosmetics, and the formulation thereof is not particularly limited.
The present invention can also provide a skin patch, a skin external preparation, a mask, etc. containing the skin probiotic mass composition. The skin patch or the external preparation for skin can be prepared by mixing various excipients contained in a general patch composition or external preparation composition.
In the present invention, a spherical skin probiotic block composition in which inactive probiotics are captured using hyaluronic acid can be formed by heating hyaluronic acid or a chitosan lactic acid bacteria fermented product of different pH produced by fermenting skin functional lactic acid bacteria in a medium of known composition containing hyaluronic acid or chitosan to produce inactive probiotics and then reacting the same. When the skin probiotic block composition is applied to the skin, the expression of aquaporin 3(AQP-3) in epidermal cells, the expression of hyaluronan synthase 2(HAS-2) in dermal cells and the wound healing (wound healing) effect of inactive probiotics can be maximized, thereby improving skin moisturizing and skin regeneration functions. The skin probiotic mass composition formed in such a manner as described above exhibits beneficial effects on skin moisturization and skin regeneration, and thus can be effectively used as a top-end biological raw material such as a functional cosmetic composition, a functional skin patch, and a functional mask.
Drawings
Fig. 1 is a scanning microscope photograph taken after centrifugal separation recovery and freeze-drying of a skin probiotic bar composition by dropping hyaluronic acid inactive probiotics of pH 4.0 while stirring chitosan inactive probiotics of pH 5.5 of examples 1-3-2.
Fig. 2 is a graph illustrating the skin moisturizing effect of hyaluronic acid non-live probiotic of pH 4.0 of example 1-1 and chitosan non-live probiotic of pH 5.5 of example 1-2 and the skin probiotic cake composition of example 1-3-2 on epidermal cells.
Fig. 3 is a graph illustrating skin moisturizing effects of dermal cells of hyaluronic acid non-viable probiotic bacteria of pH 4.0 of example 1-1 and chitosan non-viable probiotic bacteria of pH 5.5 of example 1-2 and skin probiotic cake composition of example 1-3-2.
Fig. 4 is a graph illustrating the skin moisturizing effect of the skin probiotic pellet compositions of examples 1-3-2 and of example 2 on dermal cells.
FIGS. 5 to 12 are photographs showing the effect of wound healing (wound healing) associated with skin regeneration of various composition compositions (including comparative conditions) such as hyaluronic acid-inactive probiotic of pH 4.0 of example 1-1, chitosan-inactive probiotic of pH 5.5 of example 1-2, and skin probiotic of example 1-3-2 (cell density confirmation).
Detailed Description
Next, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein, and may be embodied in other forms. Rather, the described embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.
< example 1. determination of optimal manufacturing conditions for a skin probiotic Bar composition >
In order to produce a skin probiotic block composition, a culture having a certain number of lactic acid bacteria and a pH of 3.0 to 4.5 is produced using a medium having known components of hyaluronic acid and glucose, and as another method, a culture having a certain number of lactic acid bacteria and a pH of 5.0 to 6.5 is produced using a medium having known components of chitosan and glucose, and then the cultures having different pH values are mixed, and the principle that hyaluronic acid forms a sphere by ionic bonding is used.
That is, carboxyl groups (pka: 3.19) of hyaluronic acid in the ferment cultured in the medium of known composition of hyaluronic acid and glucose are negatively charged, and chitosan (pka: 6.5) in the ferment cultured in the medium of known composition of chitosan and glucose is positively charged, thereby inducing ionic bonding of the two substances and capturing the non-active probiotic culture and thereby producing a spherical skin probiotic pellet.
Example 1-1 preparation of hyaluronic acid Inactive probiotic with pH3.0 to 4.5
The culture was carried out at 37 ℃ for 20 hours after inoculating Lactobacillus (L.paracasei SKB1192) lactic acid bacteria into MRS medium, and the number of produced bacteria was 109～1010CFU/ml。
Lactobacillus (l.paracasei SKB1192) cultured in MRS medium was recovered by centrifugation, and washed 2 times with sterilized water.
After preparing a first known component medium by dissolving 0.5% (w/v) of medium molecular weight hyaluronic acid (1,500-2,000 kDa) and 2.0% (w/v) of glucose in tertiary distilled water and sterilizing (121 ℃, 15 minutes), the washed lactobacillus (L.paracasei SKB1192) lactic acid bacteria was inoculated into the first known component medium and fermented at 37 ℃ for 24 hours, and during the fermentation, fermented products of different pH values were recovered at 0.5 unit from pH 4.5 to pH 3.0. The recovered fermentations of different pH were subjected to heat treatment at 100 ℃ for 10 minutes and then filtered using a 0.45 μm filter, thereby producing hyaluronic acid inactive probiotics of different pH.
Example 1-2 preparation of Chitosan-Inactive Probiotics having pH5.0 to 6.5
The culture was carried out at 37 ℃ for 20 hours after inoculating Lactobacillus (L.paracasei SKB1192) lactic acid bacteria into MRS medium, and the number of produced bacteria was 109～1010CFU/ml。
Lactobacillus (l.paracasei SKB1192) cultured in MRS medium was recovered by centrifugation, and washed 2 times with sterilized water.
After a second known ingredient medium was prepared by dissolving 0.4% (w/v) chitosan and 2.0% (w/v) glucose in tertiary distilled water and sterilizing (121 ℃, 15 minutes), the washed lactobacillus (l.paracasei SKB1192) was inoculated into the second known ingredient medium and fermented at 37 ℃ for 24 hours, and fermented products of different pH were recovered at 0.5 unit from pH 6.5 to pH5.0 during the fermentation. The recovered fermentates with different pH values were subjected to a heat treatment at 100 ℃ for 10 minutes and then filtered through a 0.45 μm filter, thereby producing chitosan inactive probiotics with different pH values.
Examples 1-3 confirmation of optimal mixing conditions of hyaluronic acid non-live probiotic and chitosan non-live probiotic at different pH's for forming skin probiotic pieces
Example 1-3-1.Adding chitosan with pH value of 5.0-6.5 into hyaluronic acid inactive probiotics with pH value of 3.0-4.5 Yield characteristics of skin probiotic pieces formed when sugar is inactive probiotic
Each of the hyaluronic acid non-viable probiotics having pH values of 3.0 to 4.5 prepared in example 1-1 was stirred at 100rpm, and each of the chitosan non-viable probiotics having pH values of 5.0 to 6.5 prepared in example 1-2 was dropped (driping). Thereby, a skin probiotic mass-forming reaction was caused, and then the skin probiotic mass formed by centrifugal separation at 4,000rpm for 20 minutes was recovered and the yield thereof was measured. It was confirmed that the optimum condition was 42% of the maximum yield when chitosan-inactive probiotic bacteria having a pH of 6.0 were added dropwise to hyaluronic acid-inactive probiotic bacteria having a pH of 4.0 [ table 1 ]. The skin probiotic mass yield is calculated as the percentage of the total solid content of the skin probiotic mass reaction liquid divided by the total solid content of the skin probiotic mass recovered by centrifugation (%, w/w).
[ TABLE 1 ]
Examples 1 to 3 to 2.Adding hyaluronic acid with the pH value of 3.0-4.5 into chitosan inactive probiotics with the pH value of 5.0-6.5 Yield characteristics of skin probiotic pieces formed upon acid inactivation of probiotic bacteria
Each of the chitosan non-viable probiotic bacteria having pH of 5.0 to 6.5 produced in example 1-2 was stirred at 100rpm, and each of the hyaluronic acid non-viable probiotic bacteria having pH of 3.0 to 4.5 produced in example 1-1 was dropped (driping). Thereby, a skin probiotic mass-forming reaction was caused, and then the skin probiotic mass formed by centrifugal separation at 4,000rpm for 20 minutes was recovered and the yield thereof was measured. It was confirmed that the optimum condition was 58% of the maximum yield when hyaluronic acid-inactive probiotic of pH 4.0 was added dropwise to chitosan-inactive probiotic of pH 5.5 [ table 2 ]. The skin probiotic mass yield is calculated as the percentage of the total solid content of the skin probiotic mass reaction liquid divided by the total solid content of the skin probiotic mass recovered by centrifugation (%, w/w).
[ TABLE 2 ]
As can be seen from the above-described results of forming the skin probiotic lumps of examples 1-3-1 and 1-3-2, it was confirmed that the optimal conditions were found to be the formation yield of the skin probiotic lumps of examples 1-3-2, which was obtained when the chitosan inactive probiotic of pH 5.5 was added dropwise to the hyaluronic acid inactive probiotic of pH 4.0 while stirring the chitosan inactive probiotic, reached 58%.
In the following examples, the skin probiotic cake obtained by dropping the chitosan inactive probiotic of pH 5.5 with stirring and centrifuging at 4,000rpm for 20 minutes in examples 1-3-2 was collected and used as a standard sample for confirming morphological properties, and the collected skin probiotic cake was suspended with an equal amount of tertiary distilled water and used as a standard sample for confirming skin moisturizing and skin regenerating functions.
Comparative example 1 lactic acid bacteria fermentation product mix alone >
The respective hyaluronic acid non-viable probiotics of pH3.0 to 4.5 manufactured through example 1-1 were rapidly mixed while stirring the respective chitosan non-viable probiotics of pH5.0 to 6.5 manufactured through example 1-2 at 100rpm, thereby causing a skin probiotic cake forming reaction. As a result, it was confirmed that the skin probiotic bacteria cake could not be formed due to the coagulation phenomenon.
< example 2. production of a composition of a skin probiotic bar using non-active probiotic bacteria of different lactic acid bacteria >
Example 2-1 preparation of a skin probiotic Block composition Using Bifidobacterium longum
Hyaluronic acid non-viable probiotic bacteria and chitosan non-viable probiotic bacteria produced using Bifidobacterium longum (Bifidobacterium longum) were manufactured into a skin probiotic block composition according to the optimized method for manufacturing a skin probiotic block composition of examples 1-3-2 (hyaluronic acid non-viable probiotic bacteria having a pH of 4.0 was dropped while stirring chitosan non-viable probiotic bacteria having a pH of 5.5, the same applies below).
Example 2-2 preparation of skin probiotic Block composition Using Streptococcus thermophilus (Streptococcus thermophilus)
Hyaluronic acid-inactive probiotics and chitosan-inactive probiotics produced using Streptococcus thermophilus (Streptococcus thermophilus) were manufactured into skin probiotic block compositions according to the optimized method for manufacturing skin probiotic block compositions of examples 1-3-2.
Examples 2-3 preparation of skin probiotic Block composition Using lactococcus lactis (Lactococcus lactis)
Hyaluronic acid non-viable probiotic bacteria and chitosan non-viable probiotic bacteria manufactured using lactococcus lactis (lactococcus lactis) were manufactured into skin probiotic pellet compositions according to the optimized methods of manufacturing skin probiotic pellet compositions of examples 1 to 3 to 2.
Examples 2-4 preparation of skin probiotic Block composition Using Enterococcus faecium (Enterococcus faecalis)
Hyaluronic acid non-active probiotic bacteria and chitosan non-active probiotic bacteria manufactured using Enterococcus faecium (Enterococcus faecium) were manufactured into skin probiotic pellet compositions according to the optimized method for manufacturing skin probiotic pellet compositions of examples 1-3-2.
Examples 2-5 preparation of skin probiotic Block compositions Using Pediococcus pentosaceus (Pediococcus pentosaceus)
Hyaluronic acid non-reactive probiotic bacteria and chitosan non-reactive probiotic bacteria manufactured using Pediococcus pentosaceus (Pediococcus pentosaceus) were manufactured into the skin probiotic pellet composition according to the optimized method for manufacturing the skin probiotic pellet composition of examples 1-3-2.
Examples 2-6 manufacture of skin probiotic Block compositions Using Leuconostoc lactis
Hyaluronic acid non-active probiotics and chitosan non-active probiotics prepared by using Leuconostoc lactis are prepared into the skin probiotic block composition according to the optimized preparation method of the skin probiotic block composition of the embodiment 1-3-2.
Examples 2-7 manufacture of skin probiotic bar compositions utilizing Weissella confusa (Weissla convusa)
Hyaluronic acid non-active probiotic bacteria and chitosan non-active probiotic bacteria manufactured using weisssla comfusa (weissla comfusa) were manufactured into a skin probiotic bar composition according to the optimized method of manufacturing the skin probiotic bar composition of example 1.
< comparative example 2. production of culture Medium for hyaluronic acid first known ingredient and culture Medium for Chitosan second known ingredient >
When the skin moisturizing and skin regeneration functionality were confirmed, a hyaluronic acid first known component medium and a chitosan second known component medium were produced for comparison purposes and used as comparative examples.
Comparative example 2-1 preparation of hyaluronic acid first known component Medium
A medium containing hyaluronic acid as a first known component was prepared by dissolving 0.5% (w/v) of hyaluronic acid (1,500-2,000 kDa) having a medium molecular weight and 2.0% (w/v) of glucose in tertiary distilled water, correcting the pH to 4.0 with lactic acid, and sterilizing the solution (121 ℃ C., 15 minutes).
Comparative example 2-2 preparation of culture Medium for second known composition of Chitosan
A second known component culture medium of chitosan was prepared by dissolving 0.4% (w/v) of chitosan and 2.0% (w/v) of glucose in tertiary distilled water, correcting the pH to 5.5 with lactic acid, and then sterilizing (121 ℃, 15 minutes).
< comparative example 3 preparation of Block composition of hyaluronic acid first known component Medium and Chitosan second known component Medium >
The hyaluronic acid first known ingredient medium of comparative example 2-1 was dropped (driping) while stirring the chitosan second known ingredient medium of comparative example 2-2 at 100 rpm. Thereby, a chitosan-hyaluronic acid mass-forming reaction is caused. In order to confirm the yield of the block composition formed as described above, the chitosan-hyaluronic acid block composition formed by centrifugation at 4,000rpm for 20 minutes was recovered, and it was confirmed that the yield of the chitosan-hyaluronic acid block was 54%.
< test example 1. confirmation of morphological Properties of skin probiotic Bar >
The morphological properties of the skin probiotic pellet produced in examples 1-3-2 were confirmed. The standard sample block composition was centrifuged at 4,000rpm for 20 minutes and freeze-dried after only the formed block was recovered. The spherical particles were photographed by a Scanning Electron Microscope (SEM), and the morphology thereof was confirmed to have the shape of spherical particles [ FIG. 1-1 ].
In addition, for comparative experiments, the substance in which the aggregation phenomenon occurred in comparative example 1 was freeze-dried and photographed by a scanning electron microscope [ fig. 1-2 ].
< test example 2 skin moisturizing effect of probiotic Block composition for skin >
The skin moisturizing effect of the skin probiotic patch composition was confirmed by measuring the effect of expression of moisturizing genes present in the epidermis and dermis of the skin.
Test example 2-1 aquaporin 3 messenger ribonucleic acid expression (AQP) -3mRNA expression) effect of skin probiotic pellet composition
Aquaporin (AQP) is a membrane pore protein that functions as a water channel (water channel) that selectively induces the entry and exit of water molecules in the skin while blocking the passage of other ions or solutes, wherein aquaporin 3(AQP-3) allows glycerol (glycerol) to pass through with water, and thus is also called aquaglycerol channel (aquaglycoopening), is present in human keratinocytes (human keratinocyte), and functions such as skin moisturization (skin hydration), wound healing (wound healing), and homeostasis (homeostatis) by preventing the loss of water molecules. Therefore, the moisturizing efficacy of the raw material can be confirmed by the increased expression of aquaporin 3 (AQP-3).
Human normal keratinocytes (gibco) were seeded in a culture dish (culture dish) and allowed to stand for 24 hours (37 ℃ C., CO) for the purpose of performing Real-time fluorescent quantitative polymerase chain reaction (Real time PCR)25%) was replaced with Serum-free (Serum-free) medium (Epilife, gibco), and then samples of the hyaluronic acid non-viable probiotic of example 1-1 (pH 4.0), the chitosan non-viable probiotic of example 1-2 (pH 5.5), and the skin probiotic cake composition of example 1-3-2 (prepared by dropping hyaluronic acid non-viable probiotic of pH 4.0 while stirring the chitosan non-viable probiotic of pH 5.5) were treated at a concentration of 3% (w/v) and cultured for 5 days, respectively. The cultured cells were collected by TRIzol and ribonucleic acid (RNA) was isolated according to the manufacturer's method. After quantifying the isolated ribonucleic acid (RNA), a complementary deoxyribonucleic acid (cDNA) was synthesized using 1. mu.g of ribonucleic acid (RNA) and a Real-time fluorescent quantitative polymerase chain reaction (Real time PCR) was performed. Aquaporin 3(aquaporin (AQP) -3) and β Actin (β -Actin) primers (primer) used in Polymerase Chain Reaction (PCR) were synthesized by COSMO genech (Korea), and the primer sequence (primer sequence) of aquaporin 3(AQP-3) is shown in table 3.
For comparative experiments, control treatment was performed at a concentration of 3% (w/v) using comparative example 2-1, comparative example 2-2 and comparative example 3 in the same manner, and 50nM retinoic acid (retinoic acid) was used as a positive control group.
[ TABLE 3 ]
As shown in fig. 2, it can be confirmed that the skin probiotic pellet compositions of examples 1-3-2 can significantly induce the expression of aquaporin 3(AQP-3) compared to the comparative examples and examples performed at the same concentration. This indicates that the skin moisturizing effect of the non-viable probiotics, either chitosan or hyaluronic acid, is delivered by being manufactured into a skin probiotic block shape, and the effect of acting on human keratinocytes (human keratinocytes) is further enhanced. Thus, it was confirmed that the skin probiotic pellet composition of the present invention can enhance skin moisturizing (skin hydration) effect by activating water channels (water channels) in epidermal cells of the skin, and belongs to a composition that can contribute to maintenance of the moisturization of the epidermis, and thus, dermal cells.
Test example 2-2. hyaluronic acid synthase 2 messenger ribonucleic acid expression (HAS-2mRNA expression) Effect of skin probiotic pellet composition
It has been reported that the amount of hyaluronic acid in human skin gradually decreases with aging, and this is considered to be one of the direct causes of the decrease in skin elasticity and the decrease in moisture content accompanying aging. The synthesis of Hyaluronic Acid as described above is performed by hyaluronan Synthase (HAS), and skin moisturizing and skin regenerating effects can be confirmed by the increase in the expression of hyaluronan Synthase 2 (HAS-2).
In order to perform Real-time fluorescent quantitative polymerase chain reaction (Real time PCR), human normal fibroblasts (human normal fibroblasts, gibco) were seeded in a culture dish (culture dish) and were subjected to 24 hours (37 ℃ C., CO)25%) were replaced with Serum-free (Serum-free) medium (Epilife, gibco) and then the hyaluronic acid inactive probiotic of example 1-1 (pH 4.0), the chitosan inactive probiotic of example 1-2 (pH 5.5) and the skin probiotic pellet composition of example 1-3-2 were used (by passing through the skin probiotic pellet composition of example 1-3-2), respectivelyPrepared by dropping hyaluronic acid non-viable probiotic bacteria of pH 4.0 while stirring chitosan non-viable probiotic bacteria of pH 5.5) was treated at a concentration of 3% (w/v) and cultured for 5 days. The cultured cells were collected by TRIzol and ribonucleic acid (RNA) was isolated according to the manufacturer's method. After quantifying the isolated ribonucleic acid (RNA), a complementary deoxyribonucleic acid (cDNA) was synthesized using 1. mu.g of ribonucleic acid (RNA) and a Real-time fluorescent quantitative polymerase chain reaction (Real time PCR) was performed. The hyaluronan synthase 2(HAS-2) and β Actin (β -Actin) primers (primer) used in the Polymerase Chain Reaction (PCR) were synthesized by COSMO GENTECH (Korea), and the primer sequence (primer sequence) of the hyaluronan synthase 2(HAS-2) was as shown in [ Table 4 ].
For comparative tests, control treatment was carried out in the same manner at a concentration of 3% (w/v) using comparative examples 1-1, 1-2 and 2, and 50nM retinoic acid (retinoic acid) was used as a positive control.
In addition, in order to confirm whether similar properties of the skin probiotic block could be maintained in the case of production using other lactic acid bacteria, treatment was performed at a concentration of 3% (w/v) using examples 2-1 to 2-7 according to the same method, and as a positive control group, 50nM retinoic acid (retinic acid) was used.
[ TABLE 4 ]
The results of confirming the expression of the gene by Real-time fluorescent quantitative polymerase chain reaction (Real time PCR) are shown in fig. 3, and it can be confirmed that the skin probiotic pellet composition of examples 1-3-2 can significantly induce the expression of hyaluronan synthase 2(HAS-2) compared to the comparative example and examples performed at the same concentration. This indicates that the skin moisturizing effect of the non-active probiotics, namely chitosan or hyaluronic acid, is transferred by being manufactured into the form of skin probiotic block, and the effect of acting on dermal cells, namely human fibroblasts (human fibroplasts), is further improved. Thus, it was confirmed that the skin probiotic mass composition of the present invention is an excellent composition capable of promoting skin moisturizing effect through skin regeneration process of hyaluronic acid synthesis in dermal cells.
Furthermore, as shown in FIG. 4, it was confirmed that the same effect of inducing the expression of hyaluronan synthase 2(HAS-2) as that of examples 1-3-2 was observed in the skin probiotic pellet composition prepared using other lactic acid bacteria-inactive probiotics. As described above, it was confirmed that all of the skin probiotic blocks produced using the lactic acid bacteria-inactive probiotics had skin moisturizing and regenerating functions in dermal cells.
< test example 3 skin regeneration Effect of skin probiotic Bar composition >
To confirm the skin regeneration effect of the skin probiotic pellet, a wound healing (wound healing) test was performed. After seeding Human normal fibroblasts (Human normal fibroblasts) into cell dishes (cell dish) and performing culture for 24 hours, scratches were formed in the cells (cells) using a splsca scortcher. After washing with Hank's balanced Salt Solution (HBSS, Hank's balanced Salt Solution, Gibco, USA), samples of hyaluronic acid inactive probiotic of example 1-1, chitosan inactive probiotic of example 1-2, and skin probiotic pellet composition of example 1-3-2, example 2 were treated at a concentration of 3% (w/v), respectively, and incubated for 48 hours. After the culture, the number of the cells in the scratch (scratch) portion was measured by imaging with a microscope, and the cell density (%) of the examples and comparative examples was calculated as compared with the number of the cells in the control group.
For comparative tests, control treatment was carried out at a concentration of 3% (w/v) according to the same method using comparative example 2-1, comparative example 2-2 and comparative example 3, and adenosine (adenosine) at 7. mu.g/ml was used as a positive control group. As shown in fig. 5 to 12, it was confirmed from the results of comparing the cell density of the scratch (scratch) part using the photomicrographs that the cell proliferation of the skin probiotic bacteria mass of examples 1-3-2 exhibited a more dense increase effect than the control group (control) and the comparative example, and exhibited a cell density similar to that of the positive control group, i.e., adenosine (adenosin) of 7 μ g/ml. It was confirmed that the cell probiotic pellets of examples 1-3-2 induced the proliferation of damaged skin cells and exhibited an improved skin regeneration effect.
The present invention has been described in detail with reference to the embodiments described above, but this is for exemplary purposes only, and it will be understood by those having ordinary skill in the art that the present invention may be embodied in many different forms and equivalents. Therefore, the true technical scope of the present invention should be defined by the technical matters in the appended claims.
Claims (7)
1. A method of making a skin probiotic pellet composition comprising:
a step of preparing a first medium of known composition containing hyaluronic acid and glucose and a second medium of known composition containing chitosan and glucose;
(second step) a step of preparing a fermentation product of hyaluronic acid bacteria by inoculating lactic acid bacteria to the first known component medium and fermenting the same, and preparing a fermentation product of chitosan lactic acid bacteria by inoculating lactic acid bacteria to the second known component medium and fermenting the same;
(third step) a step of producing inactive probiotics by heating the fermented product of hyaluronic acid bacteria and the fermented product of chitosan lactic acid bacteria, respectively; and the number of the first and second groups,
(fourth step) a step of manufacturing a skin probiotic bar composition by drop-mixing the hyaluronic acid non-viable probiotic and the chitosan non-viable probiotic.
2. The method of manufacturing a skin probiotic pellet according to claim 1, characterized in that:
the lactic acid bacteria in the second step are selected from the group consisting of Lactobacillus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), Streptococcus (Streptococcus sp.), Lactococcus (Lactococcus sp.), Enterococcus (Enterococcus sp.), Pediococcus (Pediococcus sp.), Leuconostoc (Leuconostoc sp.) and Weissella (Weissella sp.).
3. The method of manufacturing a skin probiotic pellet according to claim 1, characterized in that:
in the second step, when the first known component medium is inoculated with lactic acid bacteria and fermented, a fermentation product of the hyaluronic acid bacteria is produced at a pH of 3.0 to 4.5.
4. The method of manufacturing a skin probiotic pellet according to claim 1, characterized in that:
in the second step, when the second known component medium is inoculated with lactic acid bacteria and fermented, a chitosan lactic acid bacteria fermented product is produced at a pH of 5.0 to 6.5.
5. The method of manufacturing a skin probiotic pellet according to claim 1, characterized in that:
in the third step, the heating treatment of the lactic acid bacteria fermented product for producing each of the non-active probiotics is performed at 90 to 100 ℃ for 5 to 15 minutes.
6. A skin probiotic block composition is provided, which comprises a probiotic pellet,
produced by the method according to claim 1, exhibiting skin moisturizing or skin regenerating functionality to dermal cells.
7. A cosmetic composition, a patch or a mask for skin, characterized in that:
containing a mass of skin probiotics made by the method of claim 1.
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| CN114736941B (en) * | 2021-12-30 | 2024-03-29 | 云南白药集团健康产品有限公司 | Sodium hyaluronate fermented product, external preparation containing the same for skin, and preparation method and application thereof |
| CN114767922B (en) * | 2022-03-15 | 2023-09-12 | 青岛大学 | Hyaluronic acid hydrogel carrying probiotics and preparation method and application thereof |
| CN115581636B (en) * | 2022-09-08 | 2024-03-22 | 广东馨柯生命科学有限公司 | Formula containing oat glucan Essenpure CBG capable of resisting photodamage and preparation method of formula |
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