Phospholipid-based surfactant using skin flora and preparation method thereof

Abstract

The present invention relates to a phospholipid-based surfactant prepared using skin flora in an environmentally friendly and human-friendly manner, and to a method for preparing the same. The present invention can provide an environmentally friendly and human friendly composition that can replace synthetic surfactants. In addition, the surfactant disclosed by the invention can be widely applied to the fields of foods, detergents, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to a phospholipid-based surfactant prepared in an environmentally friendly and human-friendly manner using skin flora, and a method for preparing the surfactant. This application claims priority to Korean Patent Application No. 10-2023-0070150, filed May 31, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] The human body serves as a habitat for a variety of microorganisms, which form symbiotic relationships with the host and influence its functions. Among these, the skin microbiota consists of microorganisms living on the skin's surface, primarily distributed in the outermost layer of the epidermis and the upper part (upper layer) of hair follicles. There are approximately 1000 species of skin microorganisms. Among these, aerobic microorganisms can secrete lipases that utilize lipids from the epidermis.

[0003] Phospholipids are a fundamental component of cell membranes and are naturally found in animals, plants, and microorganisms. Generally, phospholipids in cell membranes can be extracted by disrupting the cells. However, due to the chemical and physical disruption methods used in this process, it is difficult to consider phospholipids obtained in this way to be environmentally friendly and human-friendly. The composition of extracellular phospholipids in Gram-positive aerobic bacteria has been studied, but this research involved analyzing extracellular vesicles contained in biofilms cultured on the surface, and extracellular phospholipids obtained through liquid culture without cell disruption have not yet been investigated.

[0004] Generally, phospholipids are used as emulsifiers, and the functional component with phospholipid groups is called lecithin. The phospholipids contained in commercially available lecithin mainly include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA).

[0005] In recent years, there has been a growing demand for environmentally friendly and human-friendly products in sectors such as food, detergents, and cosmetics. Therefore, research is needed to replace synthetic surfactants with environmentally friendly and human-friendly surfactants. Summary of the Invention

[0006] Technical issues The object of this invention is to provide an environmentally friendly and human-friendly phospholipid-based surfactant by utilizing the skin microbiota. This application claims priority to Korean Patent Application No. 10-2023-0070150, filed on May 31, 2023, the entire contents of which are incorporated herein by reference.

[0007] Technical solution To address the aforementioned problems, this invention provides a phospholipid-based surfactant prepared from skin flora microorganisms with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology).

[0008] In one embodiment, the surfactant may comprise a phospholipid with or without an amino or glycosyl group. Specifically, the surfactant may comprise one or more of phosphatidylglycerol, ceramide phosphoethanolamine, phosphatidylethanolamine, lysophosphatidylethanolamine, and branched-chain fatty acid esters of hydroxy fatty acids.

[0009] According to another embodiment of the present invention, a method for preparing a phospholipid-based surfactant is provided, comprising: The seed culture step involves culturing the skin flora strain with the preservation number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology) to prepare seed cultures. The pre-culture step involves culturing seed cultures in a medium containing vegetable oil to prepare pre-cultures. The main fermentation step involves adding the pre-culture to a culture medium containing vegetable oil to prepare the main fermentation product; The steps for separating the precipitate from the fermentation products; and The step of extracting phospholipids from precipitates.

[0010] According to one implementation scheme, the culture medium in the seed culture step may contain casein, soybean, glucose, sodium chloride, and dipotassium phosphate. Specifically, the culture medium in the seed culture step may contain 5 g / L to 30 g / L casein, 1 g / L to 10 g / L soybean, 1 g / L to 6 g / L glucose, 1 g / L to 10 g / L sodium chloride, and 1 g / L to 6 g / L dipotassium phosphate.

[0011] According to one embodiment, the culture medium in the pre-culture step may contain soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, and vegetable oil. Specifically, the culture medium in the pre-culture step may contain 0.01 g / L to 3 g / L soybean, 0.1 g / L to 5 g / L yeast extract, 0.1 g / L to 5 g / L glycerol, 1 g / L to 15 g / L monopotassium phosphate, 1 g / L to 10 g / L dipotassium phosphate, and 10 mL / L to 100 mL / L vegetable oil.

[0012] According to one embodiment, the culture medium in the main fermentation step may contain soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, magnesium sulfate, potassium nitrate, calcium chloride, and vegetable oil. Specifically, the culture medium in the main fermentation step may contain 0.01 g / L to 3 g / L soybean, 0.1 g / L to 5 g / L yeast extract, 0.01 g / L to 5 g / L glycerol, 1 g / L to 10 g / L monopotassium phosphate, 1 g / L to 10 g / L dipotassium phosphate, 0.1 g / L to 5 g / L ammonium sulfate, 0.01 g / L to 5 g / L magnesium sulfate, 0.01 g / L to 3 g / L potassium nitrate, 0.01 g / L to 3 g / L calcium chloride, and 10 mL / L to 800 mL / L vegetable oil.

[0013] According to one embodiment, the seed culture step may include culturing under aerobic conditions at 25°C to 35°C, the pre-culture step may include introducing 10 g / L to 500 g / L of the seed culture and culturing under aerobic conditions at 25°C to 35°C, and the main fermentation step may include introducing 10 g / L to 500 g / L of the pre-culture and culturing under aerobic conditions at 25°C to 35°C.

[0014] According to one embodiment, the method may include: in the seed culture step, when the absorbance measured at a wavelength of 600 nm is 0.05 to 0.2, a step of subculturing to a pre-culture medium; In the pre-culture step, when the absorbance measured at a wavelength of 600 nm is 0.2 to 1, the culture is subcultured to the main fermentation step; or The primary fermentation process is completed 60 to 150 hours after the start of the primary fermentation.

[0015] According to one implementation, the step of separating the precipitate may include centrifuging the fermentation product to recover the precipitate.

[0016] According to one embodiment, the phospholipid extraction step may include mixing the recovered precipitate with a solvent to obtain a filtrate, and concentrating the filtrate.

[0017] Specific details of other embodiments of the present invention are contained in the following detailed description.

[0018] Invention Effects The phospholipid-based surfactants utilizing skin microbiota according to the present invention can replace synthetic surfactants. The surfactants of the present invention can be applied in fields such as food, detergents, and cosmetics to provide environmentally friendly and human-friendly products. Attached Figure Description

[0019] Figure 1A photograph of the filtrate obtained according to the example.

[0020] Figure 2 Photographs of phospholipid-based surfactants recovered according to examples.

[0021] Figure 3 The image shows the TLC chromatogram of the phospholipid-based surfactants in the examples.

[0022] Figure 4 The chromatogram is the result of TLC analysis based on fermentation time.

[0023] Figure 5 Visual observation photographs showing the difference in the presence or absence of phospholipids in emulsions according to the embodiments.

[0024] Best way to carry out the invention This invention can be modified in various ways and has multiple implementation schemes, which will be illustrated and described in detail below. However, this is not intended to limit the invention to the specific implementation schemes, but should be understood to cover all variations, equivalents, or substitutions included within the spirit and scope of the invention. In explaining this invention, if a detailed description of relevant known technologies is deemed likely to obscure the spirit of the invention, such a detailed description will be omitted.

[0025] The following will describe in detail the phospholipid-based surfactants utilizing skin flora and their preparation methods according to the present invention.

[0026] The phospholipid-based surfactant of the present invention can be prepared by microorganisms (e.g., skin flora). Specifically, the present invention can utilize the genus Epidermophyton (…). Epidermidibacterium keratini sp The strain, specifically the strain with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology).

[0027] Phospholipid-based surfactants can be produced by culturing and fermenting skin microbiota under specific conditions. Phospholipid-based surfactants are environmentally friendly and human-friendly, thus they can overcome various shortcomings and problems of synthetic surfactants.

[0028] According to one aspect, the present invention provides a phospholipid-based surfactant prepared from skin flora microorganisms with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology). The surfactant of the present invention may comprise phospholipids with or without amine or glycosyl groups. For example, it may include glycerophospholipids, sphingolipids, glycerophosphoethanolamine, fatty acid esters, sterols, sphingosine bases, etc. Specifically, the surfactant of the present invention may include one or more of phosphatidylglycerol (PG), ceramide phosphoethanolamine (sphingolipid, Cer-PE), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), and fatty acid derivatives (e.g., branched-chain fatty acid esters of hydroxy fatty acids (FAHFA)).

[0029] According to another embodiment of the present invention, a method for preparing a phospholipid-based surfactant is provided, comprising: a seed culture step, namely, culturing a skin flora strain with accession number KCTC15741BP (Korea National Institute of Bioscience and Biotechnology, Bioresource Center) to prepare a seed culture; a pre-culture step, namely, culturing the seed culture in a culture medium containing vegetable oil to prepare a pre-culture; a main fermentation step, namely, adding the pre-culture to a culture medium containing vegetable oil to prepare a main fermentation product; a step of separating a precipitate from the fermentation product; and a step of extracting phospholipids from the precipitate.

[0030] The vegetable oils used in this invention are not particularly limited, as long as they are non-cytotoxic. Specifically, for example, the vegetable oils may include, but are not limited to, one or more edible or human-friendly oils, such as macadamia oil, sunflower seed oil, grape seed oil, canola seed oil, rice bran oil, olive oil, soybean oil, argan oil, brown rice oil, perilla seed oil, sesame oil, almond oil, peanut oil, corn oil, red ginseng oil, avocado oil, coconut oil, rosehip oil, vitamin tree seed oil, shea butter, oil palm oil, bergamot oil, camellia seed oil, safflower seed oil, almond kernel oil, evening primrose seed oil, castor seed oil, green tea seed oil, and meadowfoam seed oil.

[0031] According to one embodiment, the culture medium in the seed culture step may contain casein, soybean, glucose, sodium chloride, and dipotassium phosphate. Specifically, the culture medium in the seed culture step may contain 5 g / L to 30 g / L (e.g., 10 g / L to 25 g / L, or 15 g / L to 20 g / L) of casein, 1 g / L to 10 g / L (e.g., 2 g / L to 8 g / L, or 2 g / L to 4 g / L) of soybean, 1 g / L to 6 g / L (e.g., 1 g / L to 4 g / L, or 2 g / L to 3 g / L) of glucose, 1 g / L to 10 g / L (e.g., 2 g / L to 8 g / L, or 4 g / L to 6 g / L) of sodium chloride, and 1 g / L to 6 g / L (e.g., 2 g / L to 5 g / L, or 2 g / L to 3 g / L) of dipotassium phosphate.

[0032] According to one implementation plan, the culture medium in the pre-culture step may contain soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, and vegetable oil. Specifically, for example, the culture medium in the pre-culture step may contain 0.01 g / L to 3 g / L (e.g., 0.05 g / L to 2 g / L, or 0.05 g / L to 1 g / L) of soybean, 0.1 g / L to 5 g / L (e.g., 0.1 g / L to 3 g / L, or 0.5 g / L to 2 g / L) of yeast extract, 0.1 g / L to 5 g / L (e.g., 0.1 g / L to 3 g / L, or 0.5 g / L to 2 g / L) of glycerol, 1 g / L to 15 g / L (e.g., 1 g / L to 10 g / L, or 3 g / L to 6 g / L) of monopotassium phosphate, 1 g / L to 10 g / L (e.g., 1 g / L to 8 g / L, or 2 g / L to 6 g / L) of dipotassium phosphate, and 10 mL / L to 100 mL / L (e.g., 20 mL / L to 80 mL / L, or 30 mL / L to 100 mL / L) of glycerol, or 1 g / L to 15 g / L (e.g., 1 g / L to 10 g / L, or 3 g / L to 6 g / L) of monopotassium phosphate, or 1 g / L to 10 g / L (e.g., 20 mL / L to 80 mL / L, or 30 mL / L to 100 mL / L) of monopotassium phosphate, or 1 g / L to 100 mL / L (e.g., 20 mL / L to 80 mL / L, or 30 mL / L to 100 mL / L). Vegetable oils (from 60 mL / L to 60 mL / L).

[0033] According to one implementation scheme, the culture medium in the main fermentation step may contain soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, magnesium sulfate, potassium nitrate, calcium chloride, and vegetable oil. Specifically, for example, the culture medium in the main fermentation step may contain 0.01 g / L to 3 g / L (e.g., 0.05 g / L to 2 g / L, or 0.05 g / L or 1 g / L) of soybean, 0.1 g / L to 5 g / L (e.g., 0.1 g / L to 3 g / L, or 0.5 g / L to 2 g / L) of yeast extract, 0.01 g / L to 5 g / L (e.g., 0.05 g / L to 3 g / L, or 0.05 g / L to 2 g / L) of glycerol, 1 g / L to 10 g / L (e.g., 1 g / L to 10 g / L, or 3 g / L to 6 g / L) of monopotassium phosphate, 1 g / L to 10 g / L (e.g., 1 g / L to 8 g / L, or 2 g / L to 6 g / L) of dipotassium phosphate, and 0.1 g / L to 5 g / L (e.g., 0.5 g / L to 3 g / L) of glycerol, 0.01 g / L to 5 g / L (e.g., 0.05 g / L to 3 g / L) of glycerol, 0.05 g / L to 3 g / L of monopotassium phosphate, 0.05 g / L to 3 g / L of dipotassium phosphate, and 0.1 g / L to 5 g / L (e.g., 0.5 g / L to 3 g / L) of glycerol, 0.01 g / L to 5 g / L (e.g., 0.05 g / L to 3 g / L) of glycerol, 0.05 g / L to 3 g / L of monopotassium phosphate, 0.05 g Ammonium sulfate (0.01 g / L or 0.5 g / L to 2 g / L), magnesium sulfate (0.01 g / L to 5 g / L, e.g. 0.05 g / L to 3 g / L, or 0.05 g / L to 1 g / L), potassium nitrate (0.01 g / L to 3 g / L, e.g. 0.05 g / L to 2 g / L, or 0.05 g / L to 1 g / L), calcium chloride (0.01 g / L to 3 g / L, e.g. 0.05 g / L to 2 g / L, or 0.1 g / L to 1 g / L), and vegetable oil (10 mL / L to 800 mL / L, e.g. 50 mL / L to 500 mL / L, or 100 mL / L to 300 mL / L, or 150 mL / L to 250 mL / L).

[0034] According to one embodiment, the seed culture step of the present invention may include culturing under aerobic conditions at 25°C to 35°C (e.g., 28°C to 32°C). In the seed culture step of the present invention, the seed culture is diluted with a sterile solution of 0.85% sodium chloride to 1% (v / v) to 10% (v / v) (e.g., 3% (v / v) to 8% (v / v)), and when the absorbance measured using a spectrophotometer at a wavelength of 600 nm is 0.05 to 0.2 (e.g., 0.05 to 0.15), the seed culture can be subcultured into a pre-culture.

[0035] According to one embodiment, the pre-cultivation step of the present invention may include adding 10 g / L to 500 g / L (e.g., 50 g / L to 300 g / L, or 50 g / L to 200 g / L) of seed culture and culturing under aerobic conditions at 25°C to 35°C (e.g., 28°C to 32°C). In this application, the aeration rate may be set to 10 NL / min to 50 NL / min (e.g., 20 NL / min to 40 NL / min), and the stirring speed may be set to 50 rpm to 300 rpm (e.g., 100 rpm to 200 rpm, or 150 rpm to 200 rpm). In the pre-culture step of the present invention, the pre-culture can be diluted with a sterile solution of 0.85% sodium chloride to 1% (v / v) to 10% (v / v) (e.g., 3% (v / v) to 8% (v / v)), and when the absorbance measured at a wavelength of 600 nm using a spectrophotometer is 0.2 to 1 (e.g., 0.3 to 0.8), the pre-culture can be subcultured into the main fermentation.

[0036] According to one embodiment, the main fermentation step of the present invention may include adding 10 g / L to 500 g / L of preculture (e.g., 50 g / L to 300 g / L, or 50 g / L to 200 g / L) and culturing under aerobic conditions at 25°C to 35°C (e.g., 28°C to 32°C). In this application, the aeration rate may be set, for example, 100 NL / min to 500 NL / min or 200 NL / min to 400 NL / min, and the stirring speed may be set, for example, 300 rpm to 900 rpm, 400 rpm to 800 rpm, or 500 rpm to 700 rpm. The main fermentation step of the present invention may last for, for example, 60 to 150 hours, 100 to 150 hours, or up to 144 hours from the start of the main fermentation.

[0037] According to one embodiment, the precipitate separation step of the present invention may include centrifuging the fermentation product to recover the precipitate. The precipitate separation step of the present invention may include centrifugation at, for example, 3000 rpm to 10000 rpm, 4000 rpm to 9000 rpm, or 5000 rpm to 8000 rpm. Since the phospholipids of the present invention exist extracellularly without cell rupture, the recovered precipitate contains both bacterial cells and extracellular phospholipids.

[0038] According to one embodiment, the phospholipid extraction step of the present invention may include mixing the recovered precipitate with a solvent to obtain a filtrate, and concentrating the filtrate. Specifically, the phospholipid extraction step of the present invention may include adding a solvent at a weight of 2 to 20 times (e.g., 5 to 15 times) that of the recovered precipitate, and stirring at a stirring speed of, for example, 100 to 200 rpm or 130 to 180 rpm at a temperature of, for example, 25°C to 35°C or 28 to 32°C, for example, for 10 to 30 hours, 15 to 25 hours, or 15 to 20 hours. Examples of solvents that can be used include water, ethanol, methanol, butanediol, pentanediol, dipropylene glycol, propylene glycol, hexanediol, butanol, ethyl acetate, etc., specifically, for example, 95% (v / v) ethanol.

[0039] According to one embodiment, the phospholipid-based surfactant of the present invention can be applied without limitation to various fields where surfactants are used, such as food, detergents, and cosmetics.

[0040] According to one embodiment, the phospholipid-based surfactant of the present invention can also be used with natural surfactants such as proteins, polysaccharides, saponins, cocobetaine, xanthan gum, and beeswax.

[0041] When the surfactant of the present invention is used as an additive in the food industry, it can replace part or all of gelatin, fatty acid esters, carrageenan, propylene glycol, triacetin, calcium stearoyl lactylate, lecithin, sodium lauryl sulfate, etc., but is not limited to the above types.

[0042] When the surfactant of the present invention is used as a surfactant in a detergent, it may replace some or all of alkylbenzene sulfonates, α-olefin sulfonates, alkyl sulfate salts, polyoxyethylene alkyl ether sulfates, alkane sulfonates, α-sulfo fatty acid ester salts, dialkyl dimethyl ammonium salts, imidazoline salts, alkyl dimethyl benzyl ammonium salts, polyoxyethylene alkyl ethers, alkyl dimethylamine oxides, fatty acid alkanolamides, alkyl polysaccharides, alkyl phenolethoxylates, alkyl betaines, alkyl sulfobetaines, etc., but is not limited to the above types.

[0043] When the surfactant of the present invention is used as a surfactant in cosmetics, it may replace some or all of alkylbenzene sulfonates, alcohol ethoxylates, alcohol ethyl oxides, alcohol ether sulfonates, acylglutamate esters, alkylamine oxides, α-olefin sulfonates, alkylphenol ethoxy compounds, alkyl polysaccharides, alkyl pentosaccharides, alcohol sulfonates, alkyl sulfobetaine, didicetyl ether sulfonate, ethylene oxide / propylene oxide block copolymers, fatty acid alkanolamides, fatty alcohol ether sulfates, fatty amine oxides, fatty alcohol sulfates, linear alkylbenzenes, linear alkylbenzene sulfonates, methyl ester ethoxylates, etc., but is not limited to the above types.

[0044] The embodiments of the present invention will be described in detail below to enable those skilled in the art to readily implement the invention. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein.

[0045] Example: Manufacturing phospholipid-based surfactants using skin microbiota Seed culture Prepare a culture medium with a total volume of 1 L containing 17.0 g / L casein (casein digested by trypsin), 3.0 g / L soybean (soybean digested by papain), 2.5 g / L glucose, 5.0 g / L sodium chloride, 2.5 g / L dipotassium phosphate, and pure water.

[0046] Microorganisms with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology) were inoculated into prepared culture medium and cultured under aerobic conditions at 30°C to prepare seed cultures.

[0047] The seed culture was diluted to 5% (v / v) with 0.85% (w / v) sterile sodium chloride solution. When the absorbance measured at 600 nm using a spectrophotometer (BioTek, EPOCH2C) was 0.1, the seed culture was passaged into the pre-culture.

[0048] Pre-culture Prepare a culture medium containing 0.1 g / L soybean (papain-digested soybean), 1.0 g / L yeast extract, 1.0 g / L glycerol, 4.5 g / L monopotassium phosphate, 3.0 g / L dipotassium phosphate, 50 mL / L olive oil, and water, with a total volume of 1 L. Olive oil is added by volume.

[0049] 100 g / L of seed culture was added to the prepared culture medium and cultured at an aeration rate of 30 NL / min, a culture temperature of 30℃, and a stirring speed of 180 rpm to prepare a preculture.

[0050] The preculture was diluted to 5% (v / v) with 0.85% (w / v) sterile sodium chloride solution. When the absorbance measured at 600 nm using a spectrophotometer was 0.5 to 0.6, the preculture was then passaged.

[0051] Primary fermentation Prepare a culture medium containing 0.1 g / L soybean (papain-digested soybean), 1.0 g / L yeast extract, 0.1 g / L glycerol, 4.5 g / L monopotassium phosphate, 3.0 g / L dipotassium phosphate, 1.0 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 0.1 g / L potassium nitrate, 0.3 g / L calcium chloride, 200 mL / L olive oil, and water, with a total volume of 1 L. Olive oil is added by volume.

[0052] A pre-culture of 100 g / L was added to the prepared culture medium and cultured at an aeration rate of 300 NL / min, a culture temperature of 30°C, and a stirring speed of 600 rpm to prepare the main fermentation product. The main fermentation process lasted for 144 hours.

[0053] extract The main fermentation product was centrifuged at 7000 rpm using a high-speed continuous centrifuge (DHC, SM-400) to recover the precipitate.

[0054] Add 95% ethanol equivalent to 10 times the weight of the recovered precipitate, and stir for 18 hours at 30°C and 150 rpm. The solution is then filtered through a glass fiber membrane with a pore size of 0.45 μm to obtain a yellow, transparent filtrate. Figure 1 A visual photograph of the resulting filtrate is shown.

[0055] The filtrate was concentrated under reduced pressure to recover the yellow, solid extracellular phospholipids. A visual photograph of the recovered phospholipids is shown below. Figure 2 As shown. After 114 hours of primary fermentation, the yield of the primary fermentation product was approximately 5% (w / w).

[0056] Comparative Example 1 In addition to using Bacillus subtilis ( Bacillus subtilis Except for the one with accession number KCCM10619, seed culture, pre-culture and master culture were performed using the same method as in the examples.

[0057] Comparative Example 2 In addition to using Staphylococcus aureus ( Staphylococcus aureus Except for the one with accession number KCTC3881, seed culture, pre-culture and master culture were performed using the same method as in the examples.

[0058] Experimental Example 1: Comparison of extracellular phospholipid production based on skin microbiota type Extracellular phospholipid production was compared between different types of skin flora and strains with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology) according to examples. Gram-positive aerobic bacteria were selected as different skin flora.

[0059] Extracellular phospholipid yield was measured as follows. First, seeds from Comparative Example 1, Comparative Example 2, and the Examples were cultured and pre-cultured, respectively. Then, the fermentation broth, after 144 hours of fermentation, was centrifuged at 13,000 rpm for 3 minutes. Next, the supernatant was removed to obtain precipitates. The precipitates of equal weight were extracted using the solvent extraction method described in the Examples, and concentrated under reduced pressure to determine the yield of the concentrate.

[0060] The results are shown in Table 1.

[0061] [Table 1]

[0062] Compared to the examples, in Comparative Example 2, there was almost no increase in precipitate and almost no extracellular phospholipids were observed. Comparative Example 1 showed an increase in precipitate over time, but the yield of extracellular phospholipids was extremely low. In contrast, the yield of extracellular phospholipids in the examples was approximately 5% (w / w).

[0063] Phospholipids are essential components of cell membranes and can be extracted from all cells with cell membranes. However, it is difficult to obtain the phospholipids that make up cell membranes without disrupting the cells through chemical and physical methods.

[0064] In the above experimental examples, only 95% ethanol was used to extract extracellular phospholipids without damaging the cells. In Comparative Example 2, although the amount of precipitate increased significantly with culture time, the observed phospholipid yield remained low, while the examples showed a higher phospholipid yield. This indicates that, unlike Comparative Example 2, the microbial precipitate according to the example with accession number KCTC15741BP (Korea National Institute of Bioscience and Biotechnology, Bioresource Center) contains not only bacterial cells but also extracellular phospholipids.

[0065] Experimental Example 2: Thin Layer Chromatography (TLC) Analysis The extracellular phospholipids obtained according to the examples were dissolved in chloroform. The solution was spotted at the starting point (baseline) of a silica gel plate (TLC silica gel 60F254, Merck; 10 cm × 5 cm) and developed to the endpoint (front) with a mobile phase containing chloroform, methanol and distilled water (6.5:2.5:0.4, v:v:v).

[0066] The components on the naturally dried TLC plates were stained and developed using different reagents. Lipid components were stained and developed using Nile Red reagent. This reagent was prepared at a concentration of 0.001 g / L in a solvent containing methanol and distilled water (8:2 ratio). The prepared reagent was sprayed onto the naturally dried TLC plates. After complete drying, the plates were immersed in a 0.0004% (w / w) sodium hypochlorite solution to bleach any unwanted staining. After the bleached TLC plates were completely dry, they were irradiated with ultraviolet light at a wavelength of 365 nm to observe the red staining spots.

[0067] For ninhydrin, it was prepared into a 5 g / L reagent by mixing it in ethanol. The prepared reagent was sprayed onto an air-dried TLC plate. After drying, it was heated at 100°C for 5 minutes, causing the components bound to the amine groups to appear as purple spots.

[0068] For molybdenum blue, after spraying it onto a naturally dried TLC plate, leave it at room temperature and in the dark for 5 minutes to allow the components bound to the phosphate groups to develop into blue spots.

[0069] For melanin, a 2 g / L reagent was prepared in 20% sulfuric acid. The prepared reagent was sprayed onto an air-dried TLC plate, and after drying, it was heated at 100°C for 5 minutes to make the sugar components appear as purple spots.

[0070] TLC results are as follows Figure 3 As shown. By TLC development and reagent staining, it was observed that the extract according to the examples contained phospholipids, phospholipids bound to amino groups, and phospholipids bound to sugar residues.

[0071] Experimental Example 3: TLC patterns changing with incubation time To observe the changes in phospholipid composition over fermentation time, seed culture and pre-culture stages were performed according to the examples, followed by main fermentation. During this process, precipitates were collected from each sample at 36, 72, and 144 hours after the start of fermentation. Equal weights of each precipitate were extracted using the solvent extraction method described in the examples to obtain extracellular phospholipids at each time point.

[0072] Following the same method as in Experiment 2, the obtained extracellular phospholipids were developed by TLC using Nile Red reagent. The results are as follows: Figure 4 As shown.

[0073] like Figure 4 As shown, the phospholipids obtained from the precipitate did not exhibit any qualitative differences in culture time. It is believed that the differences in phospholipids are reflected in quantitative differences in precipitate weight, rather than qualitative differences.

[0074] Experimental Example 4: HPLC-TOF-MS Analysis Extracellular phospholipids obtained from the precipitate collected 144 hours after the start of primary fermentation were dissolved in tetrahydrofuran, diluted with methanol, and then analyzed using high-resolution mass spectrometry (HPLC). The instrument used was an AB Sciex Triple TOF 5600+ HPLC system coupled to an HPLC system (Thermo Fisher Scientific, Ultimate 3000). Analytical conditions are shown in Tables 2 and 3.

[0075] [Table 2]

[0076] [Table 3]

[0077] The components were analyzed in both positive and negative ion modes, and the components were predicted based on a lipid library program, as shown in Table 4.

[0078] [Table 4]

[0079]

[0080] In the lipid library procedure, results with quality accuracy scores and MS scores between 0.8 and 1.00 were selected. Component analysis results showed that, in addition to triacylglycerols and diacylglycerols, phospholipids (e.g., phosphatidylglycerol (PG), ceramide phosphoethanolamine (sphingomyelin, Cer-PE), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), and fatty acid derivatives (e.g., branched-chain fatty acid esters of hydroxy fatty acids (FAHFA)) were also detected.

[0081] Experiment Example 5: Emulsification Index Test To confirm Figure 2 The emulsion stability index was determined to determine whether the extracellular phospholipids in the emulsion were suitable for formulation and exhibited surfactant properties as amphoteric substances.

[0082] First, from five solvents suitable for cosmetics, one solvent capable of dissolving the extracellular phospholipids obtained according to the examples was selected. The solvents used in this paper are 1,3-butanediol (1,3-BG), pentanediol (PG), dipropylene glycol (DPG), 1,3-propanediol (PDO), and 1,2-hexanediol (1,2-HXD).

[0083] Results from dissolution in various solvents showed that the extracellular phospholipids obtained according to the examples exhibited solubility only in pentylene glycol.

[0084] Next, the emulsification stability index of the extracellular phospholipids according to the examples was determined. The experimental group was prepared by adding 1 mL of a pentanediol solution containing 0.1% (w / v) extracellular phospholipids according to the examples, 10 mL of purified water, and 10 mL of MCT oil (WOOSUNG CNT Ltd. / Radia7104) to a 50 mL conical tube, followed by vigorous vortexing for 3 minutes. The control group was prepared in the same manner as the experimental group, except that it did not contain the extracellular phospholipids according to the examples. Specifically, 1 mL of pentanediol, 10 mL of purified water, and 10 mL of MCT oil (WOOSUNG CNT Ltd. / Radia7104) were added to a 50 mL conical tube, followed by vigorous vortexing for 3 minutes to prepare the control group.

[0085] After each mixture from the experimental and control groups was left to stand in a container at 25°C for 24 hours, the height of the emulsion layer was measured. The appearance after 24 hours of standing was as follows: Figure 5 As shown. The emulsion stability index is calculated using the following formula 1.

[0086] [Formula 1] Emulsion stability index = (Emulsion layer height) / (Total mixture height) To confirm the functional properties of extracellular phospholipids, 1 mL of pentanediol, 10 mL of pure water and 10 mL of MCT oil (WOOSUNG CNT Ltd / Radia7104) were mixed and allowed to stand as a control group.

[0087] Experimental results showed that the emulsification stability index of the experimental group was 0.54, while that of the control group was 0.22. Therefore, it was confirmed that the emulsification stability index increased by approximately 2.45 times when extracellular phospholipids according to the examples were included. This demonstrates that the extracellular phospholipids obtained according to the examples exhibit surfactant properties.

[0088] As described above, the phospholipid-based surfactant of the present invention is produced by the skin flora, is human-friendly, minimizes irritation to the human body and the environment, and contains phospholipid components with surfactant functional properties.

[0089] Although the specific embodiments of the present invention have been described in detail above, those skilled in the art should understand that the above specific description is only a preferred embodiment, and the scope of the present invention is not limited to the above specific embodiments.

[0090] [Certificate of Microbial Preservation]

Claims

1. A phospholipid-based surfactant prepared from skin flora microorganisms with accession number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology).

2. The phospholipid-based surfactant according to claim 1, wherein, The surfactant comprises phospholipids with or without amine or glycosyl groups.

3. The phospholipid-based surfactant according to claim 1, wherein, The surfactant includes one or more of phosphatidylglycerol, ceramide phosphoethanolamine, phosphatidylethanolamine, lysophosphatidylethanolamine, and branched fatty acid esters of hydroxy fatty acids.

4. A method for preparing a phospholipid-based surfactant, comprising: The seed culture step involves culturing the skin flora strain with the preservation number KCTC15741BP (Bioresource Center, Korea National Institute of Bioscience and Biotechnology) to prepare seed cultures. The pre-culture step involves culturing seed cultures in a medium containing vegetable oil to prepare pre-cultures. The main fermentation step involves adding the pre-culture to a culture medium containing vegetable oil to prepare the main fermentation product; The steps for separating precipitates from fermentation products; as well as The step of extracting phospholipids from precipitates.

5. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium used in the seed culture step contains casein, soybean, glucose, sodium chloride, and dipotassium phosphate.

6. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium in the seed culture step contains 5 g / L to 30 g / L casein, 1 g / L to 10 g / L soybean, 1 g / L to 6 g / L glucose, 1 g / L to 10 g / L sodium chloride and 1 g / L to 6 g / L dipotassium phosphate.

7. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium in the pre-culture step contains soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, and vegetable oil.

8. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium in the pre-culture step contains 0.01 g / L to 3 g / L soybean, 0.1 g / L to 5 g / L yeast extract, 0.1 g / L to 5 g / L glycerol, 1 g / L to 15 g / L monopotassium phosphate, 1 g / L to 10 g / L dipotassium phosphate, and 10 mL / L to 100 mL / L vegetable oil.

9. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium in the main fermentation step contains soybean, yeast extract, glycerol, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, magnesium sulfate, potassium nitrate, calcium chloride, and vegetable oil.

10. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The culture medium in the main fermentation step contains 0.01 g / L to 3 g / L soybean, 0.1 g / L to 5 g / L yeast extract, 0.01 g / L to 5 g / L glycerol, 1 g / L to 10 g / L monopotassium phosphate, 1 g / L to 10 g / L dipotassium phosphate, 0.1 g / L to 5 g / L ammonium sulfate, 0.01 g / L to 5 g / L magnesium sulfate, 0.01 g / L to 3 g / L potassium nitrate, 0.01 g / L to 3 g / L calcium chloride, and 10 mL / L to 800 mL / L vegetable oil.

11. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The seed culture step includes cultivation under aerobic conditions at 25°C to 35°C. The pre-culture step includes introducing 10 g / L to 500 g / L of the seed culture and culturing it under aerobic conditions at 25°C to 35°C. The main fermentation step includes introducing 10 g / L to 500 g / L of the preculture and culturing it under aerobic conditions at 25°C to 35°C.

12. The method for preparing a phospholipid-based surfactant according to claim 4, wherein the method comprises: In the seed culture step, when the absorbance measured at a wavelength of 600 nm is 0.05 to 0.2, the step of subculturing to a pre-culture medium is performed. In the pre-culture step, when the absorbance measured at a wavelength of 600 nm is 0.2 to 1, the culture is subcultured to the main fermentation step; or The primary fermentation process is completed 60 to 150 hours after the start of the primary fermentation.

13. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The steps for separating the precipitate include centrifuging the fermentation products to recover the precipitate.

14. The method for preparing phospholipid-based surfactants according to claim 4, wherein, The steps for extracting phospholipids include: The recovered precipitate is mixed with a solvent to obtain a filtrate; and Concentrate the filtrate.

Images