JP2006219414A - External preparation for skin, method for preventing adhesion of microorganism harmful to skin by using the same and method for preventing proliferation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which does not act on microorganisms on a healthy skin, effectively controls proliferation of microorganisms on a skin part having reduced barrier functions, is not germicidal, does not concentrate resistant microorganisms and makes the skin healthy and to obtain an external preparation for skin used therefor. <P>SOLUTION: The external preparation for skin comprises a protein that is derived from a bacterium selected from bacteria belonging the genus Lactobacillus, Bifidobacterium, Escherichia, Salmonella, Aeromonas, Streptococcus, Staphylococcus, Helicobacter, Campylobacter and Yersinia and has adhesivity to an extracellular matrix. The method for preventing adhesion of microorganisms harmful to the skin comprises using the external preparation for skin. The method for preventing proliferation of microorganisms harmful to skin comprises using the external preparation for skin. The method for producing the external preparation for skin comprising a protein having adhesivity to the extracellular matrix is provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to an external preparation for skin containing a protein derived from a microorganism having adhesion to an extracellular matrix, a method for preventing adhesion of harmful skin microorganisms using the external preparation for skin, and a method for preventing proliferation of harmful microorganisms for skin.
More specifically, the present invention is a skin external preparation containing a protein that is produced by a microorganism belonging to the genus Lactobacillus and has an adhesive property to an extracellular matrix, and a skin harmful microorganism using the skin external preparation. The present invention relates to a method for preventing adhesion and a method for preventing proliferation of harmful microorganisms on the skin.
The present invention also provides a skin external preparation characterized by containing a specific protein or a protein produced by a microorganism transformed with a DNA encoding the protein, and a skin harmful microorganism using the skin external preparation. The present invention relates to a method for preventing adhesion and a method for preventing proliferation of harmful microorganisms on the skin.

  In recent years, troubles involving the skin, such as atopic dermatitis and chemical sensitivity, have been increasing. As a factor, various studies have been made, such as deterioration of the original skin barrier function due to excessive cleanliness and the occurrence of rough skin caused by drying by complete air conditioning. As one of such skin trouble factors or pathological deterioration factors, the action of skin resident microorganisms has attracted attention.

  Various bacteria and fungi are known as skin resident microorganisms, and their relationship with disease states is becoming clear. Usually, skin resident microorganisms are harmless on the skin where a healthy barrier function is maintained, and rather are considered to work beneficially such as antagonizing harmful pathogenic microorganisms. However, once the barrier function is reduced due to external factors such as those mentioned above, excessive immune reactions due to the growth of harmful microorganisms that occupy a small part of these skin resident bacteria, proteolytic enzymes produced by microorganisms, and sebum degradation products When stimulated by fatty acids derived from the skin, itching causes itchiness, which causes a vicious cycle of skin trouble that further deteriorates the state of the skin with a reduced barrier function.

  Regarding the usefulness of these resident microorganisms and the relationship between the deterioration of skin condition and microorganisms, for example, “Properties of resident skin bacteria and ability to eliminate pathogens” (Satoshi Ushijima “Human resident bacteria”, Pharmaceutical Journal, p. 156-170: Non-patent document 1), "Control by skin resident bacteria and cosmetics" (Tomoko Fukubayashi, Fragrance Journal, March 2003, p23-28: Non-patent document 2) and many other books・ It is discussed in detail in papers.

Furthermore, extensive studies have been conducted on harmful resident microorganisms that actually cause skin troubles and their control. Adverse skin indigenous bacteria, resulted in dandruff and itching of the scalp, also recently, there is also reported to exhibit high antibody titers in patients with atopic dermatitis Malassezia (Malassezia) genus, which is a typical fungus mycosis Candida ( Candida ), Staphylococcus genus isolated frequently from diseased skin, Propionibacterium genus, which is the cause of acne and pimples, and the so-called odor-causing fungus Corynebacterium genus corresponding to “Diphtheroid” is known. It is well known to use various bactericidal and antibacterial agents for controlling and treating infections caused by these microorganisms, and it is also common to control and treat microorganisms that are causing pathological conditions with such antibacterial agents. .

  However, these days, there are problems such as side effects due to frequent infection control and treatment with these drugs and long-term continuous use, and a decrease in efficacy due to the emergence of resistant bacteria. In particular, resistant bacteria are problems related to the principle of bactericidal drugs, in which only mutants that have acquired resistance survive due to the bactericidal drug, and a few resistant mutants are concentrated. Although various improved drugs have been proposed and used for the emerging resistant bacteria, they are essentially difficult to solve. In addition, the bactericidal agent also acts on the resident bacteria group having the beneficial action described above, and reduces the antagonistic action against the harmful bacteria. So-called mycosis, such as candidiasis, which occurs in patients who have been taking antibacterial agents for a long time is well known. It has also been suggested that the use of bactericides, particularly in skin trauma, results in the death of epithelial fibroblasts, which are the natural defense mechanisms of the skin, and the impediment to migration, resulting in delayed healing.

Against this background, attempts have been made to eliminate harmful skin bacteria by utilizing the interaction between skin resident bacteria instead of these drugs. As such an example, 1994 Patent Publication No. 135843 discloses a dermatological agent containing microbial cells or extracts of microorganisms having adhesion to the skin surface, and normal skin resident bacteria. A method using an extract of a microorganism belonging to the genus Staphylococcus is exemplified (Patent Document 1).
However, simply applying microorganisms and their extracts that adhere to the skin in this way will not only eliminate harmful bacteria but also beneficial normal microorganisms on normal skin. There is a possibility that the barrier function of the skin, which includes the interaction of various resident bacteria, is reduced.

In addition, Ouwehand et al. Are examining the inhibitory effect of Propionibacterium , a kind of skin resident bacteria, on the adhesion of keratin to keratin (Letters in Applied Microbiology, 36, 327-331). (2003)) (Non-Patent Document 3). Aiming to inhibit the adhesion of some commonly known skin pathogens to keratin, the main component of the skin, by the antagonism of a culture of Propionibacterium, which is considered to be a harmless skin resident bacteria It is a thing. However, sufficient effects have not been achieved.

  In addition, these adherent microorganisms and their extracts isolated from normal skin are considered to show their action on normal skin covered with keratin, but rough skin, itching due to itching, atopic dermatitis, etc. In principle, no action can be expected on the epidermis barrier, that is, the site where the keratinous material is impaired in various skin conditions. That is, there has been a big problem that the action cannot be expected at a site where the harmful bacteria control is most desired, the barrier is damaged and the infection is easily affected by the harmful bacteria.

On the other hand, it is known that various microorganisms have adhesion mechanisms and components for infection, symbiosis, and normalization of human bodies and animals. In some of them, the substance of the adhesion mechanism is extracellular. It is being elucidated that it is a kind of protein having adhesion to matrix components. Examples of microorganisms having an extracellular matrix adhesion protein include those of the genus Lactobacillus (Toba et al., Journal of Bacteriology, vol. 182, No. 22, p. 6440-6450 (2000)) (Non-patent Document 4). In addition, Bifidobacterium (Bifidobacterium) genus (Mukai et al., Current Microbiology, vol.34, 326-331 ( 1997)) ( non-Patent Document 5), Escherichia (Escherichia) genus Salmonella (Salmonella) genus Aeromonas (Aeromonas ) genus Streptococcus (Streptococcus) species, Staphylococcus (Staphylococcus) genus, Helicobacter (Helicobacter) genus Campylobacter (Campylobacter) belonging to the genus (Ofec et al., "Bacterial Adhesion To Cells And Tissues ", Chapman & Hall (1994)) ( non-patent literature 6), Yersinia (Yersinia) genus (Skurnik et al, Infection and Immunity, vol.62, 1252-1261 (1994)) ( non-Patent Document 7) and is known like, specific and preparation of adherent proteins, adhesion How to evaluate sex And extracellular matrix to be deposited respectively, such as fibronectin, collagen type I · II type · V type, laminin, heparin, and that vitronectin is a like has been studied in detail in the respective reports. However, these studies mainly focus on the mechanism of microbial adhesion / infection or normalization in the digestive epithelium. Gastrointestinal epithelium has a structure where the extracellular matrix is exposed, and it is thought that the adhesion action by the extracellular matrix adhesion component produced by microorganisms appears. The effect on the skin, which is considered to be covered with a quality barrier, was not considered at all, and its application to dermatological agents was not studied.

1994 Patent Publication No. 135843 "Properties of skin resident bacteria and the ability to eliminate pathogens", Kaoru Ushijima "Human resident bacteria", Pharmaceutical Journal, p.156-170 “Control by skin resident bacteria and cosmetics” Tomoko Fukubayashi, Fragrance Journal, March 2003, p23-28 Letters in Applied Microbiology, 36, 327-331 (2003) Toba et al., Journal of Bacteriology, vol. 182, No. 22, p. 6440-6450 (2000) Mukai et al., Current Microbiology, vol.34, 326-331 (1997) Ofec et al., “Bacterial Adhesion To Cells And Tissues”, Chapman & Hall (1994) Skurnik et al., Infection and Immunity, vol.62, 1252-1261 (1994)

  The present invention does not act on harmless microorganisms or microorganisms on healthy skin where the barrier function is not reduced, effectively suppresses the growth of microorganisms on the skin site where the barrier function is reduced, and is bactericidal. It is an object of the present invention to provide a method for healthy skin that does not concentrate resistant microorganisms, and a skin external preparation used therefor.

The inventor does not act on harmless microorganisms or microorganisms on healthy skin whose barrier function is not reduced, effectively suppresses the growth of microorganisms on the skin site where the barrier function is reduced, and is bactericidal. We sought to find materials useful for skin health that do not concentrate resistant microorganisms. As a result, a protein having adhesion to an extracellular matrix derived from a microorganism, particularly preferably, can be obtained by a simple method from a culture of a microorganism belonging to the genus Lactobacillus, which is a lactic acid bacterium widely known as a safe bacterium. The present invention was completed by finding that the protein has an excellent skin-healing action. That is, the present invention has the following configuration.

1. A skin external preparation containing a protein derived from a microorganism and having adhesion to an extracellular matrix.
2. Microorganisms, Lactobacillus (Lactobacillus) genus Bifidobacterium (Bifidobacterium) genus Escherichia (Escherichia) genus Salmonella (Salmonella) genus, Aeromonas (Aeromonas) genus Streptococcus (Streptococcus) species, Staphylococcus (Staphylococcus) genus , Helicobacter (Helicobacter) genus Campylobacter (Campylobacter) genus, and Yersinia (Yersinia) external skin preparation according to the 1 selected from microorganisms belonging to the genus.
3. A protein having an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted, deleted or inserted by another amino acid; 2. The external preparation for skin according to 1 above, characterized in that
4). The protein encodes an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 1 is encoded, or an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, deleted or inserted by other amino acids 2. The external preparation for skin according to 1 above, which is produced by a microorganism transformed with DNA.
5. 2. The skin external preparation according to 1 above, wherein the protein having adhesion to the extracellular matrix is obtained from Lactobacillus crispatus .
6). 6. The skin external preparation according to 5 above, wherein the protein having adhesion to the extracellular matrix is obtained from Lactobacillus crispatus JCM5810.
7). 5. A method for preventing the adhesion of harmful skin microorganisms, comprising using the external preparation for skin according to any one of 1 to 4 above.
8). 5. A method for preventing the growth of harmful skin microorganisms, comprising using the external preparation for skin according to any one of 1 to 4 above.
9. Lactobacillus (Lactobacillus) genus Bifidobacterium (Bifidobacterium) genus Escherichia (Escherichia) genus Salmonella (Salmonella) genus Aeromonas (Aeromonas) genus Streptococcus (Streptococcus) genus, Staphylococcus (Staphylococcus) genus Helicobacter ( Helicobacter) genus Campylobacter (Campylobacter) genus, and Yersinia (Yersinia) adherent to the extracellular matrix, characterized in that the urea and / or guanidine hydrochloride than cultured cells of a microorganism belonging to the genus comprising the step of extracting solubilize The manufacturing method of the skin external preparation containing the protein which has.
10. A protein having adhesion to the extracellular matrix is substituted, deleted, or deleted by DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 by other amino acids Alternatively, the protein having adhesion to the extracellular matrix according to 9 above, which comprises a step of solubilizing and extracting with urea and / or guanidine hydrochloride from a cultured microorganism of a microorganism transformed with a DNA encoding the inserted amino acid sequence A method for producing an external preparation for skin.

In many microorganisms, it is known that there is an adhesion mechanism for colonizing the human body / animal, especially the intestinal epithelium, and the substance is a kind of protein that adheres to extracellular matrix components. Is also being elucidated. In particular, Lactobacillus genus microorganisms belong to so-called lactic acid bacteria, and are known to have fixing properties in the human body and animals, particularly in the intestinal tract, and are also important for the production of familiar fermented foods such as yogurt and pickles. It is a highly safe microorganism that plays an important role. Based on the fact that the adhesion mechanism of these colonized microorganisms interacts with the human body, more specifically, with the extracellular matrix, the protein derived from these microorganisms is applied to the skin with a reduced barrier function. We focused on the possibility of having an antagonistic action on the adhesion of harmful microorganisms. Furthermore, the fact that a skin external preparation containing an extracellular matrix-adhering protein derived from a microorganism has a control effect against many known harmful resident bacteria and provides an excellent skin care effect has never been expected. The headline and the present invention were completed.

  Various microorganisms and extracellular matrix adhesion proteins derived from microorganisms have been suggested so far in various papers and the like, as described above, as described above, which may competitively inhibit the adhesion of harmful microorganisms to the intestinal epithelium. However, regarding the skin, unlike the intestinal epithelium, there is a stratum corneum mainly composed of keratin and a secreted sebum layer, and it is not normally thought that interaction with extracellular matrix adhesion protein occurs. . However, the present inventors paid attention to this point. That is, the present invention does not act on a healthy skin site, that is, does not disturb the healthy microflora at all, and on the other hand, in the site where the barrier property is lowered due to various factors such as dryness, itchiness, rough skin, etc. It aims to keep the skin healthy by effectively antagonizing the pathogenic microorganisms that are trying to grow and infect as the main adhesion target and suppress the adhesion of harmful bacteria.

[Extracellular matrix adhesion protein]
In the present invention, “extracellular matrix” means a generally known extracellular matrix, that is, a structural protein such as collagen or elastin, a cell adhesion protein such as fibronectin, laminin or vitronectin, or a glycoprotein such as hyaluronic acid or chondroitin. Saminoglycan, a structure existing outside of cells in animal tissues, such as proteoglycan, which is a complex of these, refers to biopolymers synthesized by cells and secreted / accumulated outside the cells, and complexes thereof. In the present invention, the extracellular matrix-adhesive protein applied to the external preparation for skin is not particularly limited as long as it has adhesiveness to the extracellular matrix to which harmful skin bacteria adhere, but for the purpose, Collagen, elastin, laminin, hyaluronic acid, which are extracellular matrix components present in the tissue immediately below, in particular, type I collagen, type III collagen, type V collagen, which are extracellular matrix components present in large amounts in the skin and subcutaneous tissue, It is preferable that it has adhesiveness to elastin.

  In the present invention, “extracellular matrix adhesion” means that any method for detecting extracellular matrix adhesion protein can actually detect adhesion to the extracellular matrix as described above, and the extracellular adhesion can actually be detected by the adhesion. What is necessary is that it can reduce the adherence of harmful bacteria to the matrix, and it does not matter to what extent, but it is usually present in many living organisms, and is a non-acting standard protein that is frequently used as a control in this technical field. It is preferably a protein that adheres more to the extracellular matrix than to the outside. Evaluation of adhesion to the extracellular matrix can be performed, for example, by the following method. That is, generally known substrates to which extracellular matrix components bind, such as glass, plastic, protein-adsorbing gel, immobilization gel introduced with -CNBr group, water, physiological saline, and buffer solution, respectively. The above-mentioned extracellular matrix component solution or bovine serum albumin solution, which has been diluted to a constant concentration with the above-described method, is smeared or dipped, and these components are bonded to the substrate surface. After washing away the unbound components on the surface of the substrate, the solution, suspension, etc. of the component to be evaluated next are applied or dipped in the same manner and washed out in the same manner. By comparing the amount of adherence to the surface of the substrate treated with the extracellular matrix and the surface treated with albumin, the substance with more adhesion to the former can be determined as the substance adhering to the extracellular matrix. In addition, the amount of binding is determined by a method in which the component to be evaluated is previously modified with a fluorescent dye and the total fluorescence intensity of the adhering portion is measured, and a method using a labeled antibody of the component to be evaluated, etc. Can be selected.

In the present invention, the extracellular matrix-adhesive protein used for the external preparation for skin can be prepared from a microorganism having the protein, for example, as follows. A culture obtained by culturing a microorganism having an extracellular matrix adhesion protein, for example, a microorganism belonging to the genus Lactobacillus according to a conventional method, is collected by centrifugation or membrane separation. This is exposed to 0.5 to 8M, preferably 1 to 4M guanidine hydrochloride aqueous solution for several minutes to 24 hours, and the solubilized fraction is collected by membrane filtration or centrifugation. In order to increase the efficiency of solubilization, stirring and heating from room temperature to 80 ° C, preferably from room temperature to 40 ° C can be performed. The solubilized protein fraction obtained in this way is used as a growth factor, according to the purpose of use as a growth factor, as it is, or as a desalination treatment by dialysis or ion exchange, concentration treatment such as evaporation, freeze drying or spray drying. Various treatments can be added and used.

  The extracellular matrix-adhering protein of the present invention has the property that it can be obtained as a soluble fraction by at least such an operation, but the method of fractionation is not particularly limited. For example, the protein such as solubilization with a high concentration of urea can be used. It may be fractionated by other conventional methods of solubilization. If necessary, the obtained protein fraction should be separated, purified and used by various methods such as salting out, solvent precipitation, gel filtration, affinity, etc. so that the components that adhere to the extracellular matrix are not lost. You can also.

In the present invention, the microorganism for obtaining the extracellular matrix adhesion protein is not particularly limited. Microorganisms known to have extracellular matrix adhesion proteins include the genus Lactobacillus (Toba et al., Journal of Bacteriology, vol.182, No.22, p.6440-6450 (2000)), Bifidobacterium (Bifidobacterium) genus (Mukai et al., Current Microbiology, vol.34, 326-331 ( 1997)), Escherichia (Escherichia) genus Salmonella (Salmonella) genus Aeromonas (Aeromonas) genus Streptococcus (Streptococcus) genus , Staphylococcus (Staphylococcus) genus, Helicobacter (Helicobacter) genus Campylobacter (Campylobacter) belonging to the genus (Ofec et al., "Bacterial Adhesion To Cells And Tissues ", Chapman & Hall (1994)), Yersinia (Yersinia) belonging to the genus (Skurnik, et al., Infection and Immunity, vol. 62, 1252-1261 (1994)). Specific methods of adhesion protein identification and adhesion evaluation, and the extracellular matrix to be attached, for example, fibronectin, collagen type I, type II, type V, laminin, heparin, vitronectin, etc. It is discussed in detail in the report. Of these, the human body its simplicity and Lactobacillus (Lactobacillus) attribute from easy availability of the culture, Bifidobacterium (Bifidobacterium) genus Escherichia (Escherichia) is particularly preferred microorganisms, the more in view of safety, such as edible A microorganism belonging to the genus Lactobacillus, which has been known for a long time, is most preferably used.

Further, the extracellular matrix-adhesive protein applied to the present invention identifies the extracellular matrix-adhesive protein from the protein fraction obtained from the culture of the microorganism having the extracellular matrix-adhesive protein, and encodes the protein. A gene is identified by PCR amplification reaction or hybridization using a mix primer based on the N-terminal analysis result, and other microorganisms such as E. coli are transformed using the gene, and the protein obtained from the culture of the microorganism is included. The form as a protein fraction is also included. In a transformed microorganism, the productivity of the protein can be increased by adding an expression factor of the promoter of the expression vector to the culture. Extraction from the transformed microorganism can be performed according to the above-described example of the extraction method from the microorganism belonging to the genus Lactobacillus , and the form of utilization is also the same. Moreover, generally known proteins such as introduction of a histidine continuous sequence (so-called His tag) having an affinity for a metal ligand into a structural gene and fusion with GST (glutathione-S-transferase) having an affinity for glutathione. Modifications suitable for expression / purification can be added to express the complex as a complex, which can be purified and recovered from the solubilized product of the expressed microorganism by the affinity chromatography method. In this case, the modified moiety can be used in the same manner as it is added as long as the function is maintained, or may be used by adding a treatment for removing the modified moiety such as degradation by protease.

Examples of the extracellular matrix adhesion protein according to the present invention include, for example, SEQ ID NO: 1 discovered by the present inventor from Lactobacillus genus microorganisms, Lactobacillus crispatus JCM 5810 (distributed from the Institute of Physical and Chemical Research Microorganisms). And the protein represented by the amino acid sequence shown in FIG. Report of Toba et al. (Journal of Bacteriology, vol.182, No.22, 6440-6450 (2000), etc.) on the characteristics and extraction method of the protein, the production method using transformants, and the adhesion of the resulting protein to the extracellular matrix Are discussed in detail.

  In addition, due to recent advances in molecular biology and genetic engineering, by directly referring to the molecular biological properties and amino acid sequences of proteins, it has functions equivalent to those of proteins by means of PCR and hybridization. It is relatively easy for those skilled in the art to search for and obtain proteins from separate microbial strains. Therefore, the extracellular matrix-adhering protein according to the present invention is most conveniently obtained as a protein fraction by extraction from the microorganism culture having the extracellular matrix-adhesive property exemplified above. The microorganism to be derived is not particularly limited as long as it has the property, and an embodiment using an adhesive protein of a bacterium that can be searched using general techniques and information such as sequence information of the protein and PCR is also included in the present invention. include.

  In addition, by the advancement of recombinant DNA technology, one or more of the constituent amino acids can be replaced, deleted, deleted or inserted with other amino acids relatively easily without substantially changing the function of the protein. Is well known. Therefore, the extracellular matrix-adhesive protein referred to in the present invention has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing as it is, and one or several amino acids are substituted, deleted, deleted or deleted by other amino acids. A partially mutated protein having an inserted amino acid sequence and having an extracellular matrix adhesion property is also encompassed in the present invention.

  The external preparation for skin of the present invention contains 0.001% to 100% of the extracellular matrix adhesion protein prepared as described above. Examples of the external preparation for skin of the present invention include emulsions, skin creams, foundation creams, massage creams, cleansing creams, shaving creams, cleansing foams, lotions, lotions, packs, shampoos, rinses, hair restorers, hair nourishing agents, hair dyes. Hair styling agents, toothpastes, gargles, permanent wave agents, ointments, bathing agents, body soaps and the like can be exemplified. The present invention imparts an excellent skin care function to these external preparations for skin, and contributes to prevention of various troubles caused by the skin microbiota and skin health.

Moreover, the component generally used for a skin external preparation can be mix | blended with the skin external preparation of this invention in the range which does not impair the effect of this invention.
Examples of such components include ozokerite, α-olefin oligomer, light isoparaffin, light liquid isoparaffin, squalene, squalane, synthetic squalane, plant squalane, ceresin, paraffin, polyethylene powder, polybutene, microcrystalline wax, liquid isoparaffin, Hydrocarbons such as liquid paraffin, mineral oil, petrolatum;

  Jojoba oil, carnauba wax, candelilla wax, rice bran wax, shellac, lanolin, mink sebum wax, whale wax, sugarcane wax, sperm whale oil, beeswax, montan wax and other natural waxes, avocado oil, almond oil, olive oil, extra virgin olive oil, Sesame oil, rice bran oil, rice oil, rice germ oil, corn oil, safflower oil, soybean oil, corn oil, rapeseed oil, persic oil, palm kernel oil, palm oil, castor oil, sunflower oil, high oleic sunflower oil, Grape seed oil, cottonseed oil, palm oil, hydrogenated palm oil, beef tallow, hardened oil, horse oil, mink oil, egg yolk oil, egg yolk fat oil, rosehip oil, cucumber nut oil, evening primrose oil, wheat germ oil, peanut oil, camellia oil Oil, sasanqua oil, cacao butter, molasses, beef bone fat, beef leg oil, pork fat, horse fat, sheep fat, sheabata , Natural fats and oils such as macadamia nut oil and meadow home oil; lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, γ-linolenic acid, isostearic acid, 12-hydroxystearic acid , Fatty acids such as undecylenic acid and coconut oil fatty acid;

Higher alcohols such as isostearyl alcohol, octyldodecanol, hexyldecanol, cholesterol, phytosterol, lauryl alcohol, myristyl alcohol, cetanol, stearyl alcohol, oleyl alcohol, behenyl alcohol, cetostearyl alcohol;
Alkyl glyceryl ethers such as batyl alcohol, chimyl alcohol, ceralkyl alcohol, isostearyl glyceryl ether;

  Isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, butyl stearate, ethyl oleate, ethyl linoleate, isopropyl linoleate, cetyl caprylate, hexyl laurate, isooctyl myristate, decyl myristate, myristic acid Myristyl, cetyl myristate, octadecyl myristate, cetyl palmitate, stearyl stearate, decyl oleate, oleyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecyl myristate, isocetyl myristate, isostearyl myristate, myristic acid Octyldodecyl, 2-ethylhexyl palmitate, isocetyl palmitate, isostearyl palmitate, 2-ethyl stearate Ruhexyl, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl ricinoleate, ethyl isostearate, isopropyl isostearate, cetyl 2-ethylhexanoate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, Hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprylate, propylene glycol di (capryl / capric acid), propylene glycol dicaprate, propylene glycol dioleate, neopentyl glycol dicaprate, neopentyl glycol dioctanoate , Glyceryl tricaprylate, glyceryl tri-2-ethylhexanoate, tri (capryl caprin ) Glyceryl, tri (caprylic acid / capric acid / stearic acid) glyceryl, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, tetra-2 -Pentaerythrityl ethylhexanoate, pentaerythrityl tetramyristate, pentaerythrityl tetraisostearate, diglyceryl tetraisostearate, octyldodecyl neopentanoate, isocetyl octoate, isostearyl octoate, 2-ethylhexyl isoperargonate, Hexyldecyl dimethyloctanoate, octyldodecyl dimethyloctanoate, 2-ethylhexyl isopalmitate, isocetyl isostearate, Isostearyl isostearate, octyldodecyl isostearate, lauryl lactate, myristyl lactate, cetyl lactate, octyldodecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, triisocetyl citrate, trioctyl dodecyl citrate , Diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisopropyl adipate, diisobutyl adipate, dioctyl adipate, diheptylundecyl adipate, diethyl sebacate, diisopropyl sebacate, sebacic acid Dioctyl, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate Dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate, isostearyl 12-stearoylhydroxystearate, polyoxyethylene acetate (3) polyoxypropylene (1) ) Esters such as cetyl ether, polyoxyethylene acetate (3) polyoxypropylene (1) isocetyl ether, isononyl isononanoate, octyl isononanoate, tridecyl isononanoate, isotridecyl isononanoate;

  Methylpolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, methylcyclopolysiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexasiloxane, octamethyltrisiloxane, decamethyltetrasiloxane, tetradeca Methyl hexasiloxane, highly polymerized methyl polysiloxane, dimethyl siloxane methyl (polyoxyethylene) siloxane methyl (polyoxypropylene) siloxane copolymer, dimethyl siloxane methyl (polyoxyethylene) siloxane copolymer, dimethyl siloxane methyl (Polyoxypropylene) siloxane copolymer, dimethylsiloxane / methylcetyloxysiloxane copolymer, dimethylsiloxane / Chill stearoxysiloxane copolymer, polyether-modified silicones, alcohol-modified silicones, alkyl-modified silicones, silicone oils such as amino-modified silicone;

Ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, 3-methyl-1,3-butanediol, 1,3-butanediol, 1, Polyhydric alcohols such as 2-pentanediol and 1,2-hexanediol;
Sugars such as mannitol, sorbitol, xylitol, maltitol, erythritol, pentaerythritol, glucose, sucrose, fructose, lactose, maltose, xylose, trehalose;

Sodium alginate, carrageenan, agar, fur celerin, guar gum, quince seed, konjac mannan, tamarind gum, tara gum, dextrin, starch, locust bean gum, gum arabic, gati gum, karaya gum, tragacanth gum, arabinogalactan, pectin, quince, chitosan, starch , Curdlan, xanthan gum, gellan gum, cyclodextrin, dextran, pullulan, microcrystalline cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxy starch, cationized cellulose, starch phosphate ester, cation Guar gum, carboxymethyl Roxypropylated guar gum, hydroxypropylated guar gum, albumin, casein, gelatin, sodium polyacrylate, polyacrylate amide, carboxyvinyl polymer, polyethyleneimine, highly polymerized polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl ether, polyacrylamide, Acrylic acid copolymer, methacrylic acid copolymer, maleic acid copolymer, vinyl pyridine copolymer, ethylene / acrylic acid copolymer, vinyl pyrrolidone polymer, vinyl alcohol / vinyl pyrrolidone copolymer, nitrogen-substituted acrylamide system Polymer, amino-modified silicone, cationized polymer, dimethylacrylammonium polymer, acrylic acid anion polymer, methacrylic acid anion polymer Modified silicones, alkyl methacrylate acrylate acid (C ₁₀ ~ ₃₀₎ copolymers, polymers such as polyoxyethylene / polyoxypropylene copolymer;

Alcohols such as ethanol, isopropyl alcohol, 1-butanol, 2-butanol, benzyl alcohol;
Palm oil fatty acid potassium, coconut oil fatty acid sodium, coconut oil fatty acid triethanolamine, potassium laurate, sodium laurate, triethanolamine laurate, potassium myristate, sodium myristate, isopropanolamine myristate, potassium palmitate, palmitic acid Sodium, isopropanolamine palmitate, potassium stearate, sodium stearate, triethanolamine stearate, potassium oleate, sodium oleate, castor oil fatty acid sodium, zinc undecylenate, zinc laurate, zinc myristate, magnesium myristate, Zinc palmitate, zinc stearate, calcium stearate, magnesium stearate, aluminum stearate, myristi Calcium oxide, magnesium myristate, aluminum dimyristate, aluminum isostearate, polyoxyethylene lauryl ether acetic acid, sodium polyoxyethylene lauryl ether acetate, polyoxyethylene tridecyl ether acetic acid, sodium polyoxyethylene tridecyl ether acetate, stearoyl lactic acid Sodium, sodium isostearoyl lactate, sodium lauroyl sarcosine, coconut oil fatty acid sarcosine, coconut oil fatty acid sarcosine sodium, coconut oil fatty acid sarcosine triethanolamine, lauroyl sarcosine, lauroyl sarcosine potassium, lauroyl sarcosine triethanolamine, oleoyl sarcosine, myristoyl sarcosine sodium , Sodium stearoyl glutamate, Oil fatty acid acyl glutamic acid, coconut oil fatty acid potassium acyl glutamate, coconut oil fatty acid acyl glutamic acid sodium, coconut oil fatty acid acyl glutamic acid triethanolamine, lauroyl acyl glutamic acid, lauroyl acyl glutamic acid potassium, lauroyl acyl glutamic acid sodium, lauroyl acyl glutamic acid triethanolamine, myristoyl Acyl glutamate, potassium myristoyl acyl glutamate, sodium myristoyl acyl glutamate, stearoyl acyl glutamate, potassium stearoyl acyl glutamate, disodium stearoyl acyl glutamate, sodium cured beef tallow fatty acid acyl glutamate, coconut oil fatty acid / cured tallow fatty acid sodium acyl glutamate, coconut oil fatty acid me Sodium tilalanine, lauroylmethylalanine, sodium lauroylmethylalanine, lauroylmethylalanine triethanolamine, sodium myristoylmethylalanine, sodium lauroylmethyltaurine, coconut oil fatty acid methyltaurine potassium, coconut oil fatty acid methyltaurine sodium, coconut oil fatty acid methyltaurine magnesium , Myristoyl methyl taurine sodium, palmitoyl methyl taurine sodium, stearoyl methyl taurine sodium, oleoyl methyl taurine sodium, sodium alkane sulfonate, sodium tetradecene sulfonate, dioctyl sodium sulfosuccinate, disodium lauryl sulfosuccinate, palm oil fatty acid ethyl ester sulfone Acid sodium, lauryl sulfate Thorium, triethanolamine lauryl sulfate, sodium cetyl sulfate, alkyl (11,13,15) triethanolamine sulfate, alkyl (12,13) sodium sulfate, alkyl (12,13) triethanolamine sulfate, alkyl (12,14) , 16) Ammonium sulfate, alkyl (12-13) diethanolamine sulfate, alkyl (12-14) triethanolamine sulfate, alkyl (12-15) triethanolamine sulfate, palm oil alkyl magnesium sulfate / triethanolamine, ammonium lauryl sulfate, lauryl Potassium sulfate, magnesium lauryl sulfate, monoethanolamine lauryl sulfate, diethanolamine lauryl sulfate, sodium myristyl sulfate, sodium stearyl sulfate, sodium oleyl sulfate, Rail ethanol triethanolamine, polyoxyethylene lauryl ether sodium sulfate, polyoxyethylene lauryl ether triethanolamine sulfate, polyoxyethylene (1) alkyl (11,13,15) ether sodium sulfate, polyoxyethylene (1) alkyl ( 11,13,15) ether sulfate triethanolamine, polyoxyethylene (3) alkyl (11-15) sodium ether sulfate, polyoxyethylene (2) alkyl (12,13) sodium ether sulfate, polyoxyethylene (3) Alkyl (12-14) ether sodium sulfate, polyoxyethylene (3) alkyl (12-15) sodium ether sulfate, polyoxyethylene (2) sodium lauryl ether sulfate, polyoxyethylene (3) Sodium listyl ether sulfate, sodium higher fatty acid alkanolamide sulfate, lauryl phosphate, sodium lauryl phosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate, polyoxyethylene oleyl ether phosphate, polyoxyethylene lauryl ether phosphate, polyoxyethylene Sodium lauryl ether phosphate, polyoxyethylene cetyl ether phosphate, polyoxyethylene cetyl ether sodium phosphate, polyoxyethylene stearyl ether phosphate, polyoxyethylene oleyl ether phosphate, polyoxyethylene oleyl ether sodium phosphate, polyoxy Ethylene alkyl phenyl ether phosphate, polyoxyethylene alkyl phenyl ether sodium phosphate, polyoxy ester Lenalkylphenyl ether phosphate triethanolamine, polyoxyethylene octyl ether phosphate, polyoxyethylene (10) alkyl (12,13) ether phosphate, polyoxyethylene alkyl (12-15) ether phosphate, polyoxyethylene Anionic surfactants such as alkyl (12-16) ether phosphate, polyoxyethylene lauryl ether phosphate triethanolamine, polyoxyethylene oleyl ether phosphate diethanolamine;

Dioctylamine, dimethylstearylamine, trilaurylamine, diethylaminoethylamide stearate, lauryltrimethylammonium chloride, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, cetyltrimethylammonium saccharin, stearyltrimethylammonium chloride, alkyl chloride (20-22) Trimethylammonium, lauryltrimethylammonium bromide, alkyl (16,18) trimethylammonium chloride, stearyltrimethylammonium bromide, stearyltrimethylammonium saccharin, alkyl (28) trimethylammonium chloride, di (polyoxyethylene) oleylmethylammonium chloride (2EO) ), Dipolyoxyethylene stearylmethylammonium chloride Polyoxyethylene chloride (1) Polyoxypropylene (25) Diethylmethylammonium chloride, tri (polyoxyethylene) stearylammonium chloride (5EO), distearyldimethylammonium chloride, dialkyl (12-15) dimethylammonium chloride, dialkylchloride (12 -18) Dimethyl ammonium, dialkyl chloride (14-18) dimethyl ammonium, dicocoyl dimethyl ammonium chloride, dicetyl dimethyl ammonium chloride, isostearyl lauryl dimethyl ammonium chloride, benzalkonium chloride, myristyl dimethyl benzyl ammonium chloride, lauryl dimethyl chloride ( Ethylbenzyl) ammonium, stearyldimethylbenzylammonium chloride, laurylpyridinium chloride, cetylpyridinium chloride Lauroyl kola amino formyl methyl pyridinium chloride, stearoyl colaminoformyl methylpyridinium bromide alkyl isoquinolizinyloxy potassium, chloride methyl benzethonium, cationic surfactants such as benzethonium chloride;

  2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, alkyldiaminoethylglycine hydrochloride, sodium lauryldiaminoethylglycine, sodium undecylhydroxyethylimidazolium betaine, undecyl-N-carboxymethylimidazolium betaine Coconut oil fatty acid acyl-N-carboxyethyl-N-hydroxyethylethylenediamine disodium, coconut oil fatty acid acyl-N-carboxyethoxyethyl-N-carboxyethylethylenediamine disodium, coconut oil fatty acid acyl-N-carboxymethoxyethyl-N -Carboxymethylethylenediamine disodium, sodium laurylaminopropionate, sodium laurylaminodipropionate, laurylaminopropyl Triethanolamine pionate, palm oil fatty acid acyl-N-carboxyethyl-N-hydroxyethylethylenediamine sodium, lauryldimethylaminoacetic acid betaine, coconut oil alkyldimethylaminoacetic acid betaine, stearyldimethylaminoacetic acid betaine, stearyldimethylbetaine sodium, coconut oil Amphoteric surfactants such as fatty acid amidopropyl betaine, palm oil fatty acid amidopropyl betaine, lauric acid amidopropyl acetic acid betaine, ricinoleic acid amidopropyl betaine, stearyl dihydroxyethyl betaine, lauryl hydroxysulfo betaine;

Polyoxyethylene (10) alkyl (12,13) ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene (3, 7, 12) alkyl ( 12-14) ether, polyoxyethylene tridecyl ether, polyoxyethylene myristyl ether, polyoxyethylene-sec-alkyl (14) ether, polyoxyethylene isocetyl ether, polyoxyethylene cetostearyl ether, polyoxyethylene (2 , 10, 20) Isostearyl ether, polyoxyethylene oleyl cetyl ether, polyoxyethylene (20) aralkyl ether, polyoxyethylene octyldodecyl ether Polyoxyethylene behenyl ether, polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether, polyoxyethylene dinonyl phenyl ether, polyoxyethylene (1) polyoxypropylene (1,2,4,8) cetyl ether, poly Oxyethylene (5) polyoxypropylene (1,2,4,8) cetyl ether, polyoxyethylene (10) polyoxypropylene (1,2,4,8) cetyl ether, polyoxyethylene (20) polyoxypropylene (1,2,4,8) cetyl ether, polyoxyethylene polyoxypropylene lauryl ether, polyoxyethylene (3) polyoxypropylene (34) stearyl ether, polyoxyethylene (4) polyoxypropylene (30) Allyl ether, polyoxyethylene (34) polyoxypropylene (23) stearyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene decyl tetradecyl ether, polyethylene glycol monolaurate, ethylene glycol monostearate, mono Polyethylene glycol stearate, polyethylene glycol monooleate, ethylene glycol fatty acid ester, self-emulsifying ethylene glycol monostearate, diethylene glycol laurate, polyethylene glycol myristate, polyethylene glycol palmitate, diethylene glycol stearate, polyethylene monostearate self-emulsifying Glycol (2), polyethylene isostearate Recall, ethylene glycol dioctanoate, diethylene glycol dilaurate, polyethylene glycol dilaurate, polyethylene glycol dipalmitate (150), ethylene glycol distearate, diethylene glycol distearate, polyethylene glycol distearate, ethylene glycol dioleate, polyethylene glycol dioleate, diglycol Polyethylene glycol ricinoleate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (6) sorbitan monostearate, polyoxyethylene (20) sorbitan monostearate, tristearin Acid polyoxyethylene (20) sorbitan, monooleic acid polyoxy Tylene (6) sorbitan, polyoxyethylene monooleate (20) sorbitan, polyoxyethylene trioleate (20) sorbitan, polyoxyethylene (20) coconut oil fatty acid sorbitan, polyoxyethylene monolaurate (10-80) sorbitan , Polyoxyethylene sorbitan tristearate, polyoxyethylene (20) sorbitan isostearate, polyoxyethylene (150) sorbitan tristearate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene (10) cured Castor oil, polyoxyethylene (20) hydrogenated castor oil, polyoxyethylene (40) hydrogenated castor oil, polyoxyethylene (50) hydrogenated castor oil, polyoxyethylene (60) hydrogenated castor oil, lipophilic monos Glyceryl allate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl coconut oil, glyceryl laurate, glyceryl myristate, glyceryl isostearate, glyceryl ricinoleate, glyceryl monohydroxystearate, glyceryl oleate , Glyceryl linoleate, glyceryl erucate, glyceryl behenate, wheat germ oil fatty acid glyceride, safflower oil fatty acid glyceryl, hydrogenated soybean fatty acid glyceryl, saturated fatty acid glyceride, cottonseed oil fatty acid glyceryl, monoisostearic acid monomyristate glyceryl, mono cow fatty acid Glycerides, monolanolin fatty acid glyceryl, glyceryl sesquioleate, glyceryl distearate, glyceryl diisostearate, diarachin Glyceryl acid, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monoisostearate, sorbitan monooleate, sorbitan sesquistearate, sorbitan sesquioleate, sorbitan tristearate, sorbitan trioleate, coconut fatty acid Sorbitan, sorbitan isostearate, sorbitan sesquiisostearate, sorbitan distearate, diglyceryl isopalmitate, poly (4-10) glyceryl monolaurate, poly (10) glyceryl monomyristate, poly (2-10) glyceryl monostearate , Poly (2-10) glyceryl monoisostearate, poly (2-10) glyceryl monooleate, diglyceryl sesquioleate, diisostearate Poly (2-10) glyceryl acid, poly (6-10) glyceryl distearate, diglyceryl triisostearate, poly (10) glyceryl tristearate, poly (10) glyceryl trioleate, poly (2) tetraisostearate ) Glyceryl, decaglyceryl pentastearate, poly (6-10) glyceryl pentaoleate, poly (10) glyceryl heptearate, decaglyceryl decastearate, poly (10) glyceryl dekaoleate, poly (6) condensed ricinoleate Glyceryl, sucrose fatty acid ester, coconut oil fatty acid sucrose ester, alkyl glucoside, coconut oil alkyl dimethylamine oxide, lauryl dimethylamine oxide, dihydroxyethyl lauryl dimethylamine oxide, stearyl dimethyl ester Le amine oxide, oleyl dimethyl amine oxide, and nonionic surfactants such as polyoxyethylene coconut oil alkyldimethylamine oxide;

  Saponin, lecithin, soybean phospholipid, hydrogenated soybean phospholipid, soybean lysophospholipid, hydrogenated soybean lysophospholipid, egg yolk lecithin, hydrogenated egg yolk lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingophospholipid, sphingomyelin, ganglioside Natural surfactants such as bile acid, cholic acid, deoxycholic acid, sodium cholate, sodium deoxycholate, spicrispolic acid, rhamnolipid, trehalose lipid, sophorolipid, mannosylerythritol lipid;

  Paraaminobenzoic acid, ethyl paraaminobenzoate, glyceryl paraaminobenzoate, amyl paradimethylaminobenzoate, paraaminobenzoic acid derivatives such as 2-ethylhexyl paradimethylaminobenzoate, benzyl cinnamate, monoparamethoxycinnamic acid mono-2 -Glyceryl ethylhexanoate, methyl 2,4-diisopropylcinnamate, ethyl 2,4-diisopropylcinnamate, potassium paramethoxycinnamate, sodium paramethoxycinnamate, isopropyl paramethoxycinnamate, paramethoxycinnamate Cinnamic acid derivatives such as 2-ethylhexyl cinnamate, 2-ethoxyethyl paramethoxycinnamate, ethyl paraethoxycinnamate, urocanic acid derivatives such as urocanic acid, ethyl urocanate, 2,4-dihydroxybenzophenone, 2, 2 ', 4, '-Tetrahydroxybenzophenone, 2-hydroxy-4-methoxy-5-sulfobenzophenone sodium, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 2-hydroxy-4-methoxybenzophenone, 2,2'-dihydroxy- Benzophenone derivatives such as sodium 4,4′-dimethoxybenzophenone and sodium 2,2′-dihydroxy-4,4′-dimethoxy-5-sulfobenzophenone, ethylene glycol salicylate, 2-ethylhexyl salicylate, phenyl salicylate, benzyl salicylate, salicylic acid p -Salicylic acid derivatives such as tert-butylphenyl, homomenthyl salicylate, salicylic acid-3,3,5-trimethylcyclohexyl, 2- (2'-hydroxy-5'-methoxyphenyl) benzoto Azole, 4-tert-butyl-4'-methoxybenzoyl ultraviolet absorbers such as methane;

  Kaolin, anhydrous silicic acid, magnesium aluminum silicate, sericite, talc, boron nitride, mica, montmorillonite, hemp cellulose powder, wheat starch, silk powder, corn starch, nitro dye, azo dye, nitroso dye, triphenyl Methane dyes, xanthene dyes, quinoline dyes, anthraquinone dyes, indigo dyes, pyrene dyes, phthalocyanine dyes, flavonoids, quinone, porphyrins, water-soluble anato, squid powder, caramel, guaiazulene, gardenia blue, gardenia yellow , Cochineal, shikonin, copper chlorophyllin sodium, paprika dye, safflower red, safflower yellow, lacqueric acid, riboflavin butyrate, and other natural dyes, carbon black, yellow iron oxide, black iron oxide, red rose, conger, group , Zinc oxide, chromium oxide, titanium oxide, black titanium oxide, zirconium oxide, chromium hydroxide, alumina, magnesium oxide, barium sulfate, aluminum hydroxide, calcium carbonate, lithium cobalt titanate, manganese violet, pearl pigment powder, etc. And coloring materials;

  Ashitaba extract, Acacia catechu extract, Avocado extract, Achacha extract, Achachazul extract, Altea extract, Arnica extract, Oil-soluble Arnica extract, Almond extract, Aloe extract, Anchovy extract, Ginkgo biloba extract, Nettle extract, Iris root extract, Fennel extract , Turmeric extract, Ages extract, Echinacea leaf extract, Ogon extract, Oat extract, Prunus extract, Barley extract, Okra extract, Hypericum extract, Oil-soluble Hypericum extract, Adriatic extract, Oil-soluble extract , Orange flower water, seaweed extract, oyster tannin, cuckoo extract, valerian extract, cattail extract, chamomile extract, oil-soluble chamomile extract, chamomile Water, oat extract, carrot extract, oil-soluble carrot extract, carrot oil, pearl millet extract, licorice extract, licorice extract powder, licorice flavonoids, cantalis tincture, raspberry extract, kiwi extract, quina extract, cucumber extract, kyonin extract, quince seed extract , Gardenia extract, Kumazasa extract, Clara extract, walnut shell extract, grapefruit extract, clematis extract, brown sugar extract, chlorella extract, mulberry extract, cinnamon extract, gentian extract, gentian pepper extract, black tea extract, citrus extract, burdock extract, oil-soluble burdock Extract, wheat germ extract, hydrolyzed wheat powder, rice bran extract, rice bran fermented extract, comfrey extract, saicin extract, saffron extract, savonso Kiss, Oil-soluble Salvia extract, Hawthorn extract, Salamander extract, Shiitake extract, Shiitake extract powder, Ginger extract, Shikon extract, Oil-soluble shikon extract, Perilla extract, Linden extract, Oil-soluble Linden extract, Shimotake extract, Peonies extract, Jujudama extract, Pepper extract Extract, Oil-soluble Pepper extract, Pepper tincture, Ginger root extract, Birch extract, Oil-soluble birch extract, Birch sap, Honeysuckle extract, Japanese horsetail extract, Oil-soluble Japanese horse chestnut extract, Scorginin, Stevia extract, Atlantic kizuta extract, Hawthorn extract , Elderberry extract, black rat extract, yarrow extract, oil-soluble yarrow extract, mint extract, sage extract, oil-soluble Di extract, sage water, mallow extract, celery extract, nematode extract, nematode water, assembly extract, soy extract, tiso extract, thyme extract, tea extract, tea dry extract, tea seed extract, clove extract, chimpi extract, camellia extract, camellia extract, oil Soluble Teuchigurumi extract, Duke extract, Terminaria extract, Capsicum tincture, Toki extract, Oil-soluble Toki extract, Toki water, Toki-senka extract, Oil-soluble Toki-senka extract, Soy milk powder, Tonin extract, Spruce extract, Dokudami extract, Tomato extract, Toll Mentilla extract, natto extract, carrot extract, oil-soluble carrot extract, garlic extract, wild rose extract, oil-soluble wild rose extract, buckthorn extract, buckthorn root extract, buckthorn extract, parsley extract Concentrated leaf juice, distilled mint water, hamamelis water, hamamelis extract, rose extract, parietalia extract, toad extract, loquat leaf extract, oil-soluble loquat leaf extract, dandelion extract, butterfly extract, butcher bloom extract, butcher bloom extract powder , Grape extract, Grape leaf extract, Grape water, Hayflower extract, Loofah extract, Loofah water, Safflower extract, Oil soluble bodai extract, Bodaiju water, Button extract, Hop extract, Oil soluble hop extract, Pine extract, Maria thistle extract, Maronier Extract, Oil-soluble Maronnier extract, Mukuroji extract, Melissa extract, Merirot extract, Peach leaf extract, Oil-soluble Peach leaf extract, Peach extract, Cornflower extract, Cornflower water, Eucalyptus extract, Yukinoshita Eki , Lily extract, Yokuinin extract, oil soluble Yokuinin extract, mugwort extract, mugwort water, lavender extract, lavender water, apple extract, litchi extract, lettuce extract, lemon extract, lotus extract, rose water, rosemary extract, oil soluble rosemary extract , Plant extracts such as Roman chamomile extract, bitumen extract;

  Glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, proline, hydroxyproline, aspartic acid, asparagine, glutamic acid, glutamine, arginine, histidine, lysine, γ-aminobutyric acid Amino acids and peptides such as DL-pyrrolidone carboxylic acid, ε-aminocaproic acid, hydrolyzed elastin, water-soluble elastin, hydrolyzed collagen, water-soluble collagen, casein, glutathione, wheat peptide, soybean peptide;

  Vitamin A such as retinol, retinal, retinoic acid, retinol acetate, retinol palmitate, etc., carotenoids such as α-carotene, β-carotene, γ-carotene, δ-carotene, lycopene, zeaxanthin, cryptoxanthine, echinone, astaxanthin, Vitamin B1 such as thiamine, vitamin B2 such as riboflavin, vitamin B6 such as pyridoxine, pyridoxal, pyridoxamine, vitamin B12 such as cyanocobalamin, folic acid, nicotinic acid, nicotinamide, pantothenic acid, biotins, L -Ascorbic acid, sodium L-ascorbate, L-ascorbyl stearate, L-ascorbyl palmitate, L-ascorbyl dipalmitate, L-ascorbyl tetraisopalmitate, L-ascorb Vitamin Cs such as disodium acid sulfate ester, L-ascorbyl magnesium, sodium L-ascorbyl phosphate, L-ascorbic acid-2-glucoside, vitamins D such as ergocalciferol, cholecalciferol, d-α- Vitamin E such as tocopherol, DL-α-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol succinate, β-tocopherol, γ-tocopherol, d-δ-tocopherol, ubiquinones, vitamin K, carnitine Vitamins such as ferulic acid, γ-oryzanol, α-lipoic acid, orotic acid and vitamin-like agents;

  Benzoic acid, sodium benzoate, undecylenic acid, salicylic acid, sorbic acid, potassium sorbate, dehydroacetic acid, sodium dehydroacetate, isobutyl paraoxybenzoate, isopropyl paraoxybenzoate, ethyl paraoxybenzoate, butyl paraoxybenzoate, propyl paraoxybenzoate , Preservatives such as benzyl paraoxybenzoate, methyl paraoxybenzoate, methyl sodium paraoxybenzoate, phenoxyethanol, photosensitive element 101, photosensitive element 201, photosensitive element 401;

Antioxidants such as butylhydroxyanisole, butylhydroxytoluene, propyl gallate, erythorbic acid, sodium erythorbate, parahydroxyanisole, octyl gallate;
Metal ion sequestering agents such as trisodium ethylenediaminehydroxyethyl triacetate, edetic acid, disodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, gluconic acid, phytic acid, sodium polyphosphate, sodium metaphosphate ;
Moisturizers such as hyaluronic acid, sodium hyaluronate, sodium chondroitin sulfate, sodium lactate, sodium pyrrolidonecarboxylate, betaine, lactic acid bacteria culture, yeast extract, ceramide;

Anti-inflammatory agents such as glycyrrhizic acid, trisodium glycyrrhizinate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, β-glycyrrhetinic acid, glyceryl glycyrrhetinate, stearyl glycyrrhetinate, lysozyme chloride, hydrocortisone, allantoin;
PH adjusters such as sodium hydroxide, potassium hydroxide, triethanolamine;
Salts such as sodium chloride, potassium chloride, magnesium chloride, sodium sulfate;
Α-hydroxy acids such as citric acid, glycolic acid, tartaric acid, lactic acid;
Whitening agents such as arbutin, α-arbutin, placenta extract;

Angelica oil, Ylang-Ylang oil, Elemi oil, Orange oil, Chamomile oil, Roman chamomile oil, Cardamom oil, Calamus oil, Galvanum oil, Camphor oil, Carrot seed oil, Clarage sage oil, Grapefruit oil, Clove oil, Keihi oil, Coriander oil , Cypress oil, Sandalwood oil, Cedarwood oil, Citronella oil, Cinnamon leaf oil, Jasmine absolute, Juniper berry oil, Ginger extract, Spearmint oil, Sage oil, Cedar oil, Geranium oil, Thyme oil, Tea tree oil, Nutmeg oil , Near uri oil, neroli oil, pine oil, basil oil, peppermint oil, patchouli oil, palmarosa oil, fennel oil, petitgren oil, black pepper oil, frankincense oil, vetiver oil, peppermint oil, bergamot oil Zoin oil, Boad Rose oil, Marjoram oil, Mandarin oil, Myrrh oil, Melissa oil, Eucalyptus oil, Yuzu oil, Lime oil, Lavensara oil, Lavandin oil, Lavender oil, Linden oil, Lemon oil, Lemongrass oil, Rose oil, Rose Essential oils such as wood oil, rosemary oil, lobe oil;
Terpenes such as limonene, pinene, terpinene, terpinolene, myrcene, longiferin, etc .;
A fragrance | flavor, water, etc. are mentioned.

Furthermore, in cosmetics that are one embodiment of the present invention, existing cosmetic raw materials can be added at a general concentration. For example, Cosmetic Material Standards Second Edition Annotation, Japan Official Church Church, 1984 (Pharmaceutical Daily Report), Cosmetic Raw Material Standards Independent Component Standards, Ministry of Health and Welfare Pharmaceutical Affairs Bureau Examination Division Supervision, 1993 (Pharmaceutical Daily Report) Standards supplement, supervised by Ministry of Health and Welfare Pharmacy Examination Division, 1993 (Pharmaceutical Daily Report), permission standards by cosmetic type, supervision by Ministry of Health and Welfare Pharmacy Examination Division, 1993 (Pharmaceutical Daily Report), formulation standard by cosmetic variety, Ministry of Health and Welfare Pharmacy Examination Division All cosmetic raw materials described in Supervision, 1997 (Pharmaceutical Daily), Cosmetics Material Dictionary, 1991 (Nikko Chemicals), etc. can be used.
These preferable contents are 0.01-90 mass% with respect to cosmetics whole quantity, More preferably, it is 0.1-25 mass%, More preferably, it is 0.3-10 mass%.

Example 1: Preparation of extracellular matrix adhesion protein
50 mL of the culture solution of Lactobacillus crispatus JCM5810 (distributed from RIKEN Microorganism Storage Facility) cultured for 48 hours at 37 ° C. in MRS broth was inoculated into 500 mL of the same medium and cultured at 37 ° C. for 48 hours. The culture solution was subjected to centrifugation at 8,000 × g for 10 minutes to recover the cells. The collected cells were suspended in a guanidine hydrochloride solution (2M, 100 mL) and stirred at 37 ° C. for 2 hours. The suspension was subjected to centrifugation at 10,000 × g for 10 minutes, and the supernatant was collected. This was dialyzed twice against 50 volumes of distilled water, and the resulting dialyzed internal solution was freeze-dried. A part of the obtained dried product was dissolved in distilled water, and the amount of protein was quantified using Protein Assay DC kit (manufactured by Bio-Lad). The product obtained from 500 mL of the culture solution was 15.3 mg of protein. This was used for the following experiment as extracellular matrix adhesion protein A.

Example 2: Preparation of extracellular matrix adhesion protein
100 mL of the culture solution cultured at 37 ° C. for 48 hours in MRS broth was collected by centrifugation at 8,000 × g for 10 minutes, and the cells of Lactobacillus crispatus JCM5810 washed with sodium phosphate buffer (50 mM) were added to guanidine hydrochloride solution ( 6M, 3 mL) and stirred at 20 ° C. for 20 minutes. The cells were collected by centrifugation at 8,000 × g for 10 minutes, washed with Tris buffer (pH 8.2, 20 mM), and resuspended in 2.5 mL of the same buffer. Polyethylene glycol 20000 (5 mL) and a lysozyme solution (2.5 mL, prepared to 4 mg / mL, Boehringer Mannheim) were added, and the solution was gently shaken at 37 ° C. for 60 minutes. After adding an EDTA solution (0.2 M, 5 mL), the cells were collected by centrifugation at 4 ° C. and 3,000 × g for 15 minutes. The obtained cells were suspended in a Tris buffer solution (pH 8.2, 10 mL) containing a mutanolysin solution (50 μL, 15,000 U / mL, manufactured by Sigma), and gently shaken at 37 ° C. for 1 hour. % Sarcosyl (1.5 mL, manufactured by Sigma) and sodium chloride solution (5 M, 3 mL) were added to solubilize the cells. Further, sodium perchlorate (5M, 2.9 mL) was added, followed by extraction with chloroform / isoamyl alcohol (24: 1). The solution after extraction was subjected to ethanol precipitation, and the resulting precipitate was used as genomic DNA and used in the following experiments.
Based on the nucleotide sequence disclosed in GenBank Accession No. AF001313, using the obtained genomic DNA as a template, it contains the full length of the mature part, and the restriction enzyme BamHI site on the 5 ′ side of the forward and the 3 ′ side of the reverse A primer was designed and synthesized so as to include a restriction enzyme SacI site downstream of the stop codon, and PCR amplification reaction was performed.

The approximately 800 base pair fragment obtained as a result of the amplification reaction was connected to the BamHI and SacI sites of the expression vector pQE30 (manufactured by Qiagen) according to a conventional method, and Escherichia coli M15 (pREP4) strain (manufactured by Qiagen) was used using this plasmid. ) Was transformed according to the manual.
This transformant was cultured with shaking in 200 mL of LB medium at 35 ° C. for 24 hours, after which IPTG having a final concentration of 1 mM was added and further cultured with shaking for 6 hours. The cultured cells were collected by centrifugation, washed with sodium phosphate buffer (50 mM), resuspended in an aqueous guanidine hydrochloride solution (6 M, 50 mL), and stirred at room temperature for 12 hours. This solution was centrifuged at 10,000 × g for 10 minutes to recover the supernatant, and the entire amount was passed through an affinity column (HiTrap Chelating HP, bed volume 5 mL, manufactured by Amersham Bioscience).
The column was washed with a sodium phosphate buffer (50 mM, pH 8.0, 50 mL) containing NaCl (0.1 M), and then passed through a sodium phosphate buffer (50 mM, pH 4.0, 50 mL) containing NaCl (0.1 M). The eluate was sequentially fractionated into 5 mL × 10-point fractions, and the protein elution fraction was collected by absorbance at 280 nm. A portion of the recovered protein fraction, about 10 μg of the protein amount, is subjected to SDS-polyacrylamide (12%) electrophoresis, and an electroblotter (Electro-Blot Unit for 10 × 10 cm Gel, manufactured by Funakoshi) is used. And blotted on ProBlott PVDF membrane (Applied Biosystems). After the membrane was stained with CBB and the main component was confirmed to be a protein of about 43 kilodaltons, the band part of the membrane was cut out, and the N-terminal amino acid sequence was cut out according to the manual using a Procise 494A protein sequencer (Applied Biosystems). Was analyzed. The obtained amino acid sequence was represented by SEQ ID NO: 2 and reflected the designed base sequence.
N-Asp Ala Val Val Ser Ala Asn Asn Ser Asn Leu Gly C (SEQ ID NO: 2)

  The remainder of the protein fraction was dialyzed twice against 100 volumes of distilled water, subjected to lyophilization, and the resulting dialyzed internal solution was lyophilized. A part of the obtained dried product was dissolved in distilled water, and the amount of protein was quantified using Protein Assay DC kit. The product obtained from 200 mL of the culture solution was 20.4 mg of protein. This was used for the following experiment as extracellular matrix adhesion protein B.

Example 3: Microbial adhesion inhibition test to extracellular matrix Collagen I, III, IV, V type, laminin, fibronectin, bovine serum albumin (BSA), fetuin on Teflon-printed slide glass (ER-208L, manufactured by Funakoshi) An aqueous solution (manufactured by Sigma) was dropped to 2.5 pmol / well and air-dried. This slide glass was immersed in a sodium phosphate buffer solution (50 mM, pH 7.0) containing 2% BSA for 2 hours at room temperature, the remaining slide glass surface was masked, lifted again, and air-dried. This was used as a test slide.

Malassezia furfur NBRC 0656 and Candida albicans NBRC 1594 (both from the National Institute of Technology and Evaluation, Biotechnology Headquarters, Biogenetic Resources Division (NBRC)) cultured in advance under the following culture conditions 1 × 10 ⁸ / It was suspended in a sodium phosphate buffer (50 mM, pH 7.0) so as to be equivalent to mL. The bacterial concentration was adjusted in advance by obtaining the number of bacteria per turbidity by the plating method so that it could be estimated from the turbidity, and the actual dilution suspension was performed in terms of turbidity. Further, extracellular matrix adhesion proteins A and B prepared in Examples 1 and 2 were added to 100 μg / mL of this bacterial cell suspension and dissolved. The test slide coated with each extracellular matrix was immersed in the bacterial cell suspension containing the extracellular matrix-adhering protein for 2 hours at room temperature. The slide glass was pulled up from the suspension and washed by repeating immersion for 3 minutes in sodium phosphate buffer (50 mM, pH 7.0) three times. The slide glass was immersed in 70% ethanol for 1 minute and then air-dried to fix the attached microorganism, and then immersed in Gram Crystal Violet solution for 1 minute to stain the attached microorganism. Twelve randomly selected visual fields (each 4.38 × 10 ³ μm ² ) on the coated surface were photographed with an optical microscope, and the attached microorganisms observed in the visual field were counted, and the average value was obtained. The result of Malassezia furfur NBRC 0656 is shown in FIG. 1, and the result of Candida albicans NBRC 1594 is shown in FIG.

<Culture conditions>
Malassezia furfur NBRC 0656
Freeze-dried cells ↓ Suspended in physiological saline ↓ Streaked to Modified Dixon's agar ↓ Static culture 30 ° C for 5 days Scraped with platinum ears, suspended in phosphate buffer and supplied to the test

Modified Dixon's agar Composition:
Malt extract 36g
Peptone 6g
Bile powder 20g
Tween 40 10ml
Glycerin 2ml
Oleic acid 2ml
Agar 12g
/ 1L distilled water pH 6.0

Candida albicans NBRC 1594
Freeze-dried cells ↓ Suspended in physiological saline ↓ Streaked to YM agar ↓ Static culture 25 ° C for 3 days ↓ Inoculated into YM broth in platinum ears ↓ Shaking culture 25 ° C 18 hours ↓ Centrifugal 8,000 rpm, 10 minutes Phosphoric acid Suspend in buffer and donate to test

Comparative example:
The test slide prepared in the same manner as in Example 3 was treated with a buffer solution excluding the matrix-adhering protein, and the attached cells were counted in the same manner as in Example 3 to obtain the average value. The result of Malassezia furfur NBRC 0656 is shown in FIG. 1, and the result of Candida albicans NBRC 1594 is shown in FIG.

It is a figure which shows the result of the microorganisms (Malassezia furfur NBRC 0656) adhesion inhibition test by the extracellular matrix adhesion protein of Example 3 and Comparative Example 1. It is a figure which shows the result of the microorganisms (Candida albicans NBRC 1594) adhesion inhibition test by the extracellular matrix adhesion protein of Example 3 and Comparative Example 1.

Claims (10)

  A skin external preparation containing a protein derived from a microorganism and having adhesion to an extracellular matrix. Microorganisms, Lactobacillus (Lactobacillus) genus Bifidobacterium (Bifidobacterium) genus Escherichia (Escherichia) genus Salmonella (Salmonella) genus, Aeromonas (Aeromonas) genus Streptococcus (Streptococcus) species, Staphylococcus (Staphylococcus) genus , Helicobacter (Helicobacter) genus Campylobacter (Campylobacter) genus, and Yersinia (Yersinia) skin external agent of claim 1 selected from the microorganisms belonging to the genus.   A protein having an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted, deleted or inserted by another amino acid; The external preparation for skin according to claim 1, characterized in that it is characterized by the following.   The protein encodes an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 1 is encoded, or an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, deleted or inserted by other amino acids The external preparation for skin according to claim 1, which is produced by a microorganism transformed with DNA. The skin external preparation according to claim 1, wherein the protein having adhesion to the extracellular matrix is obtained from Lactobacillus crispatus . The skin external preparation according to claim 5, wherein the protein having adhesion to the extracellular matrix is obtained from Lactobacillus crispatus JCM5810.   A method for preventing the adhesion of harmful microorganisms to the skin, comprising using the external preparation for skin according to any one of claims 1 to 4.   A method for preventing the growth of harmful skin microorganisms, comprising using the external preparation for skin according to any one of claims 1 to 4. Lactobacillus (Lactobacillus) genus Bifidobacterium (Bifidobacterium) genus Escherichia (Escherichia) genus Salmonella (Salmonella) genus Aeromonas (Aeromonas) genus Streptococcus (Streptococcus) genus, Staphylococcus (Staphylococcus) genus Helicobacter ( Helicobacter) genus Campylobacter (Campylobacter) genus, and Yersinia (Yersinia) adherent to the extracellular matrix, characterized in that the urea and / or guanidine hydrochloride than cultured cells of a microorganism belonging to the genus comprising the step of extracting solubilize The manufacturing method of the skin external preparation containing the protein which has.   A protein having adhesion to the extracellular matrix is substituted, deleted, or deleted by DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 by other amino acids Or having an adhesive property to the extracellular matrix according to claim 9, comprising a step of solubilizing and extracting with urea and / or guanidine hydrochloride from cultured cells of a microorganism transformed with DNA encoding the inserted amino acid sequence. The manufacturing method of the skin external preparation containing protein.

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