CN113355377A - Mixed bacteria cellulose facial mask with skin microbial population regulating effect and preparation method thereof - Google Patents
Mixed bacteria cellulose facial mask with skin microbial population regulating effect and preparation method thereof Download PDFInfo
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- CN113355377A CN113355377A CN202010720205.2A CN202010720205A CN113355377A CN 113355377 A CN113355377 A CN 113355377A CN 202010720205 A CN202010720205 A CN 202010720205A CN 113355377 A CN113355377 A CN 113355377A
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- bacterial cellulose
- lactobacillus
- bacteria
- cellulose
- probiotic
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- 239000011787 zinc oxide Substances 0.000 description 1
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Abstract
The present invention relates to a method for preparing bacterial cellulose containing a probiotic having a skin microbiome regulating effect, which comprises the step of mixing a fermentative bacterial cellulose-producing bacterium with a probiotic having a skin microbiome regulating effect, a bacterial cellulose composition containing a probiotic having a skin microbiome regulating effect prepared by the above method, and a mask sheet containing the same. In the case of using the method for preparing bacterial cellulose of the present invention, the bacterial cellulose is high in yield and excellent in quality as compared with the case of culturing the bacterial cellulose-producing bacteria alone. Also, the probiotics trapped by the bacterial cellulose have the effect of regulating the skin microbiota, and thus are useful for skin health.
Description
Technical Field
The invention relates to a mixed bacterial cellulose facial mask with skin microbial flora regulating effect and a preparation method thereof.
Background
The bacterial cellulose is (C)6H10O5)nOrganic compounds expressed by the formula of (a.j. brown, 1886) were originally discovered and produced by fermentation of microorganisms of the genus Acetobacter (Acetobacter), Sarcina ventriculi (Sarcina ventriculi), Agrobacterium (Agrobacterium). Unlike plant cellulose, bacterial cellulose has a β -1,4glucan chain (β -1,4glucan chain) as a chain in which branches are not formed as a basic unitThe polymer of (1).
In the prior art, the yield of bacterial cellulose sheets is low, and thus mass production is difficult. Accordingly, the present inventors invented a strain of Acetobacter gluconicum KOSS-15 (Komagataibacter rhaeticus KOSS-15) (accession No. KCCM12270P) that produces bacterial cellulose at a high yield (Korean patent application No. 10-1970439), and invented a method for improving the yield by using glucose as a monosaccharide and sucrose as a disaccharide (glucose + fructose ) as a complex carbon source, using glucose in a medium for the growth of microorganisms, and using glucose decomposed from the sucrose as a substrate glucose for the production of bacterial cellulose (Korean patent application No. 10-1970440).
However, even when a strain having a high yield of bacterial cellulose is used and the ratio of the concentrations of both sugars is optimized, the bacterial concentration of the bacterial cellulose-forming strain is sensitively increased by the change of pH, temperature, oxygen amount, glucose concentration, etc. during the culture, and the substrate glucose for producing bacterial cellulose is used instead due to the rise of pH, the rise of temperature caused by abnormal fermentation, the consumption of dissolved oxygen, and the consumption of growing glucose, so that there is still a problem that an incomplete bacterial cellulose sheet (incomplete bacterial cellulose sheet) having a non-uniform thickness of the sheet is produced. Therefore, it is required to develop a method for preparing a bacterial cellulose tablet having high yield and excellent quality.
On the other hand, the skin surface of a healthy person has skin microbiome (skin microbiome) as skin microbiota, and it is known that the balance of these skin microbiota contributes to the maintenance of a healthy skin state. Specifically, a known skin-beneficial bacterium is Staphylococcus epidermidis (Staphylococcus epidermidis), and a known skin-harmful bacterium is Staphylococcus aureus (Staphylococcus aureus). Recently, cosmetics and the like using probiotics containing lactic acid bacteria have been proposed for the purpose of maintaining such facial skin microbiome (facial skin microbiome), and products are mostly proposed in the form of topical creams (facial cream) or milk in which the probiotics are diluted.
However, it is difficult to achieve the intended effect of such dairy-like products according to personal hygiene conditions and skin regeneration cycles (skin turn over). Therefore, it is required to study other formulations, not to adjust the skin microbiome by cosmetic formulations, and in the process, a mask sheet formulation of a bacterial cellulose material containing moisture for a certain period of time and having excellent skin adaptability is studied. The invention provides a novel facial mask, which takes probiotics with skin microbiome regulating effect as an object, is easy to generate the existing bacterial cellulose and is trapped by the bacterial cellulose to realize the skin microbiome regulating effect.
Disclosure of Invention
The present inventors have made an effort to develop a method for producing a bacterial cellulose tablet with high yield and excellent quality. As a result, the present invention has been completed by finding out the effect that bacterial cellulose having excellent productivity and quality is produced in the case of mixed fermentation (mixed culture or symbiotic culture) of bacterial cellulose-forming strains and probiotics, and that the probiotics contained in these bacterial celluloses have the effect of regulating the balance of skin microbiota.
Accordingly, an object of the present invention is to provide a method for preparing bacterial cellulose containing probiotics, which comprises the step of mixing a fermentative bacterial cellulose-producing bacterium and a probiotic.
It is a further object of the present invention to provide a bacterial cellulose composition comprising probiotic bacteria.
Another object of the present invention is to provide a mask sheet comprising a bacterial cellulose composition, wherein the bacterial cellulose comprises the probiotic.
According to an embodiment of the present invention, there is provided a method for preparing bacterial cellulose containing probiotics, including a step of mixing a fermentative bacterial cellulose-producing bacterium and a probiotic.
In the present invention, "bacterial cellulose" is cellulose produced by bacteria of the genus Acetobacter (Acetobacter), Rhizobium (Rhizobium), Agrobacterium (Agrobacterium), or the like, and has high mechanical strength and a three-dimensional network structure composed of extremely fine and pure cellulose. In the case of ordinary plant fibers, the fibers are composed of cellulose, hemicellulose, lignin, etc., and if only cellulose is produced, the hemicellulose must be removedCellulose and lignin, however, bacterial cellulose is composed of pure cellulose only. The reticular involucra formed by the bacterial cellulose is ribbon-shaped fibrillated cellulose formed randomly, the width of the banded fibrillated cellulose is less than 100nm, the banded fibrillated cellulose is formed by countless micro-microfibers with the radius of 2-4 nm, and the micro-microfibers have the weight of 1600kg/m3The density of (c). And, has inherent characteristics of high crystallinity (84-89%) and 78MPa, thus generally exhibiting characteristics of higher than that of the reported natural fiber of Macro-scale (Macro-scale) or similar to the elastic modulus (70GPa) of glass fiber, and also exhibiting characteristics of having sufficient porosity and being very excellent in biocompatibility. Bacterial cellulose is excellent in biocompatibility and mechanical properties and low in density, and is used as a medical material for artificial skin, artificial cartilage, wound protection agents, artificial blood vessels, burn treatment agents, etc., and is excellent in skin adhesion and wettability, and therefore, it is also useful as a cosmetic material, and also as an edible material such as coconut (Nata de coco).
In one embodiment of the present invention, the bacterial cellulose-producing strain is Acetobacter gluconicum KOSS15(Komagataeibacter rhaeticus KOSS15) (accession No. KCCM 12270P).
The strain Acetobacter gluconicum KOSS15 of the present invention may be further designated as Acetobacter gluconicum KOSS-15 strain. The strain acetobacter gluconicum KOSS15 is derived from kombucha (kombucha), also called conpu tea, black tea mushroom yeast, red mushroom, and jonquil mushroom, and is known as one of organic acid-producing bacteria (lactic acid bacteria) in which bacteria and yeast coexist with military such as silky military as a parent.
In the present invention, the probiotics (probiotics) to be mixed with the above-mentioned bacterial cellulose-producing bacteria and fermented include lactic acid bacteria and non-lactic acid bacteria.
In the present invention, the lactic acid bacteria are selected from the group consisting of Lactobacillus casei (Lactobacillus casei), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus sake (Lactobacillus sakei), Lactobacillus acidophilus (Lactobacillus acidophilus), Bifidobacterium longum (Bifidobacterium longum), and Enterococcus faecalis (Enterococcus faecalis), but not limited thereto.
In a specific embodiment of the present invention, the lactic acid bacteria are lactobacillus rhamnosus, lactobacillus plantarum or a combination thereof. The lactobacillus rhamnosus is more specifically lactobacillus rhamnosus SKB1253 strain with the deposit number of KCCM12452P, and the lactobacillus plantarum is more specifically lactobacillus plantarum SKB1234 strain with the deposit number of KCCM 12450P.
In the present invention, the non-lactic acid bacteria are selected from the group consisting of Bacillus subtilis (Bacillus subtilis), Bacillus coagulans (Bacillus coagulousns), Bacillus licheniformis (Bacillus licheniformis), Bacillus indicus (Bacillus indicus), Streptococcus salivarius (Streptococcus salivarius), and Streptococcus thermophilus (Streptococcus thermophilus), but not limited thereto.
The method for preparing bacterial cellulose of the present invention is described according to the steps.
(a) First, a bacterial cellulose-producing bacterium (acetic acid bacterium) and a probiotic bacterium are cultured and propagated, respectively.
The above-mentioned bacterial cellulose-producing bacteria and probiotics are as described above.
The proliferation step can be repeated for the first generation, the second generation or more than the third generation, and the large-volume culture medium is replaced along with repeated passage. For example, after culturing the initial medium in a culture vessel and medium having a volume of 10L or less, the first generation grows in a culture vessel and medium having a scale of 100L or less, 1000L or less, and 10000 to 30000L or less.
(b) Next, when a sufficient amount of bacterial cellulose-producing bacteria (acetic acid bacteria) and probiotics are prepared, the bacterial cellulose-producing medium is inoculated with each of the bacteria, and mixed fermentation is performed.
In one embodiment of the present invention, in order to prepare the bacterial cellulose in a sheet (sheet), a bacterial cellulose-producing bacterium (acetobacter) and a probiotic bacterium are cultured in a tray-shaped culture container.
In a specific embodiment of the present invention, the above culture is performed on an uncovered tray (open system).
In one embodiment of the present invention, the initial pH of the medium for the above-mentioned cultivation is 3.5 to 9. As demonstrated in one embodiment of the present invention, the initial pH of the culture medium affects the yield and quality of bacterial cellulose. The culture medium preferably has an initial pH of 3.5 to 6, more preferably an initial pH of 3.5 to 5 or 3.5 to 4, and most preferably an initial pH of 4.
In another embodiment of the present invention, the final pH of the medium for the above-mentioned culture (pH of the medium after the end of the culture) is 3 to 4.5. The final pH value of the culture medium is more preferably 3-4.
In one embodiment of the present invention, the culturing is performed at a temperature of 25-35 ℃. As demonstrated in one embodiment of the present invention, the yield of bacterial cellulose varies depending on the culture temperature. The preferable temperature for the above culture is 25 to 33 ℃, 25 to 30 ℃, 25 ℃, 28 ℃ or 30 ℃, but is not limited thereto. However, in the case of culturing under the temperature condition of 37 ℃, the yield of bacterial cellulose can be reduced.
In one embodiment of the present invention, the culture medium is cultured in a concentration of 0.5 to 5% (w/v) glucose. As demonstrated in one embodiment of the present invention, the concentration of glucose contained in the culture medium of the present invention affects the yield of bacterial cellulose and the quality of the sheet. In an embodiment of the present invention, the culture medium of the present invention preferably contains glucose at a concentration of 1 to 5%, 1.5 to 5%, 2 to 5%, 3 to 5%, 4 to 5%, 0.5 to 4%, 1 to 4%, 1.5 to 4%, 2 to 4%, 3 to 4%, 0.5 to 3%, 1 to 3%, 1.5 to 3%, 2%, 3%, 4%, or 5%, and most preferably 2%, but is not limited thereto.
In one embodiment of the present invention, the cultivation time for producing the above bacterial cellulose is 24 hours or more. As demonstrated in one embodiment of the present invention, the bacterial cellulose of the present invention exhibits increased linearity of yield versus culture time. In addition, the capturing rate of probiotics by the mixed fermentation of the bacterial cellulose of the present invention is also increased in proportion to the culture time. Preferably, the culture time is 24 to 120 hours, 24 to 96 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 48 to 120 hours, 48 to 96 hours, 48 to 72 hours, 48 to 60 hours, 48, 60 hours, 72 hours, or the like, but is not limited thereto.
(c) Then, the bacterial cellulose produced by mixing the fermentative bacterial cellulose-producing bacteria (acetic acid bacteria) and the probiotic bacteria is washed and pressurized.
The bacterial cellulose thus produced is washed in order to remove excess bacteria that produce bacterial cellulose (acetic acid bacteria), foreign matter, and the like. Before and after the above washing, a step of immersing in an aqueous NaOH solution (0.1%) may be further performed. The above-mentioned step of immersing in an aqueous NaOH solution has the effect of reinforcing the strength of the sheet.
The washed bacterial cellulose sheet is subjected to a pressurizing and/or sterilizing step in order to adjust the thickness of the sheet to be uniform and to remove moisture contained in the sheet.
When the above washing, pressing (dehydration) and/or sterilization process is completed, the bacterial cellulose sheet is completed.
(d) Finally, the prepared bacterial cellulose sheet is prepared into a bacterial cellulose facial mask sheet through an additional processing step of cutting the bacterial cellulose sheet in a predetermined size and shape or is used as a cosmetic raw material through a pulverization step.
According to yet another embodiment of the present invention, there is provided a bacterial cellulose composition comprising a probiotic.
According to another embodiment of the present invention, there is provided a mask pack comprising a bacterial cellulose composition, wherein the bacterial cellulose comprises the probiotic.
The definition of the above probiotics is the same as that in the method for preparing bacterial cellulose containing probiotics according to an embodiment of the present invention as described above.
According to still another embodiment of the present invention, there is provided a probiotic mixed dough sheet comprising bacterial cellulose prepared by mixing a fermentative bacterial cellulose-producing bacterium and a probiotic.
In this specification, the term "probiotic mixing" or "mixing" refers to i) preparation by a symbiotic cultivation process of probiotics and bacterial cellulose-producing bacteria; or ii) an article comprising bacterial cellulose or a bacterial cellulose sheet prepared by a symbiotic culture procedure.
In the present specification, terms of a mixed probiotic pellet, a mixed probiotic dough sheet, mixed probiotic bacteria cellulose and the like have the same meaning.
In a specific example of the present invention, the probiotic bacteria include lactic acid bacteria, and the mixed probiotic bacteria tablet is further expressed in terms of a mixed lactic acid bacteria tablet, a mixed lactic acid bacteria dough sheet, a mixed lactic acid bacteria cellulose, or a mixed lactic acid bacteria cellulose tablet.
In the present specification, the term "the above-mentioned mixed fermentation" has the same meaning as that of co-culture or co-culture (co-culture).
As demonstrated in one embodiment of the present invention, the bacterial cellulose prepared by mixing the fermentative bacterial cellulose-producing bacteria and the probiotics of the present invention is entrapped with the probiotics. Also, the bacterial cellulose trapping the above probiotics has the effects of increasing the survival rate of staphylococcus epidermidis as skin beneficial bacteria and inhibiting the proliferation of staphylococcus aureus as skin harmful bacteria.
In one embodiment of the present invention, the probiotic bacteria trapped by the bacterial cellulose of the present invention are live bacteria or fire-extinguishing bacteria. Even in the case of dead cell bodies, the above-described effect is exhibited by the cytoplasmic components of the remaining probiotic bacteria.
Therefore, the bacterial cellulose composition comprising probiotics and the mask sheet comprising the same of the present invention can be effectively used for skin beauty or make-up. In particular, the bacterial cellulose comprising probiotics of the present invention has the effects of increasing the survival rate of staphylococcus epidermidis, which is a skin-beneficial bacterium, and inhibiting the proliferation of staphylococcus aureus, which is a skin-harmful bacterium, and thus, has the effect of improving skin health by regulating skin microbiota.
As demonstrated in one embodiment of the present invention, the thickness, water content, and maximum load of the facial film sheet prepared by the above-described mixed fermentation of the present invention are increased as compared to a facial film sheet containing bacterial cellulose prepared by fermenting bacterial cellulose-producing bacteria alone.
In an embodiment of the present invention, the mask sheet of the present invention has a thickness of 0.7 to 1.0mm, 0.75 to 1.0mm, 0.8 to 1.0mm, or 0.85 to 1.0mm, when measured after soaking in distilled water for 15 hours or more, but is not limited thereto.
In still another embodiment of the present invention, the mask sheet of the present invention has a water content of 110 to 160, 120 to 160, 130 to 160, 135 to 160, 140 to 160, 110 to 150, 120 to 150, 130 to 150, 135 to 150, or 140 to 150, when the water content according to formula 3 is calculated after soaking in distilled water for 15 hours or more, but is not limited thereto.
In another example of the present invention, in the mask sheet of the present invention, when the elongation according to the above 5 is calculated after soaking in distilled water for 15 hours or more, the elongation is 16 to 30%, 18 to 30%, 19 to 30%, 20 to 30%, 22 to 30%, 16 to 28%, 18 to 28%, 19 to 28%, 20 to 28%, 22 to 28%, 16 to 26%, 18 to 26%, 19 to 26%, 20 to 26%, or 22 to 26%, but not limited thereto.
The conditions for measuring the thickness, water content and elongation of the present invention are based on the mixed fermentation of acetobacter gluconicum (k.rhaeticus) and lactobacillus rhamnosus (l.rhamnosus) at a temperature of 30 ℃, an initial glucose concentration of 2% (w/v) and an initial pH of 4.0 for 72 hours, in order to prepare the facial mask sheet of the present invention, and the prepared bacterial cellulose sheet is cultured in the following steps.
According to an embodiment of the present invention, there is provided a mask for skin beauty or make-up comprising the probiotic mixed-mask sheet as described above.
According to still another embodiment of the present invention, there is provided a mask pack for skin beauty or make-up comprising the probiotic mixed facial mask sheet and the cosmetic and dairy industry as described above.
According to another embodiment of the present invention, there is provided a cosmetic composition comprising the bacterial cellulose containing probiotic bacteria or the probiotic mixed bacterial cellulose as described above.
In the case where the bacterial cellulose containing probiotics of the present invention is used as a cosmetic composition, the above cosmetic composition may be prepared into a dosage form selected from the group consisting of a solution, a topical ointment, a cream, a foam, a nutritional lotion, a softening lotion, a mask sheet, a soothing lotion, a cream, a pre-makeup emulsion, an essence, a soap, a liquid detergent, a bathing agent, a sunscreen cream, a sunscreen oil, a suspension, an emulsion, a paste, a gel, an emulsion, a powder, a surfactant-containing detergent, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.
The cosmetic composition of the present invention may further comprise one or more cosmetically acceptable carriers to be combined with general skin cosmetics, for example, conventional ingredients may be properly combined with oil, water, surfactant, humectant, low alcohol, thickener, chelating agent, pigment, preservative, perfume, etc., but not limited thereto.
The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation. In the case where the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, amine gum, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, etc. can be used as the carrier component, but the formulation is not limited thereto. These may be used alone or in combination of two or more. In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or the like can be used as the carrier component, and particularly, in the case of a spray, a filler such as chlorofluorocarbon, propane/butane, or dimethyl ether may be further included, but the present invention is not limited thereto. These may be used alone or in combination of two or more. In the case where the dosage form of the present invention is a solution or emulsion, the carrier component may be a solvent, solubilizer, emulsifier, or the like, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol oil, or the like, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil, sesame oil, glycerol fatty ester, polyethylene glycol, or fatty acid ester of sorbitol, but is not limited thereto. These may be used alone or in combination of two or more. When the formulation of the present invention is a suspension, the carrier component may be, for example, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth, but is not limited thereto. These may be used alone or in combination of two or more. When the formulation of the present invention is a soap, the carrier component may be an alkali metal salt of a fatty acid, a fatty acid half-ester salt, a fatty acid protein hydrolysate, a isethionate salt, a lanolin derivative, a fatty alcohol, a vegetable oil, glycerol, a sugar, or the like, but is not limited thereto. These may be used alone or in combination of two or more.
The invention provides a preparation method of bacterial cellulose containing probiotics, which comprises the step of mixing and fermenting bacterial cellulose generating bacteria and probiotics. Also, the present invention provides a bacterial cellulose composition comprising probiotic bacteria and a mask sheet comprising the same.
Compared with the case of culturing bacterial cellulose-producing bacteria alone, the bacterial cellulose preparation method of the invention has high yield and excellent quality. Also, the probiotics trapped by the bacterial cellulose have the effect of regulating the skin microbiota, and thus are useful for skin health.
Drawings
FIG. 1 is a graph showing the production amount of mixed bacterial cellulose pieces and pH stability in a fermentation system according to the mixed fermentation temperature.
Fig. 2 is a graph showing the production amount of mixed bacterial cellulose pieces according to the initial pH of the mixed fermentation system and the pH stability in the fermentation system.
FIG. 3 is a graph of mixed bacterial cellulose flake production and pH stability in a fermentation system according to the initial glucose concentration of the mixed fermentation system.
Fig. 4 is a graph showing the linearity of the production amount of mixed bacterial cellulose pieces as a function of time when mixed fermentation is performed.
Fig. 5a and 5b are graphs showing that the mixed bacterial cellulose sheet of the present invention affects the survival rate of skin beneficial bacteria.
Fig. 6a and 6b are graphs showing that the mixed bacterial cellulose sheet of the present invention affects the survival rate of harmful bacteria on the skin.
FIG. 7 is a graph showing comparison of a bacterial cellulose tablet of acetic acid bacteria alone as a control group with a mixed bacterial cellulose tablet of the present invention prepared by mixing fermented acetic acid bacteria and lactic acid bacteria (left: lactic acid bacteria, center: bacterial cellulose alone, right: mixed fermented bacterial cellulose).
Fig. 8 is a schematic diagram showing a step of preparing a mixed bacterial cellulose tablet by mixed fermentation of a bacterial cellulose-producing bacterium and a lactic acid bacterium according to the present invention.
Fig. 9 is a view showing the appearance of bacterial cellulose alone which produces bacterial cellulose-producing bacteria (left) and a mixed bacterial cellulose tablet produced by mixing fermented bacterial cellulose-producing bacteria and lactic acid bacteria according to the present invention (right).
FIG. 10 is a graph showing the elongation percentage of a bacterial cellulose sheet prepared by culturing a bacterial cellulose-producing microorganism alone according to a conventional method and a bacterial cellulose sheet prepared by mixed fermentation according to the present invention in comparison.
Fig. 11 is a graph showing the thickness or density uniformity of a bacterial cellulose sheet prepared by culturing bacterial cellulose-producing bacteria alone according to the prior art and a bacterial cellulose sheet prepared by mixed fermentation according to the present invention in comparison according to the date of culture.
FIG. 12 is a specification chart of a test piece.
Detailed Description
The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples in light of the spirit of the present invention.
Examples
Throughout the present specification, "%" used to indicate the concentration of a specific substance when no separate volume is used means: solids/solid (wt./wt.)%; solid/liquid (weight/volume)%; and liquid/liquid is (volume/volume)%.
Culture method
Preparation of cellulose tablets from acetic acid bacteria according to the prior art (comparative example 1)
The amount of biocellulose synthase (BCS), which is an enzyme that biosynthesizes bacterial cellulose, was expressed as the amount of proliferation of Acetobacter gluconicum KOSS-15 (Komagataibacterium Rheiceus KOSS-15) in the logarithmic growth phase (exotic phase). Therefore, the culture process of Acetobacter gluconicum KOSS-15 (accession No. KCCM12270P) used in common in the examples later was carried out in 2-step culture of the preculture step and the main culture step. To perform the preculture, 1.0X 10 inoculation of 1.0X 10 medium containing 1% glucose, 1% yeast extract, 1% magnesium sulfate, 0.02% calcium chloride, 2% fermented alcohol8The strain of CFU/ml Acetobacter gluconicum KOSS-15 (with the preservation number of KCCM12270P) is stirred at 80-160 rpm under the temperature condition of 30 ℃ and cultured for 48 hours with shaking. To carry out the main culture, the above-mentioned preculture final solution was inoculated in a main culture medium containing 1% of glucose, 1% of granulated sugar, 0.1% of proline, 0.02% of magnesium sulfate, 0.1% of sodium acetate and 0.2% of acetic acid in an amount of 10% (v/v) based on the volume of the main culture, and the mixture was cultured with stirring at 80rpm and an aeration rate of 0.3v/v/m for 48 hours. The final culture solution of Acetobacter gluconicum KOSS-15 was inoculated into a culture medium, mixed, dispensed onto a tray, and subjected to static culture at 0rpm and 0.3vvm for 3 days at a rate of 5 to 10% (v/v) relative to the culture medium, thereby inducing transformation of a bacterial cellulose sheet. After completion of the culture, 0.1% NaOH was added thereto, and the mixture was washed and dehydrated.
Method for producing Mixed bacterial cellulose tablet of the present invention (preparation example)
Different from the growth of the Acetobacter gluconicum KOSS-15 strain used in comparative example 1, the strain was inoculated with a lactic acid bacterium culture solution stored in an MRS liquid medium so as to be 10% (v/v), and the culture was allowed to stand at 37 ℃ for 24 hours, and the 24-hour standing culture was an overnight (overnight) culture. The final cell count was maintained at 1.0X 1010Level of CFU/ml.
The final main culture solution of Acetobacter gluconicum KOSS-15 and the final lactic acid bacterium culture solution were inoculated into a culture medium, mixed, and dispensed onto a tray, and the mixture was subjected to static culture at 0rpm and 0.3vvm for 72 hours in an amount of 5 to 10% (v/v) and 0.5 to 2% (v/v) relative to the culture medium, thereby inducing transformation (generation) of a mixed bacterial cellulose sheet. The obtained bacterial cellulose mixed sheet was finally prepared by washing with 0.1% NaOH, dehydrating the washed mixture, and then sterilizing the mixture under pressure at 121 ℃ and 1.2MPa for 60 minutes.
Example 1: screening mixed fermentation lactic acid bacteria
When bacterial cellulose is produced solely by Acetobacter gluconicum KOSS-15 which is an Aerobic bacterium (Aerobic bacterium), a predetermined number of bacteria is required. Therefore, Acetobacter gluconicum KOSS-15 was proliferated in a closed fermentation environment (closed system) for proliferation, and switched to an uncovered tray environment (open system) to produce bacterial cellulose by whole cell reaction (whole cell reaction). Therefore, in the whole cell reaction step for producing bacterial cellulose, the activity of bacterial cellulose-producing enzymes (bacterial cellulose synthase) or biocellulose synthase (BCS) is reduced due to the falling of external bacteria and the change of dissolved oxygen and the like caused by the uncovered tray environment, and it is difficult to produce a complete sheet (perfect sheet). Therefore, in the open whole cell reaction step of producing bacterial cellulose using a heterotypic lactic acid bacterium sharing the pentose acid pathway (PPP) for propagation using the acetobacter gluconicum KOSS-15 strain, it is important to control metabolism not to progress toward the cell growth side, and to provide an acidic environment that maximizes the activity of the Bacterial Cellulose Synthase (BCS) as lactic acid and acetic acid produced by the lactic acid bacterium itself of the anaerobic bacterium (anaerobic bacterium), thereby screening of the lactic acid bacterium designed for the mixed step of contributing to the cellulose synthesis reaction of the Bacterial Cellulose Synthase (BCS).
In order to select lactic acid bacteria that were co-fermented with the Acetobacter gluconicum KOSS-15 strain of the present invention, the present inventors purchased 8 kinds of lactic acid bacteria from SK bioland own cell bank (cell bank). The mixed bacterial cellulose tablets were produced by mixed fermentation of each lactic acid bacterium and Acetobacter gluconicum KOSS-15 strain by preparative examples, and the yields were compared by measuring the dry weight. As a control group, bacterial cellulose produced by using Acetobacter gluconicum KOSS-15 strain alone was used (comparative example 1), and among them, lactic acid bacteria having the highest yield were selected. The results are shown in Table 1.
TABLE 1
Screening mixed fermentation lactic acid bacteria
As shown in Table 1, when the fermentation mixture was mixed with a combination of Acetobacter gluconicum KOSS-15+ Lactobacillus rhamnosus SKB1253 (accession No. KCCM12452P) and a combination of Acetobacter gluconicum KOSS-15+ Lactobacillus plantarum SKB1234 (accession No. KCCM12450P), the amount of bacterial cellulose was most excellent. These exhibited about a 2.5-fold increase in yield compared to bacterial cellulose produced using the acetobacter aceti KOSS-15 alone. In the following examples, conditional experiments were performed using lactobacillus rhamnosus SKB1253 as a mixed fermentation lactic acid bacterium.
Example 2: effect of Mixed fermentation temperature on bacterial cellulose production
The suitable proliferation temperature of the gluconacetobacter gluconicum KOSS-15 strain developed by SK BIOLAND is lower than 25-30 ℃. In contrast, Lactobacillus rhamnosus (Lactobacillus rhamnosus) is known to grow at 30-37 ℃ above it. In the fermentation step, lactic acid and acetic acid produced by lactobacillus rhamnosus are used to maintain the pH range (pH 3-4) required for producing bacterial cellulose. Therefore, it was confirmed that the amount of the produced bacterial cellulose was higher when the reaction temperature range of the bacterial cellulose-producing enzyme (BCS) was set higher than the growth temperature of conventional Acetobacter gluconicum KOSS-15.
The Lactobacillus rhamnosus screened in example 1 was used as a mixed lactic acid bacterium, and mixed fermentation was performed by the method of the preparation examples under the conditions of 25 ℃, 28 ℃, 30 ℃, 33 ℃ and 37 ℃. Each of the results was compared with the amount of the bacterial cellulose produced in comparative example 1, and it was confirmed whether the bacterial enzyme was stable in the temperature range and the pH value required for fermentation was maintained. The results are shown in table 2 and fig. 1.
TABLE 2
Influence of Mixed fermentation temperature
As shown in Table 2, the dry weight of the sheet was 0.7 to 0.79 (g/18.15X 18.15 cm) at a temperature of 25 to 33 ℃2) Showing temperature stability. However, under the temperature condition of 37 ℃, the amount of the bacterial cellulose was reduced by 46.8% with respect to the maximum value. In general, it is known that acetic acid bacteria can be produced by optimizing bacterial cellulose under a temperature condition of 25 to 30 ℃, but when bacterial cellulose is produced by lactobacillus rhamnosus and a mixed fermentation system, the fermentation temperature can be raised to 33 ℃ at most. After that, the temperature condition was set to 30 ℃ in the examples to conduct the condition experiment.
Example 3: effect of initial pH of mixed fermentation on the amount of Bacterial Cellulose (BC) produced, pH, and quality of cellulose pieces
Acetobacter gluconicum KOSS-15 is an acetic acid bacterium which produces acetic acid (acetic acid) as a metabolite during its own proliferation. Therefore, all enzymes in the bacterial cells were photographed in an acidic external environment. However, since the falling of bacteria cannot be controlled in an open system (open system) for producing bacterial cellulose, the pH value is increased, the activity of an enzyme for producing bacterial cellulose is lowered, and incomplete bacterial cellulose pieces (incomplete sheets) having inconsistent thickness and quality are produced, thereby causing industrial damage. Therefore, if the pH is maintained under an acidic condition that does not increase even when the mixed fermentation is exposed to an environment such as a falling bacteria, a thin perfect cellulose sheet (perfect sheet) can be produced, and industrial loss can be prevented.
Therefore, in order to confirm this, the present inventors adjusted the initial pH of the reaction medium to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 under the conditions of the preparation examples, and then mixed-fermented at 30 ℃ for 72 hours, and compared the production amount, the final pH and the integrity of cellulose with comparative example 1. The results are shown in table 3 and fig. 2.
TABLE 3
Effect of initial pH on Mixed fermentation
As shown in table 3, when the fermentation broth was mixed with acetobacter gluconicum KOSS-15+ lactobacillus rhamnosus, the bacterial cellulose formed incomplete cellulose pieces at an initial pH of 3.0 or less, and formed complete cellulose pieces at an initial pH of 5.0 or more, but the amount of production was reduced by 40% or more compared to the condition of initial pH of 4.0. Therefore, it was confirmed that the amount produced was the highest and a perfect cellulose sheet was formed under the condition that the initial pH was 4.0. In the following examples, conditional experiments were performed using an initial pH of 4.0.
Example 4: effect of Mixed fermentation glucose concentration
Acetic acid bacteria are known to use a complex sugar of a monosaccharide and a disaccharide such as glucose, fructose, and fructose to additionally supply consumption of glucose converted into bacterial cellulose by a disaccharide. In this method, if bacteria fall and are mixed into the open fermentation system within a fermentation time of 72 hours, the pH value rises due to the consumption of glucose or the like, and the yield is lowered, thereby inducing the formation of incomplete cellulose pieces.
Therefore, in order to optimize the amount of substrate that can maintain the maximum amount of bacterial cellulose production when the amount of substrate alone is higher than that of enzyme during the fermentation time, the present inventors have changed the concentration of glucose to 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0, respectively, and have confirmed the amount and quality of bacterial cellulose pieces produced and the pH of the fermentation system. The results are shown in table 4 and fig. 3.
TABLE 4
Effect of Mixed fermentation glucose concentration
As shown in Table 4, when Acetobacter gluconicum KOSS-15+ Lactobacillus rhamnosus was mixedly fermented, bacterial cellulose was not produced in the absence of glucose as a carbon source, and the amount of production of bacterial cellulose increased as the concentration of glucose increased. Wherein, when the glucose concentration is 2-4%, the production amount of the cellulose tablet is optimal, and a complete cellulose tablet with excellent quality is formed. Further, it was confirmed that when the glucose concentration was less than 2% or 5% or more, the resulting value of the decrease in the amount of production was shown instead. Therefore, under the condition that the concentration of glucose is adjusted to be 2-4%, the mixed enzyme reaction in the same substrate is maximized, both acetic acid bacteria and lactic acid bacteria can utilize the metabolic process smoothly, and bacterial cellulose can be generated to the maximum extent in an open type fermentation system.
In the following examples, conditional experiments were performed with an initial glucose concentration of 2%.
Example 5: linearity of bacterial cellulose production and lactic acid bacteria cell trapping rate according to mixed fermentation time
The amount required for efficacy may vary depending on the lactic acid bacteria effective for the skin microbiome. Therefore, it is necessary to set the trapping rate of lactic acid bacteria in the bacterial cellulose tablet to be different depending on the kind of lactic acid bacteria and the effect, and to adjust the thickness of the mixed bacterial cellulose tablet depending on the fermentation time. In order to solve the above two industrial problems, the present inventors have confirmed the amount of time-lapse production when mixed fermentation is performed by mixing and fermenting lactobacillus rhamnosus screened in example 1 for 24 hours, 48 hours, 72 hours, etc. in comparative example 2 and comparing the amounts with those of comparative example 1. Then, the number of bacteria remaining after the trapping reaction was subtracted from the total number of bacteria according to the following formula, and the trapping rate of lactic acid bacteria was calculated as a percentage, and a direct counting method using a hemocytometer was used for each number of bacteria.
Lactic acid bacteria trapping rate (%) of bacterial cellulose (1-number of remaining bacteria/total bacteria) × 100 (%)
The results are shown in table 5 and fig. 4.
TABLE 5
Lactic acid bacteria cell trapping rate according to mixed fermentation time
As shown in Table 5 and FIG. 4, it was confirmed that the cellulose production of the cellulose flakes increased with the lapse of the culture time in the case of the mixedly fermenting Acetobacter gluconicum KOSS-15+ Lactobacillus rhamnosus, and the highest production of the bacterial cellulose was exhibited in the case of the fermentation for 72 hours, and the cellulose production was linear with the fermentation time. Further, it was confirmed that the lactic acid bacteria trapping rate also tended to increase with the increase in the amount of bacterial cellulose produced. The present inventors have made this because, as the amount of the bacterial cellulose generated increases, more pores are formed, and the lactic acid bacteria cells are trapped between the formed pores, so that the trapping rate of lactic acid bacteria increases.
Example 6: comparison of skin microbiome Steady states
In order to compare the steady state (homeostasis) maintenance effects associated with the balance regulation of the skin microbiome of the lactic acid bacteria mixed bacterial cellulose tablet of the present invention, the proliferation effects of staphylococcus epidermidis as skin beneficial bacteria and staphylococcus aureus as skin harmful bacteria on the lactic acid bacteria mixed bacterial cellulose tablet of the present invention were performed.
Specifically, a 1% (v/v) glycerol stock solution containing the beneficial skin bacteria and the harmful skin bacteria is inoculated to a Trypticase Soy Broth (TSB), and the mixture is shake-cultured at 37 ℃ for 18 to 20 hours, and the resulting culture medium is diluted with physiological saline to adjust the o.d.600nm to 0.02 to 0.04 (about 1 × 10)7CFU/ml). At a concentration of 3% (w/v) toThe lactic acid bacteria mixed tablet of the present invention is added to physiological saline. 9.9ml of the physiological saline solution to which 3% of the above mixed tablet was added was inoculated with 100. mu.l each of the dilutions of the above-mentioned beneficial bacteria and harmful bacteria (final bacterial count 1X 10)5CFU/ml), the number of viable cells at 0 th, 3 rd and 6 th days after inoculation was diluted by 10-fold, and 1ml was inoculated in tryptic soy agar medium (TSA), respectively, and cultured on a plate at 37 ℃ for 24 hours. The survival rate of each bacterium was calculated by the following formula 2. As a control group, a bacterial cellulose tablet prepared using only the acetic acid bacteria of comparative example 1 was used.
Survival (%) - (number of viable cells in experimental group on day n)/(day 0)
Number of living cells in distilled water) × 100
As shown in fig. 5a and 5b, it was confirmed that the survival rate of the skin beneficial bacteria of the control group was sharply and decreased over time, whereas the survival rate of the skin beneficial bacteria of the mixed bacterial cellulose of the present invention tended to be decreased slowly. This is because the influence of the relative decrease in the air permeability of the bacterial cellulose attachment is drastically reduced in the control group, however, in the case of the mixed bacterial cellulose sheet, the lactic acid bacteria trapped in the cellulose contribute to the growth of the beneficial bacteria, and the death rate of the beneficial bacteria becomes relatively slow.
As shown in fig. 6a and 6b, it was confirmed that the survival rate of the bacterial cellulose against harmful bacteria was measured over time, and in the case of the control group, the growth of harmful bacteria was not affected, but rather, the growth of harmful bacteria was derived over time. On the contrary, when the mixed bacterial cellulose tablet of the present invention is used, the lactic acid bacteria cell component trapped by the cellulose tablet has an effect of inhibiting the proliferation of harmful bacteria.
Example 7: electron micrograph (FE-SEM) comparison of Mixed bacterial cellulose tablets of the invention
The completion of the fifth-generation mixed bacterial cellulose tablet of the present invention containing lactic acid bacteria cells was confirmed by electron micrograph. Fig. 7 is a graph showing the results of comparing a cellulose tablet of bacteria that produce acetic acid bacteria alone as a control group with a mixed tablet of the present invention prepared by mixing fermented acetic acid bacteria and lactic acid bacteria. In the mixed tablet of the present invention, lactic acid bacteria were not confirmed due to various bacteria and foreign substances before washing, and it was confirmed that lactic acid bacteria were trapped in the bacterial cellulose tablet after washing.
Example 8: mass production of mixed bacterial cellulose tablets
Based on the step of producing the fifth-generation mixed bacterial cellulose tablets of the present invention completed in the above-described examples, mass production of 1 ton units was performed. Fig. 8 shows a process diagram. The integrity of the mixed bacterial cellulose sheet of the present invention and the bacterial cellulose sheet prepared by culturing KOSS-15 strain alone was confirmed visually (fig. 9).
As shown in fig. 9, it was confirmed that the mixed sheet of the invention (right) was thin and exhibited a uniform whole cellulose sheet, and the cellulose sheet produced by culturing KOSS-15 strain alone was relatively uneven in thickness and low in quality, as compared to the bacterial cellulose produced by KOSS-15 acetic acid bacteria alone (left).
Example 9: physical property comparison of Mixed bacterial cellulose tablets
The physical properties related to the quality of the 5 th-generation mixed bacterial cellulose tablet of the present invention prepared by the above-described example were confirmed by the following experiments. The test group used the mixed bacterial cellulose tablets of the present invention, and the control group used bacterial cellulose tablets prepared without cocultivation with lactic acid bacteria.
9-1, measuring the thickness, the water content and the dry weight of the cellulose sheet
The inventors measured the thickness, water content and dry weight of the cellulose sheet (cf. Biotechnology Letters (2005)27: 1435-.
The water content (WHC) of the cellulose sheet was measured according to the following formula 3.
Water content (WHC) ═ weight of (hydrous cellulose sheet)Quantity (W)wet) Weight of dry cellulose sheet (W)dry) Weight of dry cellulose tablet (W)dry)
Prior to measuring the above water content, each bacterial cellulose sheet was prepared to 5000mm2A round test piece (about 79.8mm in diameter) was immersed in distilled water for 15 hours or more. Removing the cellulose sheet soaked in the above distilled water for 15 hr or more, and throwing off for 2 times in a semicircle or 4 times at 90 ° to remove the water, and measuring the weight (W) of the above hydrous cellulose sheetwet). The weight (W) of the dried cellulose sheet was measured after drying at 85 ℃ overnight or at 105 ℃ for 5 hours or moredry)。
The measured weight (W) of the above sample was measured according to the following 4dry) The same area value was converted and the dry weight (basis weight per unit area) of the cellulose sheet was measured.
Basis weight per unit area (g/m)2) Dry cellulose weight (g/5000 mm)2)×200
After each of the above test pieces was immersed in distilled water for 15 hours or more, the thickness of each cellulose piece was measured by a micrometer.
The results are shown in Table 6.
TABLE 6
As shown in table 6, it was confirmed that the dry weight of the mixed bacterial cellulose sheet of the present invention was reduced compared to the bacterial cellulose sheet prepared by the conventional method, but the thickness, wet weight and water content were greatly increased. It is predicted that the reason for the increase in the water content and the increase in the wet weight and thickness relative to the weight of the cellulose is due to the interaction with lactic acid bacteria
The porosity of the cellulose sheet is increased by symbiotic culture. Water content increasing meterIt is shown that the cellulose sheet has a soft and fluffy feeling in the cosmetic industry or moisture where the cellulose sheet can absorb much water, and if the cellulose sheet has many pores.
9-2. measuring the maximum load and elongation of the cellulose sheet
The present inventors measured the maximum load and elongation of the cellulose sheet.
First, a test piece as shown in fig. 12 was prepared.
In width (b): 15mm
Thickness (h): less than or equal to 1mm, and before testing tensile strength, measuring by using micrometer
Gauge length (L0): 50.0mm
Total length: 90mm preparation
According to the above specifications, 2 test pieces cut in the transverse direction and 2 test pieces cut in the longitudinal direction in 1 sheet of face film were prepared.
The measuring device adjusts the clamping distance to the gauge distance (L)0)50mm and prepared by inputting test conditions (Separation rate)50mm/min, Preload (Preload) 0.1N).
The test piece prepared in the above was placed with filter paper (HM No.20) at the end of measurement and held according to the gauge indication. The reason for placing the filter paper is that, in the case of merely holding the facial film, slippage occurs, and when stress occurs, the holding distance of the measuring device is adjusted to solve the above-described problem. The name and size (dimension) of the test piece were entered for testing. The width and length were made equal, the thickness before gripping was measured and input, and the test was repeated until 5 data were obtained for each test piece according to the orientation. At this time, a sample broken during the holding process was discarded (refer to ASTM D882-Standard Test Method for Tensile Properties of Thin Plastic Sheeting, KS K ISO 9073 textile-nonwoven Test Method-section 3: measurement of Tensile Strength and elongation)
The elongation of the cellulose sheet was calculated from the following formula 5.
Elongation (%) (L-L) length until breakage0) Initial gauge length (L0). times.100
The results are shown in table 7 and fig. 10.
TABLE 7
As shown in Table 7 and FIG. 10 (A: conventional method, B: symbiotic culture), it is understood that in the case of the mixed bacterial cellulose sheet of the present invention, the maximum load and elongation of the cellulose sheet are both greatly increased by symbiotic culture of lactic acid bacteria. This is because the internal structure of the cellulose sheet is changed due to the presence of lactic acid bacteria, and it is predicted that entanglement between celluloses increases. When the elongation is increased, the cellulose sheet has an excellent touch, and the strength of the cellulose sheet is increased, so that the cellulose sheet can be used without tearing. Further, the difference in physical properties according to the orientation of the test piece is not significant.
9-3 comparison of the uniformity of thickness or Density of cellulose sheets
The present inventors compared the uniformity of the thickness or density of the cellulose sheet according to the number of days of cultivation. As shown in part a of fig. 11, when only bacterial cellulose-producing microorganisms were cultured by the existing method, a uniform cellulose sheet was not prepared on day 1, and thus, the cellulose sheet had a difference in color. However, as shown in part B of fig. 11, it was confirmed that when the bacterial cellulose-producing bacteria and the lactic acid bacteria were symbiotically cultured, a uniform cellulose sheet was obtained from day 1.
And (3) preserving the sample: acetobacter glucoraphanus KOSS15
The preservation unit: KCCM-Korean Collection of microorganisms
And (4) storage address: korea, Seoul, West Damen, Hongji-2 street, 45 Yulin mansion
The preservation number is: KCCM12270P
Preservation day: 20180530
And (3) preserving the sample: lactobacillus plantarum SKB1234 (Lactobacillus plantarum) A
The preservation unit: KCCM-Korean Collection of microorganisms
And (4) storage address: korea, Seoul, West Damen, Hongji-2 street, 45 Yulin mansion
The preservation number is: KCCM12450P
Preservation day: 20190305
And (3) preserving the sample: lactobacillus rhamnosus SKB1253
The preservation unit: KCCM-Korean Collection of microorganisms
And (4) storage address: korea, Seoul, West Damen, Hongji-2 street, 45 Yulin mansion
The preservation number is: KCCM12452P
Preservation day: 20190305
Claims (21)
1. A preparation method of bacterial cellulose containing probiotics is characterized by comprising the step of mixing and fermenting bacterial cellulose generating bacteria and probiotics.
2. The method for preparing bacterial cellulose containing probiotics according to claim 1, wherein the bacteria cellulose-producing strain is Acetobacter gluconicum KOSS15 strain with accession number KCCM 12270P.
3. The method of claim 1, wherein the probiotic bacteria are lactic acid bacteria selected from the group consisting of lactobacillus casei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus curvatus, lactobacillus sake, lactobacillus acidophilus, bifidobacterium longum, and enterococcus faecalis.
4. The method of claim 3, wherein the lactic acid bacteria is Lactobacillus rhamnosus, Lactobacillus plantarum, or a combination thereof.
5. The method of claim 4, wherein the lactic acid bacteria is Lactobacillus rhamnosus SKB1253 strain, Lactobacillus plantarum SKB1234 strain or a combination thereof, the Lactobacillus rhamnosus SKB1253 strain has a deposit number of KCCM12452P, and the Lactobacillus plantarum SKB1234 strain has a deposit number of KCCM 12450P.
6. The method of claim 1, wherein the culturing is performed on a tray without a cover.
7. The method for preparing bacterial cellulose containing probiotics according to claim 1, wherein the initial pH of the culture medium for the cultivation is 3.5 to 9.
8. The method for preparing bacterial cellulose containing probiotics according to claim 1, wherein the final pH value of the culture medium for the cultivation is 3-4.5.
9. The method for preparing bacterial cellulose containing probiotics according to claim 1, wherein the culturing is performed at a temperature of 25-35 ℃.
10. The method for producing bacterial cellulose containing probiotics according to claim 1, wherein the glucose concentration of the culture medium for the culture is 0.5-5% (w/v).
11. The method for preparing bacterial cellulose containing probiotics according to claim 1, wherein the culturing is performed for 24 hours or more.
12. A probiotic mixed dough sheet, characterized by comprising a bacterial cellulose sheet prepared by mixing a fermentation bacterial cellulose-producing bacterium and a probiotic.
13. The probiotic mixed dough sheet of claim 12, wherein the probiotic is a lactic acid bacteria selected from the group consisting of lactobacillus casei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus curvatus, lactobacillus sake, lactobacillus acidophilus, bifidobacterium longum and enterococcus faecalis.
14. The probiotic mixed facial patch of claim 13, wherein the lactic acid bacteria is lactobacillus rhamnosus, lactobacillus plantarum or a combination thereof.
15. The probiotic mixed facial patch as claimed in claim 14, wherein the lactic acid bacteria is lactobacillus rhamnosus SKB1253 strain, lactobacillus plantarum SKB1234 strain or a combination thereof, the lactobacillus rhamnosus SKB1253 strain has a deposit number of KCCM12452P, and the lactobacillus plantarum SKB1234 strain has a deposit number of KCCM 12450P.
16. The probiotic mixed facial patch according to claim 12, wherein the bacterial cellulose patch is used to regulate skin microbiota.
17. The probiotic mixed facial patch according to claim 12, wherein the bacterial cellulose patch is used to increase the survival rate of staphylococcus epidermidis as a skin beneficial bacterium.
18. The probiotic mixed facial patch according to claim 12, wherein the bacterial cellulose patch is used for inhibiting the proliferation of staphylococcus aureus, which is a skin harmful bacterium.
19. The probiotic mixed facial mask sheet according to claim 12, wherein the thickness, water content and maximum load of the bacterial cellulose sheet are increased as compared with a facial mask sheet containing bacterial cellulose prepared by fermenting bacterial cellulose-producing bacteria alone.
20. A mask for making up or making up the skin, comprising the probiotic mixed-mask sheet according to any one of claims 12 to 19.
21. A mask pack for making up or making up the skin, comprising the mask sheet according to any one of claims 12 to 19 and a cosmetic lotion.
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| KR20170085284A (en) * | 2016-01-14 | 2017-07-24 | (주) 본느 | Hydrogel mask pack containing biocellulose |
| CN108018240A (en) * | 2017-12-26 | 2018-05-11 | 华海科技股份有限公司 | A kind of middle gluconacetobacter and its application |
| KR101970439B1 (en) * | 2018-09-17 | 2019-04-18 | 에스케이바이오랜드 주식회사 | A novel Komagataeibacter rhaeticus Strain and Method for Producing Cellulose Sheet Using the Same |
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| KR20170085284A (en) * | 2016-01-14 | 2017-07-24 | (주) 본느 | Hydrogel mask pack containing biocellulose |
| CN108018240A (en) * | 2017-12-26 | 2018-05-11 | 华海科技股份有限公司 | A kind of middle gluconacetobacter and its application |
| KR101970439B1 (en) * | 2018-09-17 | 2019-04-18 | 에스케이바이오랜드 주식회사 | A novel Komagataeibacter rhaeticus Strain and Method for Producing Cellulose Sheet Using the Same |
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