Antimicrobial substance and method for producing same

Abstract

The present invention relates to an antimicrobial substance and a method for producing the same. The present invention also relates to the use of the antimicrobial substance. The antimicrobial substance disclosed in the present invention is composed of a complex of an acid-form sophorolipid and a Group 11 element ion. The present disclosure provides the use of the antimicrobial substance as an antimicrobial agent and as a surfactant.

Description

Antimicrobial substance and method for producing the same

[0001] This disclosure relates to antimicrobial substances and methods for producing the same. More specifically, it relates to antimicrobial substances comprising sophorolipid, a biosurfactant, as a component, and methods for producing the same. This disclosure also relates to the applications of the said antimicrobial substances.

[0002] Recent advances in medical technology have revealed that some diseases are caused by infections from viruses and microorganisms. Furthermore, global pandemics caused by novel viruses, such as novel influenza and COVID-19, which had not been previously reported, have occurred. On the other hand, focusing on bacteria, the emergence of antibiotic-resistant bacteria and deaths from fulminant hemolytic streptococcal infections, sometimes called flesh-eating bacteria, have been reported even in developed countries. For these reasons, hygiene measures in medical and public facilities, where advanced infection control is particularly necessary, are considered crucial. Antimicrobial agents used for cleaning facility environments require high bactericidal efficacy, but because they are likely to be in contact with many people, high safety and minimal impact on various materials are also required. To address these characteristics, formulations containing antimicrobial metal ions such as silver and copper ions have been marketed (Non-Patent Literature 1, 2, 3). However, it has been reported that their antimicrobial activity is significantly reduced in the presence of chloride ions, amino acids, or proteins (Non-Patent Literature 4).

[0003] Biosurfactants, which are surfactants derived from living organisms, are substances that are expected to be used industrially as next-generation surfactants due to their high biodegradability and safety. Among them, sophorolipid, one of the glycolipid-type biosurfactants, is a fermentation product obtained from yeast fermentation and has been known for its high safety, extremely low toxicity to aquatic organisms (medaka, Daphnia magna, and freshwater algae), and good biodegradability (Non-Patent Literature 5). Sophorolipid is a glycolipid composed of sophorose, a very bulky disaccharide with a hydrophilic group, and a fatty acid. It is produced from yeast as a mixture of lactone-type sophorolipid, a hydrophobic nonionic surfactant, and acid-type sophorolipid, a water-soluble anionic surfactant. Among them, acid-type sophorose lipid has low foaming properties and high cleaning properties, and has been conventionally incorporated into various detergents as an ideal low-foaming surfactant (Non-Patent Literature 6). However, acid-type sophorolipids themselves have weaker antibacterial activity compared to lactone-type sophorolipids and are not suitable for antibacterial applications (Non-patent documents 7, 8).

[0004] Patent No. 5788058

[0005] DH Niles (1999), Appl. Microbiol. Biotechnol. 51,730-750. G. Borkow et. al. (2005), Curr. Med. Chem. 12 (18), 2163-2175. DJ Weavers et. al. CHAPTER 19 In: Block SS, eds. Disinfection, sterilization, and preservation, 5th.Ed. Lippincott Williams & Wilkins. 415-430 (2001).Y. Matsumura et.al. (2003), Appl. Environ. Microbiol., 69, 4278-4281.W. Kumano et al. (2019), J Environ. Biol. 40 (4), 595-600.Hirata, Y., Ryu, M., Igarashi, K., Nagatsuka, A., Furuta, T., Kanaya, S., and Sugiura, M. (2009) J. Oleo. Sci., 58, 565-572.Zhang, X. et al. (2016), Front. Microbiol. 7, 2076.Zhang, X. et al. (2017), Biocatal. Agric. Biotechnol. 11, 176-182.

[0006] This disclosure aims to provide an antimicrobial substance and a method for producing the same. More specifically, this disclosure aims to provide an antimicrobial substance comprising sophorolipid as a component and a method for producing the same. Furthermore, this disclosure aims to provide applications for said antimicrobial substance.

[0007] The inventors of the present invention have been diligently studying to solve the aforementioned problems and have discovered that an aqueous solution prepared by converting an acid-type sophorolipid into an alkali metal salt or alkaline earth metal salt (acid-type sophorolipid salt) and then coexisting it with a group 11 element ion in an aqueous solution has a strong bactericidal effect. They have also confirmed that this bactericidal effect is significantly higher than the bactericidal effect inherently possessed by the group 11 element ions. Furthermore, through further research, they have confirmed that the acid-type sophorolipid and the group 11 element ions bind together in the aqueous solution, forming a complex.

[0008] This disclosure is the result of further research based on these findings and includes the following embodiments. Hereinafter, "sophorolipid" may be abbreviated as "SL".

[0009] (I) Antimicrobial substances and their uses (I-1) An antimicrobial substance comprising a complex of acid-type SL and a group 11 element. (I-2) The antimicrobial substance described in (I-1), characterized by having surfactant properties. (I-3) The antimicrobial substance described in (I-1) or (I-2), wherein the microorganism is a bacterium, a virus, or a fungus. (I-4) An antimicrobial agent containing an effective amount of the antimicrobial substance described in any of (I-1) to (I-3) having antimicrobial activity. (I-5) A surfactant containing an effective amount of the antimicrobial substance described in any of (I-1) to (I-3) having surfactant properties. (I-6) A composition containing the antimicrobial substance described in any of (I-1) to (I-3), the antimicrobial agent described in (I-4), or the surfactant described in (I-5). (I-7) A composition described in (I-6) which is a food or beverage, a pharmaceutical, a quasi-drug, a cosmetic, a deodorant, a disinfectant, or a cleaning agent. (I-8) An antimicrobial material treated with an antimicrobial substance described in any of (I-1) to (I-3) or an antimicrobial agent described in (I-4). The treatment includes mixing, coating, spraying, and immersion of the material with the antimicrobial agent. (I-9) An antimicrobial material described in (I-8) which is a plastic resin, a nonwoven fabric, a fiber, a film, a paint, wood, or a metal. (I-10) An antimicrobial-treated product containing an antimicrobial material described in (I-8) or (I-9). (I-11) Antimicrobial processed products as described in (I-10), which include (I-11) sanitary products (including antimicrobial masks, antimicrobial sheets, sanitary mats, and sanitary gloves), antimicrobial material products (including antimicrobial processed plastic containers, antimicrobial processed cloths, antimicrobial clothing, antimicrobial interior materials, and antimicrobial building materials), and sanitary processed products (including furniture, electrical appliances, and bathroom products that have been treated with antimicrobial materials).

[0010] (II) Method for producing an antimicrobial substance (II-1) A method for producing a composite of acidic SL and a group 11 element, comprising the following steps: (1) A step of heating and reacting acidic SL and an acid salt of a group 11 element in the presence of water; (2) A step of removing the acid from the reaction solution. (II-2) A method for producing a composite of acidic SL and a group 11 element, comprising the following steps: (i) A step of reacting acidic SL with an alkali metal salt or alkaline earth metal salt in the presence of water to form an alkali metal or alkaline earth metal salt of acidic SL; (ii) A step of reacting the alkali metal or alkaline earth metal salt of the acidic SL with an acid salt of a group 11 element in the presence of water; (iii) A step of removing the alkali metal or alkaline earth metal salt of the acid from the reaction solution.

[0011] This disclosure provides a novel antimicrobial substance and a method for producing the same. Furthermore, this disclosure provides applications for the antimicrobial substance. Specifically, since the antimicrobial substance has higher antimicrobial activity than the Group 11 element ions used as raw materials for its production, it can be used as an antimicrobial agent. Also, since the antimicrobial substance has surfactant properties similar to acid-type SL used as a raw material for its production, it can be used as a surfactant. Thus, the antimicrobial substance of this disclosure possesses both antimicrobial and surfactant properties, and can be effectively used in the manufacture of various products requiring these characteristics.

[0012] The upper left image shows each aqueous solution prepared in Experimental Example 1, taken in a transparent container (the original image is in color). From left to right: (1) an aqueous solution of the sodium salt of acidic SL (SL-Na) (colorless and transparent), (2) an aqueous solution of copper sulfate (transparent light blue), and (3) a mixed solution prepared by mixing the SL-Na aqueous solution and the copper sulfate aqueous solution (transparent dark blue). At the bottom of the figure, the chemical formula of the compound (complex of SL and copper) (complex) presumed to be formed in the mixed solution of (3) is shown. The results of Experimental Example 2 are shown. The antibacterial activity (logarithmic decrease value) against Escherichia coli (left) and Pseudomonas aeruginosa (right) was measured for CuSO4 aqueous solution (---◆---), CuSO4 + SL-Na (1 eq.) aqueous solution (---▲---), and CuSO4 + SL-Na (2.0 eq.) aqueous solution (-black square-) is shown. The results of Experimental Example 2 are shown. The left figure shows the results of measuring the antibacterial activity (logarithmic reduction) against E. coli for CuSO4 aqueous solution (---◆---), CuSO4 + SL-Na (0.5eq.) aqueous solution (---●---), CuSO4 + SL-Na (1eq.) aqueous solution (---▲---), and CuSO4 + SL-Na (2.0eq.) aqueous solution (-black square-). The right figure shows the results of measuring the antibacterial activity (logarithmic reduction) against Staphylococcus aureus for CuSO4 aqueous solution (---◆---) and CuSO4 + SL-Na (2.0eq.) aqueous solution (-black square-). The results for Experimental Example 3 are shown. Figure 4(a) shows the UV-Vis spectra of copper sulfate aqueous solution (H), SL-Na aqueous solution (A), and CuSO4 + SL-Na aqueous solutions (B-G) at various mixing ratios. Figure 4(b) shows the absorbance at 680 nm in an aqueous solution of CuSO4 + SL-Na, where Cu 2＋The graph plotted against concentration (mM) is shown. The results of Experimental Example 13 are shown. The results of measuring the antibacterial activity (logarithmic decrease) against E. coli for AgNO3 aqueous solution (---◆---) and AgNO3 + SL-Na (1eq.) aqueous solution (-▲-) are shown. The results of Experimental Example 13 are shown. The left figure shows the results of measuring the antibacterial activity (decrease) against Pseudomonas aeruginosa (left) and Staphylococcus aureus (right) for AgNO3 aqueous solution (---◆---), AgNO3 + SL-Na (0.5eq.) aqueous solution (-●-), AgNO3 + SL-Na (1eq.) aqueous solution (-▲-), and AgNO3 + SL-Na (2.0eq.) aqueous solution (-black square-). The results of Experimental Example 14(1) are shown. Figure 7(a) shows the results of measuring the static surface tension of acid-type SL and SL-copper composite (Cu2(SL)4・2H2O) (Figure 7(b)). The results of Experimental Example 14(2) are shown. The results of evaluating foaming ability (Time: foam volume at 0 min) and foam stability (Time: change in foam volume over time from 0 to 10 min) for acid-type SL and SL-copper composite (Cu2(SL)4・2H2O) are shown. Figure 8(a) shows the results for a compound concentration of 500 μM, and Figure 8(b) shows the results for a compound concentration of 50 μM. In the figures, "AC-100" means acid-type SL. The UV-Vis spectra of the pale blue solid (test sample: SL-copper composite) (A) prepared in Production Example 1, and the control copper acetate (B) are shown. The FT-IR spectra of the pale blue solid (test sample) SL-copper composite prepared in Production Example 1, as well as the control copper acetate and acid-type SL are shown. The UV-Vis spectra of the pale blue solid (test sample: SL-copper composite) (A) prepared in Production Example 2, and the control copper sulfate (B) are shown. The FT-IR spectra of the pale blue solid (test sample: SL-copper composite) prepared in Production Example 2, the precipitate removed during the reaction, and, as controls, the calcium salt of SL (SL2-Ca), calcium sulfate dihydrate (CaSO4・2H2O), and acidic SL are shown.

[0013] (This antimicrobial substance) The antimicrobial substance targeted by this disclosure consists of a complex of acidic SL and Group 11 elements. Hereinafter, this is also referred to as "this antimicrobial substance". Specifically, this antimicrobial substance is a complex formed by the binding of Group 11 element ions to the carboxyl groups of acidic SL. In other words, it is a compound (complex) in which Group 11 element ions are centered and acidic SL is bound as a ligand through its carboxyl group.

[0014] (Group 11 elements) Group 11 elements are elements belonging to Group 11 in the periodic table in IUPAC format. Preferably, they are copper, silver, and gold, which are known to have antibacterial effects as metal ions. More preferably, they are copper and silver, and even more preferably, copper. The ions of Group 11 elements are theoretically considered to be able to take +1, +2, and +3. In this disclosure, copper ions are preferably divalent copper (II) ions. Hereinafter, unless otherwise specified, copper ions mean copper (II) ions. Also, in this disclosure, silver ions are preferably monovalent silver (I) ions. Hereinafter, unless otherwise specified, silver ions mean silver (I) ions.

[0015] (SL and acidic SL) Sophorolipid (SL) is a glycolipid composed of sophorose or sophorose in which some of the hydroxy groups are acetylated or esterified, and hydroxy fatty acids. Sophorose is a sugar composed of two molecules of glucose linked by a β1→2 bond. Hydroxy fatty acids are fatty acids having a hydroxy group. SL is roughly classified into acidic SL in which the carboxyl group of hydroxy fatty acid is free and lactone-type SL bound to sophorose in the molecule. SL obtained by the fermentation of certain yeasts (SL-producing yeasts) is usually a mixture of acidic SL represented by the following general formula (1) and lactone-type SL represented by general formula (2), and fatty acid chain lengths (R ２ ) are different, those in which the 6'(R ３ ) and 6'' positions (R [[ID=eleven]] ４ ) of sophorose are acetylated or protonated, or those in which one of the 3', 4', 2'', 3'' and 4'' positions (R ５ ) of sophorose is esterified, etc., and are obtained as an aggregate of more than 30 structural homologues.

[0016] [Acidic SL]

[0017] [In formula (1), R １ represents a hydrogen atom or a methyl group; R ３ and R ４ are the same or different and represent a hydrogen atom or an acetyl group; R ５ are all hydrogen atoms, or among the five Rs ５ one is a saturated fatty acid residue that may have a hydroxy group or an unsaturated fatty acid residue that may have a hydroxy group, and the rest are hydrogen atoms; R ２ is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more substituents; R ６ represents a hydroxy group. ].

[0018] The acidic SL represented by the above formula (1) is a monomer, and may be referred to as "monomeric acidic SL" with respect to the dimeric SL described later.

[0019] [Lactone-type SL]

[0020] [In formula (2), R １ to R ４ are the same as the definitions defined in formula (1). ].

[0021] Among the acidic SLs targeted in the present disclosure, for the acidic SL represented by the above formula (1), when one of the Rs ５ is a saturated fatty acid residue that may have a hydroxy group or an unsaturated fatty acid residue that may have a hydroxy group, the R ６ at the C-1 position of the acidic SL may include a dimer formed by combining with any one of the Rs ７ of the acidic SL represented by the following formula (3) to form a single bond. That is, in the present disclosure, unless otherwise specifically mentioned, the acidic SL includes both the monomeric acidic SL described above and the following dimeric SL.

[0022] [Dimeric SL]

[0023] [In formula (3), R １’ represents a hydrogen atom or a methyl group; R３’ and R ４’ R represents a hydrogen atom or an acetyl group, either identical or different; ２’ R is a saturated aliphatic hydrocarbon chain, or an unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more substituents; ７ One of them is the R of the acid-type SL shown in formula (1). ６ It combines with another atom to form a single bond, and the rest are all hydrogen atoms.

[0024] In the above general formulas (1) and (3), R ２ or R ２’ The number of carbon atoms in the saturated or unsaturated aliphatic hydrocarbon chain shown is not limited, but is typically 9 to 20, preferably 9 to 18, more preferably 11 to 16, and particularly preferably 14 to 16. Examples of saturated aliphatic hydrocarbon chains include linear or branched alkylene groups. Linear alkylene groups are preferred. Examples of unsaturated aliphatic hydrocarbon chains include alkenylene groups having 1 to 3 double bonds. Alkenylene groups having 1 to 2 double bonds are preferred, and alkenylene groups having 1 double bond are preferred. ２ or R ２’ The substituents on the saturated or unsaturated aliphatic hydrocarbon chain shown are not particularly limited, but examples include halogen atoms, hydroxyl groups, lower (C1-6) alkyl groups, halo-lower (C1-6) alkyl groups, hydroxy-lower (C1-6) alkyl groups, and halo-lower (C1-6) alkoxy groups. Examples of halogen atoms bonded to halogen atoms or alkyl or alkoxy groups include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0025] In the above general formula (1), R ５Examples of saturated fatty acid residues include linear fatty acid residues having 12 to 20 carbon atoms (lauric acid residues, myristic acid residues, pentadecyl acid residues, palmitic acid residues, margaric acid residues, stearic acid residues, and arachidin residues). Preferably, linear fatty acid residues have 14 to 20 carbon atoms, more preferably 16 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms, with palmitic acid residues having 16 carbon atoms and stearic acid residues having 18 carbon atoms being particularly preferred. Examples of unsaturated fatty acid residues include linear fatty acid residues having 12 to 20 carbon atoms and having 1 to 3 double bonds. Preferably, the number of double bonds is 1 to 2, more preferably 1. Preferably, the number of carbon atoms is 16 to 20, more preferably 16 to 18, and particularly preferably 18. Suitable unsaturated fatty acid residues include: a 16-carbon palmitoleic acid residue with one double bond; an 18-carbon oleic acid residue or vaccenic acid residue (preferably an oleic acid residue) with one double bond; a 18-carbon linoleic acid residue with two double bonds; a 18-carbon (9,12,15) linolenic acid residue, a (6,9,12) linolenic acid residue, and an eleostearic acid residue with three double bonds; a 20-carbon (9,12,15) linolenic acid residue, a (6,9,12) linolenic acid residue, and an eleostearic acid residue with two double bonds. More preferably, a 16-carbon palmitoleic acid residue and a 18-carbon oleic acid residue with one double bond are used, and particularly preferably, a 18-carbon oleic acid residue with one double bond is used.

[0026] These fatty acid residues may or may not have hydroxyl groups. If they have hydroxyl groups, the number of hydroxyl groups can be 1 to 2, preferably 1. The position of the hydroxyl group on the fatty acid residue can be the ω position or the ω-1 position. In acidic SL(1), R ５ -OR when is a saturated fatty acid residue that may have a hydroxyl group or an unsaturated fatty acid residue that may have a hydroxyl group ５The position may be any of the 3', 4', 2", 3", and 4" positions of the sophorose ring. In other words, in the acidic SL(1), the above fatty acid residue R may be present at any one of these positions. ５ Having - OR ５ It contains SL compounds formed by the bonding of groups.

[0027] As the SL-producing yeast, Starmerella bombicola, a known SL-producing yeast that is known to produce large amounts of SL, can be preferably cited. The genus Starmerella is a yeast that was previously known as the genus Candida. Starmerella bombicola is registered in the ATCC (American Type Culture Collection), a biological resource bank, and can be obtained from there (e.g., Candida bombicola ATCC22214). Other SL-producing yeasts known to produce SL can also be used. Examples of such SL-producing yeasts, though not limited to them, include Candida apicola, Candida petrophilum, and Rhodotorula (Candida) bogoriensis.

[0028] SL can be produced by culturing SL-producing yeast under predetermined conditions using an aqueous liquid medium containing a hydrophobic substrate and a hydrophilic substrate. The hydrophobic substrate is not limited, but preferably includes fatty acids and vegetable oils composed of fatty acids. Examples of fatty acids include saturated or unsaturated fatty acids having 6 to 18 carbon atoms. These can be used individually or in combination of two or more. Preferably, it is at least one fatty acid selected from saturated or unsaturated fatty acids having 12 to 18 carbon atoms, and more preferably at least one fatty acid selected from saturated fatty acids having 16 carbon atoms (palmitic acid) and saturated or unsaturated fatty acids having 18 carbon atoms (stearic acid, oleic acid, linoleic acid, linolenic acid). The vegetable oil is not limited, but preferably includes those containing saturated or unsaturated fatty acids having 12 to 18 carbon atoms as constituent fatty acids of triglycerides. Examples of such vegetable oils include soybean oil, rapeseed oil, cottonseed oil, sunflower oil (high linol, high oleic), kapok oil, sesame oil, corn oil, rice oil, peanut oil, safflower oil (high linol, high oleic), olive oil, linseed oil, tuki oil, castor oil, palm kernel oil, palm olein, palm stearin, and coconut oil. All of these are vegetable oils with a melting point of 30°C or lower. They can be used individually or in combination of two or more.

[0029] The hydrophilic substrate is one that has a high affinity for water used as the culture medium solvent, and is not limited, but preferably includes sugars and nitrogen sources necessary for the growth of SL-producing yeast. In addition to the hydrophobic and hydrophilic substrates mentioned above, the culture medium may also optionally contain inorganic salts (e.g., phosphates, magnesium salts, sodium salts, etc.), organic acids (e.g., lactic acid, acetic acid, citric acid, propionic acid, etc.), and vitamins that are useful for the growth of SL-producing yeast.

[0030] SL can be separated by purifying the culture obtained by culturing SL-producing yeast through processes such as centrifugation, decantation, and ethyl acetate extraction. Further washing with hexane yields a brownish, syrupy substance. Since SL has a higher specific gravity than water, it can be easily separated by settling to the lower layer when the culture is allowed to stand. The SL thus obtained is a mixture of the acidic SL and lactoneic SL described above.

[0031] Acid-type SL can be prepared by alkaline treatment of a mixture of acid-type SL and lactone-type SL obtained by the above method, although this method is not limited, to hydrolyze the ester bonds in the lactone-type SL. Examples of alkaline treatment methods include alkaline reflux, but the method is not limited to this, and known alkaline treatment methods can be used. Furthermore, it may be produced according to the method described in Patent Document 1, thereby obtaining highly purified acid-type SL. Acid-type SL is also readily available commercially; for example, it is sold by Saraya Co., Ltd. under the product names "SOFORO® AC-30" or "SOFORO® AC-100".

[0032] (Method for producing this antimicrobial substance) Method 1 This antimicrobial substance can be produced by the following steps, although it is not limited to the following: (1) Step 1: Heating and reacting an acidic form of SL with an acid salt of a group 11 element in the presence of water; (2) Step 2: Removing the acid produced from the reaction solution.

[0033] Examples of salts of Group 11 elements used in Step 1 include salts of Group 11 elements with inorganic acids (such as hydrochloric acid, sulfuric acid, and nitric acid) or organic acids (such as acetic acid, citric acid, malic acid, lactic acid, and succinic acid). Although not limited, for example, when the Group 11 element is copper, copper acetate and sulfate salts are preferably exemplified. Also, when the Group 11 element is silver, nitrate salts are preferably exemplified. The salt of the Group 11 element may be an anhydrous or a solvate such as a hydrate. Examples of solvates of Group 11 element salts may preferably include copper(II) acetate monohydrate, copper(II) sulfate dihydrate, and copper(II) sulfate pentahydrate.

[0034] Step 1 can be carried out by dissolving the acid salt of a Group 11 element and the acid form SL in water, and then heating it in the range of 40 to 90°C, although this is not limited. The heating temperature is not limited, but preferably ranges of 50 to 80°C, more preferably ranges of 60 to 70°C. The reaction time is not particularly limited as long as it is the time required for the acid form SL and the Group 11 element ions to combine and form a complex in the aqueous solution. For example, it can be appropriately set in the range of 1 minute to 12 hours.

[0035] In step 1, the ratio of the acid salt of the group 11 element to the acid-type SL in the aqueous solution is not limited, but for example, if the group 11 element is a divalent metal (e.g., copper), it is preferable to adjust the ratio so that the acid-type SL is 2 molar equivalents or more relative to the acid salt of the group 11 element. Preferably, it is 2 molar equivalents, and thus a complex can be formed in which acid-type SL is bound to 2 molecules for every 1 molecule of copper(II) ion. On the other hand, if the group 11 element is a monovalent metal (e.g., silver), it is preferable to adjust the ratio so that the acid-type SL is 1 molar equivalent or more relative to the acid salt of the group 11 element. Preferably, it is 1 molar equivalent, and thus a complex can be formed in which acid-type SL is bound to 1 molecule for every 1 molecule of silver(I) ion. In step 1, the reaction between the acid salt of the group 11 element and the acid-type SL produces a complex of the group 11 element and acid-type SL, and acid is also produced as a by-product.

[0036] Step 2 is the step of removing the acids (inorganic acids, organic acids) produced as by-products in Step 1 from the reaction solution. Any method that can remove the acids from the reaction solution is acceptable, and is not limited to that extent. For example, when an acetate salt is used as the salt of a Group 11 element, the acetic acid produced in the reaction solution evaporates along with the water when the aqueous solution is heated, so it can be removed by heating the reaction solution. In this case, depending on the heating temperature and heating time used in Step 1, it is possible to carry out Step 2 simultaneously with Step 1 (Steps 1 and 2 are carried out simultaneously).

[0037] In step 2, after removing the acid, a complex remains in the reaction solution in which acid-type SL is coordinated to a group 11 element ion. By drying and solidifying the reaction solution, the "complex of acid-type SL and group 11 elements" can be recovered in solid form. This complex has high antimicrobial activity that surpasses the antimicrobial properties of the group 11 element ions, and can therefore be considered an antimicrobial substance.

[0038] Method of manufacture 2 The antimicrobial substance can also be manufactured by the following steps: (i) step I, reacting acid-type SL with an alkali metal salt or alkaline earth metal salt in the presence of water to form an alkali metal or alkaline earth metal salt of acid-type SL (acid-type SL salt); (ii) step II, reacting the acid-type SL salt with an acid salt of a group 11 element in the presence of water; (iii) step III, removing the alkali metal or alkaline earth metal salt of the acid from the reaction solution.

[0039] The alkali metal salt or alkaline earth metal salt used in step I may be water-soluble, but is not limited to that. Examples include hydroxides and chlorides of alkali metals or alkaline earth metals. Hydroxides are preferred, and specifically include hydroxides of alkali metals such as sodium hydroxide and potassium hydroxide, and hydroxides of alkaline earth metals such as calcium hydroxide and magnesium hydroxide. More preferably, an alkaline earth metal hydroxide such as calcium hydroxide that can form a water-insoluble acid salt with an acid is used.

[0040] Step I is not limited, but it can be carried out by dissolving the acid-type SL and an alkali metal salt or alkaline earth metal salt (hereinafter sometimes collectively referred to as "metal salt") in water, and then stirring at a temperature of, for example, 10 to 40°C, preferably 15 to 30°C. For convenience, room temperature can be used as the temperature. The reaction time is not particularly limited as long as it is the time it takes for the acid-type SL and the metal salt to react in the aqueous solution to form the metal salt of the acid-type SL (hereinafter also referred to as "acid-type SL salt"). For example, it can be appropriately set in the range of 1 minute to 60 minutes.

[0041] In step I, the ratio of acid-type SL to metal salt is not limited, but for example, if the metal salt is a monovalent alkali metal salt, it is preferable to adjust the ratio so that the alkali metal salt is 1 molar equivalent or more to the acid-type SL. Also, if the metal salt is a divalent alkaline earth metal salt, it is preferable to adjust the ratio so that the alkaline earth metal salt is 1 / 2 molar equivalent or more to the acid-type SL.

[0042] Examples of group 11 element salts used in step II include salts of group 11 elements with inorganic acids (hydrochloric acid, sulfuric acid, nitric acid, etc.) or organic acids (acetic acid, citric acid, malic acid, lactic acid, and succinic acid, etc.). When the group 11 element is copper, copper acetate and sulfate are preferred, and when the group 11 element is silver, nitrate is preferred. The group 11 element salt may be an anhydrous or a solvate such as a hydrate. Examples of solvates of group 11 element salts include copper(II) acetate monohydrate, copper(II) sulfate dihydrate, and copper(II) sulfate pentahydrate.

[0043] Step II can be carried out by adding a group 11 element salt to the reaction solution obtained in Step I, and stirring at a temperature of, for example, 10 to 40°C, preferably 15 to 30°C, although this is not limited. Room temperature can be used for convenience. The reaction time is not particularly limited as long as it is the time required for the group 11 element salt and the acidic SL salt to react in the aqueous solution and form a complex in which the acidic SL is bonded to the group 11 element ion. For example, it can be set appropriately in the range of 1 minute to 60 minutes.

[0044] In step II, the ratio of the group 11 element salt and the acidic SL salt in the reaction solution is not limited, but for example, when the group 11 element is a divalent metal (copper), it is preferable to adjust the ratio of acidic SL to the group 11 element salt to be 2 molar equivalents or more. Preferably, it is 2 molar equivalents, and thus a complex can be formed in which acidic SL is bonded via carboxyl groups at a ratio of 2 molecules per 1 molecule of copper(II) ion. On the other hand, when the group 11 element is a monovalent metal (silver), it is preferable to adjust the ratio of acidic SL to the group 11 element salt to be 1 molar equivalent or more. Preferably, it is 1 molar equivalent, and thus a complex can be formed in which acidic SL is bonded via carboxyl groups at a ratio of 1 molecule per 1 molecule of silver(I) ion. In step II, a reaction between the acid acid salt of a group 11 element and an acidic SL salt produces a complex of the group 11 element and the acidic SL, as well as by-products of alkali metal or alkaline earth metal salts (acid salts) of acids (inorganic acids, organic acids).

[0045] Step III is the step of removing the salt acid by-product from the reaction solution of Step II. Any method that can remove the salt acid from the reaction solution is acceptable and is not limited to that extent. For example, if calcium hydroxide is used as the metal salt in Step I and sulfate is used as the salt acid of a Group 11 element in Step II, water-insoluble calcium sulfate will be produced as a by-product in the reaction solution of Step II. In this case, the water-insoluble salt acid can be removed using a conventional solid-liquid separation method (centrifugation, filtration, etc.).

[0046] The substance recovered by removing the acid salt in step III is a complex in which acidic SL is coordinated to a group 11 element ion, and it has high antimicrobial activity that surpasses the antimicrobial properties inherent in the group 11 element ions. For this reason, it can be called an antimicrobial substance.

[0047] In this disclosure, the terms “antimicrobial,” “antimicrobial properties,” “antimicrobial activity,” and “antimicrobial effect” include both “microstatic” (inhibiting the growth or proliferation of microorganisms) and / or “microcidal” (killing microorganisms). In other words, unless otherwise specified, the term “antimicrobial” is a general term that means either one or both of the properties of microstatic and microcidal microorganisms. The term “antimicrobial” also includes the meanings of “reducing the number of microorganisms,” “demicrobial” (reducing the number of microorganisms present on an object or in a limited space, increasing cleanliness), and / or “disinfecting” (reducing pathogenic microorganisms to a harmless level, or neutralizing their toxicity). In this disclosure, when something is said to be “antimicrobial” or “having antimicrobial activity,” it simply means that the thing has antimicrobial properties and exhibits antimicrobial activity when exposed to microorganisms, regardless of its intended use (whether or not it has an antimicrobial use).

[0048] The microorganisms covered in this disclosure include prokaryotes (bacteria), eukaryotes (fungi), and viruses. Prokaryotes include, but are not limited to, Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, as well as Gram-positive bacteria such as Staphylococcus species including Staphylococcus aureus. Eukaryotes include, but are not limited to, yeast-type fungi (e.g., Candida) and filamentous fungi (e.g., molds, Aspergillus, dermatophytes). Viruses include, but are not limited to, herpesviruses (Herpesviridae), which belong to the DNA virus family, and influenza viruses (Orthomyxoviridae), which belong to the RNA virus family. These viruses are examples of human pathogenic viruses that infect humans and cause disease. It is already known that Group 11 element ions themselves, particularly copper and silver ions, possess antimicrobial activity against these microorganisms (see Non-Patent Documents 1-3, etc.).

[0049] In this disclosure, "microorganisms" is used as a general term referring to a group of organisms including prokaryotes (bacteria) and viruses. Furthermore, the term "antimicrobial" does not mean that it is effective against all of these microorganisms, but rather is used as a concept that also includes cases where it exhibits antimicrobial activity against any one type of microorganism.

[0050] (Antibacterial Properties) In this disclosure, the terms "antibacterial," "antibacterial properties," "antibacterial action," "antibacterial effect," and "antibacterial activity" include the meaning of bacteriostatic (inhibiting the growth and proliferation of bacteria) and / or bactericidal (killing bacteria). In other words, unless otherwise specified, the term "antibacterial" is a general term that means either bacteriostatic or bactericidal, or both. The term "antibacterial" also includes the meanings of sterilization (reducing the number of bacteria), disinfection (reducing the number of bacteria present on an object or in a limited space, increasing cleanliness), and / or disinfection (reducing pathogenic bacteria to a harmless level, or neutralizing their toxicity). The bacteria targeted for antibacterial action in this disclosure include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, etc. Preferably, Escherichia coli and Staphylococcus aureus. More preferably, Escherichia coli.

[0051] The antimicrobial activity of this antimicrobial substance can be measured and evaluated by the test bacterial suspension method. The test bacterial suspension method is specified in the Japanese Pharmacopoeia (Reference Information, Disinfection Table and Decontamination Methods 2.2.1 Test Bacterial Suspension Method), "ASTM E 2315", "EN 13727", "EN 13624", and "EN 17126", and any of these methods can be used for evaluation. Furthermore, antimicrobial activity can also be evaluated by determining the minimum inhibitory concentration (MIC) using the microdilution method. This antimicrobial substance exhibits antimicrobial activity by at least one of these methods. For example, in experimental examples 1-2, 4-6, and 10-12 described later, the antimicrobial activity of this antimicrobial substance is evaluated in accordance with the test bacterial suspension method (ASTM E 2315). In this evaluation method, the target bacteria are applied to the test sample for a predetermined time (10-30 minutes), the bacterial count (CFU / mL) is measured, and the logarithmic decrease (-Log) is calculated from the bacterial count before and after the application. 10 Calculate CFU. 1 Log 10When the bacterial count decreases by a certain amount, it can be evaluated that the test sample has a certain antibacterial effect against the target bacteria. Also, 2Log 10 If the above-mentioned reduction in bacterial count is observed, it can be considered to have a high antibacterial effect.

[0052] (Antiviral Properties) In this disclosure, the terms “antiviral,” “antiviral properties,” “antiviral power,” “antiviral action,” “antiviral effect,” and “antiviral activity” include the meaning of inactivating or deactivating a virus (removing its infectivity). The term “antiviral” also includes the meaning of reducing the number of infectious viruses (reducing the number of infectious viruses), removing them (reducing the number of infectious viruses present on an object or in a limited space, increasing cleanliness), and / or disinfecting (reducing pathogenic viruses to a harmless level, or neutralizing their toxicity).

[0053] The antiviral activity of this antimicrobial substance can be measured and evaluated by the test virus suspension method. The test virus suspension method is defined in "ASTM E1052," "EN14476," and "DVV&RKI guidelines," and evaluation can be performed using any of these methods. This antimicrobial substance exhibits antiviral activity by at least one of these methods, and in Experimental Example 9, described later, the antiviral activity of this antimicrobial substance is evaluated in accordance with ASTM E1052 (see Measurement Method (2-6)). In this evaluation method, the target virus is applied to the test sample for a predetermined time (10 or 30 minutes), and then the viral infectivity titer (50% Tissue Culture Infectious Dose (TCID)) is measured. 50 )) and the logarithmic decrease value (-Log) from the viral infectivity titer before and after the action. 10 TCID 50 The following calculation is performed: If the difference (ab) between the logarithmic reduction of the sample under evaluation (a) and the logarithmic reduction of the comparison sample (b) is 0 or greater, the sample under evaluation can be evaluated as having a certain level of antiviral activity against the target virus.

[0054] (Surface Activity) This antimicrobial substance is an amphiphilic polymer compound and is characterized by possessing surface activity in addition to the antimicrobial properties described above. Surfactants with surface activity are adsorbed onto gas-liquid surfaces, reducing surface tension, and forming micelles when the surfactant concentration exceeds a certain level. The surface activity of this antimicrobial substance can be evaluated by determining the static surface tension using the Wilhelmy method (plate method). Details are described in the Examples section, and the method can be implemented based on that description.

[0055] This antimicrobial substance is characterized by having a critical micelle concentration that is equal to or lower than the critical micelle concentration of the acid-type SL that constitutes the antimicrobial substance. In other words, this antimicrobial substance can exhibit surfactant activity at the same concentration as or lower than the acid-type SL that constitutes the antimicrobial substance. Preferably, as shown in Experimental Example 14(1), the critical micelle concentration is lower than that of the acid-type SL that constitutes the antimicrobial substance, and it exhibits surfactant activity at a low concentration.

[0056] The foaming properties and foam stability of this antimicrobial substance are almost equivalent to those of the acid-type SL that constitutes this antimicrobial substance (see Experimental Example 14(2)). Since acid-type SL is known as a low-foaming surfactant, this antimicrobial substance can also be used as a low-foaming surfactant in a similar manner.

[0057] (Uses of this antimicrobial substance) As described above, this antimicrobial substance, which consists of a complex of acid-type SL and a group 11 element, has antimicrobial properties (antibacterial and / or antiviral properties) and surfactant properties, and can therefore be used as an active ingredient in antimicrobial agents (antibacterial or antiviral agents), as a processing component for manufacturing antimicrobial processed products, and / or as an active ingredient in surfactants.

[0058] [Antimicrobial agents, compositions containing them, or antimicrobial processed products] The antimicrobial agents covered by this disclosure are compositions used for antimicrobial applications that contain an effective amount of the aforementioned antimicrobial substance (a complex of acid-type SL and a group 11 element) having antimicrobial activity (hereinafter also referred to as "the antimicrobial agent"). The antimicrobial agent includes antimicrobial agents used for antimicrobial applications that contain an effective amount of a complex of acid-type SL and a group 11 element having antimicrobial activity. The antimicrobial agent also includes antiviral agents used for antiviral applications that contain an effective amount of a complex of acid-type SL and a group 11 element having antiviral activity.

[0059] This antimicrobial agent may consist of 100% by mass of the antimicrobial substance, but may also contain other components, as long as they possess antimicrobial activity. These other components may include, for example, by-products generated in the manufacturing process of the complex of acid-type SL and group 11 elements described above. In other words, it is not necessary to contain a highly purified complex, as long as it possesses antimicrobial activity, and may contain a crude product obtained in the manufacturing process. Furthermore, the form of this antimicrobial agent is not particularly limited and may be in solid form (including powder and granules), liquid form, or semi-solid form (including cream form).

[0060] This antimicrobial agent can be suitably used in various products requiring antimicrobial properties (food and beverages, pharmaceuticals, quasi-drugs, cosmetics, deodorants, disinfectants, cleaning agents, daily necessities, etc.). Here, food and beverages include not only general foods and beverages, but also health supplements, functional foods, foods for specified health uses, or other foods and beverages that have specific functions and are consumed for the purpose of maintaining health. Cosmetics are intended to be used on the human body by rubbing, spraying, or other similar methods (e.g., patching) to cleanse, beautify, enhance attractiveness, alter appearance, or maintain the health of the skin or hair. Examples include makeup cosmetics (foundation, lipstick, etc.), basic cosmetics (lotion, emulsion, etc.), hair cosmetics (hair tonic, hair liquid, hair cream, etc.), and toiletries (toothpaste, mouthwash, shampoo, conditioner, soap, facial cleanser, bath additive, etc.). Furthermore, cleaning agents include laundry detergents, dishwashing detergents, and household cleaning agents for toilets, bathtubs, floors, walls, etc. Deodorizers include clothing deodorizers and air deodorizers.

[0061] Furthermore, this antimicrobial agent can impart antimicrobial properties to various materials by mixing (including kneading), applying, spraying, or immersing them. Examples of materials include, without limitation, plastic resins, nonwoven fabrics, woven fabrics, fibers, films, paints, wood, and metals. The aforementioned nonwoven fabrics, woven fabrics, fibers, and films include those used in sanitary or medical supplies. For example, by incorporating this antimicrobial agent into nonwoven fabrics, woven fabrics, fibers, or films used as wound dressings, antimicrobial wound dressings can be prepared. In addition to antimicrobial properties, this antimicrobial agent also possesses surfactant properties, making it suitable for use in products requiring both antimicrobial and surfactant properties.

[0062] Based on the above, this disclosure provides the use of the antimicrobial substance as an antimicrobial agent, and also provides various products containing the antimicrobial substance (or the antimicrobial agent) in an effective amount having antimicrobial properties. These products include, but are not limited to, antimicrobial compositions such as food and beverages, pharmaceuticals, quasi-drugs, cosmetics, deodorants, disinfectants, and cleaning agents, as well as antimicrobial processed products such as daily necessities.

[0063] Furthermore, antimicrobial materials can be manufactured by treating various materials (e.g., plastic resins, nonwoven fabrics, fibers, films, paints, wood, metals, etc.) with this antimicrobial substance (or antimicrobial agent), and antimicrobial processed products can be manufactured using such antimicrobial materials. This disclosure provides these antimicrobial materials and antimicrobial processed products containing them. Such antimicrobial processed products include, without limitation, sanitary products such as antibacterial masks, antibacterial sheets, sanitary mats, and sanitary gloves; antibacterial material products such as antibacterial treated plastic containers, antibacterial treated cloths, antibacterial clothing, antibacterial interior materials, and antibacterial building materials; and furniture, electrical appliances, and sanitary processed products used in wet areas such as toilets, bathrooms, kitchens, and washrooms that contain materials that have been treated with antimicrobial agents.

[0064] When preparing the various compositions, products, and materials described above using this antimicrobial agent, the amount of this antimicrobial agent (or its active ingredient complex) to be incorporated into them should be appropriately set within a range that exhibits the desired antimicrobial effect, depending on the purpose and properties of each composition, product, and material.

[0065] Due to its high antimicrobial activity, this antimicrobial agent is suitably used in the preparation of disinfectant compositions for use on hands, in homes, or in the environment. For example, a disinfectant in a dispensing container can be prepared and provided by filling an aqueous solution containing an effective amount of this antimicrobial agent that exhibits antimicrobial activity into a container equipped with a dispensing function. Dispensing containers include spray containers such as spray bottles, hand-operated spray pumps, or dispenser-equipped containers. Non-gas dispensing containers are preferred. By spraying the disinfectant composition onto hands, it is possible to sterilize or disinfect microorganisms (bacteria, viruses, etc.) attached to hands. It is also possible to sterilize or disinfect airborne microorganisms (bacteria, viruses, etc.) by spraying the disinfectant composition into the environment. Furthermore, by impregnating nonwoven fabric with the disinfectant composition and using it to wipe hands or environmental surfaces (kitchen areas, dining tables, furniture, toilets, bathrooms, etc.), it is possible to sterilize or disinfect microorganisms (bacteria, viruses, etc.).

[0066] [Surfactants and Compositions Containing the Same] The surfactants covered by this disclosure are anionic surfactants containing the aforementioned antimicrobial substance (a complex of acid-type SL and a Group 11 element) in an effective amount having surfactant properties. Hereinafter, this will also be referred to as "the surfactant."

[0067] This surfactant may consist of 100% by mass of the antimicrobial substance, but may also contain other components as long as they have surfactant properties. For example, it may contain by-products generated in the manufacturing process of the complex of the acid-type SL and group 11 elements described above. In other words, it is not necessary to contain a highly purified complex as long as it has surfactant properties, and may contain crude purified products obtained in the manufacturing process. Furthermore, the form of this surfactant is not particularly limited and may be solid (including powder and granules), liquid, or semi-solid (including cream).

[0068] This surfactant can be suitably used in various products requiring surfactant properties (food and beverages, pharmaceuticals, quasi-drugs, cosmetics, deodorants, detergents, daily necessities, etc.). In particular, because this surfactant possesses antimicrobial properties in addition to surfactant properties, it can be suitably used in products requiring antimicrobial action. Therefore, this disclosure provides the use of this antimicrobial substance as a surfactant, as well as various products (food and beverages, pharmaceuticals, quasi-drugs, cosmetics, deodorants, detergents, daily necessities, etc.) containing an effective amount of this antimicrobial substance that exhibits surfactant properties as a surfactant.

[0069] When this surfactant is added to various products (food and beverages, pharmaceuticals, quasi-drugs, cosmetics, deodorants, detergents, daily necessities, etc.), that is, when various products are prepared using this surfactant, the amount of this surfactant (or its active ingredient complex) to be incorporated into these products should be appropriately set within a range that exhibits the desired surfactant effect, according to the purpose and properties of each product.

[0070] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.”

[0071] The present invention will be described below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. The following experiments were conducted at room temperature (25 ± 5°C) and atmospheric pressure unless otherwise specified. Unless otherwise specified, "%" below means "mass percent" and "parts" means "parts by mass".

[0072] The materials used in the manufacturing and experimental examples described later, as well as the various measurement and evaluation methods, are as follows. (1) Raw materials Acid type SL: SOFORO (registered trademark) AC-100 (manufactured by Saraya Co., Ltd.) Copper acetate: Copper(II) acetate monohydrate (manufactured by Tokyo Chemical Industry Co., Ltd.) Copper sulfate: Copper(II) sulfate pentahydrate (manufactured by Kanto Chemical Co., Ltd.) Silver nitrate: Silver(I) nitrate (manufactured by Fujifilm Wako Pure Chemical Corporation) Calcium hydroxide: Calcium hydroxide (manufactured by Fujifilm Wako Pure Chemical Corporation) Sodium hydroxide: 0.1N sodium hydroxide (manufactured by Kanto Chemical Co., Ltd.) BSA: Albumin from bovine serum Fraction V pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Corporation) NaCl: Sodium chloride (manufactured by Kanto Chemical Co., Ltd.) EDTA-2Na: Ethylenediaminetetraacetate disodium dihydrate (manufactured by Sigma-Aldrich) Ethanol: Traceable 95 Grade 1 (manufactured by Nippon Alcohol Industry Co., Ltd.) APG: Alkyl polyglycoside raw material name: EcoSense 919 (manufactured by Dow Chemical Japan Co., Ltd.) LADO: Lauryldimethylamine oxide (Raw material name: Unisafe A-LMR, manufactured by NOF Corporation) Ammonia: 30% aqueous ammonia solution (manufactured by Kanto Chemical Co., Ltd.) Escherichia coli: Escherichia coli ATCC 25922 Pseudomonas aeruginosa: Pseudomonas aeruginosa ATCC 15442 Staphylococcus aureus: Staphylococcus aureus ATCC 6538 Herpes simplex virus: Herpes simplex Type 1 ATCC VR-733 Influenza virus: Influenza A virus (H1N1) ATCC VR1469 Vero cells: African green monkey kidney cell JCRB 9013 MDCK cells: Madin Darby canine kidney cell JCRB 9029 TSB medium: AccuDia TMTrypto-sawyer broth granules (manufactured by Shimadzu Diagnostics Corporation) Agar: Agar Code No. 01-1900-6 (manufactured by Sigma-Aldrich) TSB agar medium: Prepared by mixing the above TSB medium and the above agar MEM: Eagle's minimum essential medium (manufactured by Sigma-Aldrich) Opti-MEM: Opti-MEM (registered trademark) (manufactured by Gibco) FBS: Fetal Bovine Serum (manufactured by Corning) NCS: Newborn calf serum (manufactured by Gibco)

[0073] (2) Measurement Method (2-1) Ultraviolet-Visible Absorption Spectroscopy (UV-VIS) The amount of light absorbed by the test sample (absorbance) is measured using the UV-Vis absorption measuring device and measurement method described below.

[0074] [UV-Vis Absorption Measurement Device] Measurement device: UV2600 (Shimadzu Corporation) Measurement method: The test sample is placed in a 1.5 mL disposable cell, and ultraviolet or visible light is irradiated onto the test sample. The amount of light absorbed by the test sample (absorbance) is measured while continuously scanning the irradiation wavelength (250 nm to 900 nm).

[0075] (2-2) Fourier Transform Infrared (FT-IR) Spectroscopic Analysis The FT-IR spectrum of the test sample is obtained using the following FT-IR spectrophotometer and measurement method. [FT-IR Spectrophotometer] Measurement device: Spectrum100 (PerkinElmer) Measurement conditions: Infrared light (4000-400 cm) is applied to the test sample prepared in powder form. -1 Irradiate the area with ) and acquire the FT-IR spectrum.

[0076] (2-3) Antimicrobial activity evaluation method 1 (Test bacterial suspension method, compliant with ASTM E2315) The antimicrobial activity of the test sample was evaluated using antimicrobial activity evaluation method 1 (Test bacterial suspension method, ASTM E2315) according to the following method. Specifically, 0.5 mL of the test bacterial solution (test bacteria: Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa) was added to 4.5 mL of the test sample (sample to be evaluated, comparison sample) and the mixture was reacted at a predetermined temperature for a predetermined time. After the reaction, 0.5 mL of the reaction solution obtained in the above reaction was added to 4.5 mL of reaction stop solution (containing 10% polysorbate 80 and 3% lecithin) to stop the reaction. Then, the reaction solution that had been stopped was serially diluted with 0.1% tryptone solution, and the diluted solution was cultured on TSB agar medium in a plate smear culture (37°C for 24 hours). After culturing, colony counting was performed to measure the number of bacteria.

[0077] [Evaluation Criteria] The degree of antibacterial activity is evaluated by comparing the logarithmic reduction value (a) of the sample to be evaluated with the logarithmic reduction value (b) of the comparison sample (comparison sample), and using the difference (ab) according to the following criteria: ◎: Difference from the logarithmic reduction value of the comparison sample is "+0.5 or more" 〇: Difference from the logarithmic reduction value of the comparison sample is "0 or more and less than +0.5" △: Difference from the logarithmic reduction value of the comparison sample is "-0.5 or more and less than 0" ×: Difference from the logarithmic reduction value of the comparison sample is "less than -0.5"

[0078] (2-4) Antimicrobial activity evaluation method 2 (in accordance with the micro-liquid dilution method specified by the Japanese Society of Chemotherapy) The antimicrobial activity of the test sample was evaluated using antimicrobial activity evaluation method 2 (micro-liquid dilution method) according to the following method. Specifically, 5 μL (approximately 10) of the test bacterial suspension (test bacterium: Escherichia coli) was placed in each well of a 96-well plate. 9 After adding the CFU / mL doses, 100 μL each of the test sample (evaluation sample, comparison sample), serially diluted with TSB medium diluted to 1 / 10th of its original concentration, was dispensed into each well and mixed. After incubation at 37°C for 24 hours, the culture solution was spread onto TSB agar and incubated overnight at 37°C to visually check for the presence or absence of E. coli growth. If no E. coli colonies were detected after incubation, it was determined that no growth had occurred. The concentration of the test sample (copper concentration) at which no growth was observed was defined as the minimum inhibitory concentration (MIC).

[0079] (2-5) Antimicrobial activity evaluation method 3 (Test bacterial suspension method, chemical load, in accordance with EN13727) The antimicrobial activity of the test sample in the presence of a chemical substance was evaluated according to antimicrobial activity evaluation method 3 (Test bacterial suspension method, EN13727). BSA, NaCl, or EDTA-2Na were used as the chemical substance. Specifically, 0.5 mL of a chemical solution prepared by dissolving the chemical substance in sterile distilled water was added to 0.5 mL of the test bacterial solution, and after holding at 20°C for 2 minutes, it was mixed with 4.0 mL of the test sample (evaluation sample, comparison sample) which had been held at 20°C, and reacted for 10 or 30 minutes. After the reaction, 0.5 mL of the reaction solution obtained in the above reaction was added to 4.5 mL of reaction stop solution (containing 10% polysorbate 80 and 3% lecithin) to stop the reaction. Subsequently, the reaction solution, whose reaction had been stopped, was serially diluted with 0.1% tryptone solution and cultured on TSB agar plates (at 37°C for 1 day). After culturing, colony counting was performed to measure the number of bacteria. As a control test without chemical exposure, the same test was performed using sterile distilled water instead of the aforementioned chemical solution.

[0080] [Judgment Criteria] When the value obtained by subtracting the logarithmic reduction value obtained under chemical load conditions from the logarithmic reduction value obtained under chemical load conditions is "less than 0.10", it is judged that the antibacterial activity of the test sample is not inhibited by chemical substances.

[0081] (2-6) Antiviral Activity Evaluation Method (Test Virus Suspension Method, in accordance with ASTM E1052) The antiviral activity of the test samples was evaluated according to the test virus suspension method. Specifically, 0.9 mL of the test sample (evaluation sample, comparison sample) kept at 20°C was mixed with 0.1 mL of the test virus solution (test virus: herpesvirus or influenza virus), and the mixture was allowed to react at 20°C for 10 or 30 minutes. After the reaction, 0.1 mL of the reaction solution obtained from the above reaction was added to 0.9 mL of reaction stop solution to stop the reaction. The stopped reaction solution was serially diluted with diluents, and 0.1 mL of each diluent was dispensed into 96-well plates in which host cells had been previously cultured in cell culture medium. 34°C, 5% CO2 ２ After culturing for 1 hour under the specified conditions, remove the culture medium, add 0.1 mL of fresh cell maintenance medium to each well, and maintain at 34°C and 5% CO2. ２The cells were cultured for 5-6 days under the specified conditions. After culturing, the deformation or death of cells due to viral infection was observed under a microscope to confirm the presence or absence of cytopathic effects (CPE) due to infection. Based on this, the 50% tissue culture infectious dose (TCID) was calculated using the Behrens-Kaerber method. 50 The infectivity was calculated. This method involves inoculating cells with the virus at multiple dilution concentrations and statistically estimating the infectivity from the percentage of wells in which infection occurs. It is widely used to quantitatively evaluate the infectivity of a virus. The virus, host cells, cell culture medium, reaction stop solution, cell maintenance medium, and diluents used are shown in Table 1.

[0082]

[0083] [Evaluation Criteria] The degree of antiviral activity is evaluated by comparing the logarithmic reduction value (a) of the sample being evaluated with the logarithmic reduction value (b) of a comparison sample (comparison sample), and using the difference (ab) according to the following criteria: ◎: Difference from the logarithmic reduction value of the comparison sample is "+0.5 or more" 〇: Difference from the logarithmic reduction value of the comparison sample is "0 or more and less than 0.5" △: Difference from the logarithmic reduction value of the comparison sample is "-0.5 or more and less than 0" ×: Difference from the logarithmic reduction value of the comparison sample is "less than -0.5"

[0084] (2-7) Static Surface Tension Measurement (Wilhelmy Method) The surfactant activity of the test sample is evaluated by measuring the static surface tension. Static surface tension was measured using an automated surface tension analyzer (Kyowa Interface Science, CBVP-Z) and the Wilhelmy method (plate method). 25 mM HEPES buffer (pH 7.0) was used to dissolve and dilute the test sample. 10 mL of the prepared solution was poured into a measurement petri dish, covered, and left to stand overnight or longer. The change in surface tension over time at 20°C was measured, and the results were printed on paper every 5 minutes for a measurement time of 40,000 seconds. The value obtained when the mixture was stable for the first 10 minutes or more from the start of measurement was taken as the surface tension at that concentration. The critical micelle concentration (CMC) and γCMC were determined by drawing approximate lines to five or more odd-numbered points in the adsorption phase and three or more odd-numbered points in the equilibrium phase, and finding the intersection of these lines. If there are not enough samples, prepare samples of the required concentration each time and measure the static surface tension.

[0085] (2-8) Evaluation of Foaming Ability and Foam Stability The foaming ability and foam stability of the test sample are evaluated by measuring foaming ability. 10 mL of the test sample solution is placed in a graduated ground-joint test tube (capacity 25 mL), shaken vigorously up and down 10 times, and then immediately allowed to stand. The foam volume is measured at time intervals (1 minute, 5 minutes, 10 minutes) using the test tube's scale. Foaming ability is evaluated based on the foam volume immediately after shaking. Foam stability is evaluated based on the change in foam volume over time immediately after shaking.

[0086] Experimental Example 1: Evaluation of the antibacterial activity of a mixture of SL-Na and copper sulfate (1) (1) Preparation of test samples (1-1) Preparation of aqueous solution of acidic SL sodium salt (SL-Na aqueous solution) 0.1N sodium hydroxide was added to an aqueous solution of acidic SL to the same molar concentration and stirred at room temperature for 10 minutes. After that, it was left to stand overnight in a cool, dark place and diluted with distilled water as appropriate to prepare an aqueous solution of acidic SL sodium salt (SL-Na aqueous solution).

[0087] (1-2) Preparation of a mixture of SL-Na and copper sulfate (example samples) An aqueous solution of copper sulfate, prepared separately, was added to the SL-Na aqueous solution so that the concentrations in the mixture were as shown in Table 2, and the mixture was stirred at room temperature for 15 hours to prepare the example samples (Examples 1 to 9).

[0088] (1-3) Preparation of control and comparative examples As control examples, aqueous copper sulfate solutions (control examples 1-6) and SL-Na solutions (control examples 7-8) were prepared. As comparative examples, copper sulfate-oxidized SL mixtures (comparative examples 1-2) were prepared. The aqueous copper sulfate solutions were prepared by dissolving copper sulfate in distilled water to the concentrations shown in Table 3. The SL-Na solutions were prepared by the method described in (1-1) above. The copper sulfate-oxidized SL mixtures were prepared by adding copper sulfate and acid-type SL to distilled water in the proportions shown in Table 4, mixing at room temperature, and letting it stand overnight.

[0089] (2) Evaluation of antimicrobial activity Using E. coli as the test bacteria, the logarithmic decrease values ​​of the example samples (Examples 1-9), control example samples (Control Examples 1-8), and comparative example samples (Comparative Examples 1-2) were determined using the antimicrobial activity evaluation method 1 (test bacteria suspension method: reaction time 30 minutes) described above (Initial bacterial count (Log[CFU / mL]) of E. coli: approximately 7). The antimicrobial activity of the example samples and comparative example samples was then evaluated using the judgment criteria described above. Among the example samples, Examples 1-5 were evaluated using Control Example 5 as a comparative sample, and Examples 6-9 were evaluated using Control Example 6 as a comparative sample. Among the comparative example samples, Comparative Example 1 was evaluated using Control Example 6 and Example 8 as comparative samples, and Comparative Example 2 was evaluated using Control Example 6 and Example 9 as comparative samples. The results are shown in Tables 2-4.

[0090]

[0091]

[0092]

[0093] As shown in Table 3, an antibacterial effect was observed in a concentration-dependent manner in the copper sulfate aqueous solution (Control Examples 1-6), while no antibacterial effect was observed in the SL-Na aqueous solution (Control Examples 7 and 8). In contrast, the SL-Na and copper sulfate mixture prepared by mixing copper sulfate aqueous solution and SL-Na aqueous solution (Examples 1-9) was found to exhibit a significantly higher antibacterial effect compared to the antibacterial effect of the copper sulfate aqueous solution itself. On the other hand, as shown in Table 4, although the copper sulfate-oxidized SL mixture had an antibacterial effect, it was lower than that of the copper sulfate aqueous solution, confirming that the antibacterial effect due to copper(II) ions decreased when oxidized SL was added to the copper sulfate aqueous solution.

[0094] Figure 1 shows images of SL-Na aqueous solution (6.27 mM), copper sulfate aqueous solution (6.27 mM), and a mixture of SL-Na and copper sulfate (SL-Na 3.13 mM, copper sulfate 3.13 mM) prepared by mixing the SL-Na aqueous solution and the copper sulfate aqueous solution, taken in transparent containers. As shown in Figure 1, the mixture obtained by mixing the colorless and transparent SL-Na aqueous solution with the light blue copper sulfate aqueous solution exhibited a deep blue color. In contrast, the color of the copper sulfate-oxidized SL mixture was no different from the color of the copper sulfate aqueous solution before the addition of oxidized SL (results not shown). From these findings, it is considered that the copper ions in the SL-Na and copper sulfate mixture form a complex (SL-copper complex) with SL in the aqueous solution (see Figure 1), and that this complex exerts an antibacterial effect.

[0095] Experimental Example 2: Evaluation of the antibacterial activity of a mixture of SL-Na and copper sulfate (2) (1) Preparation of test samples An aqueous solution of SL-Na was prepared in the same manner as in Experimental Example 1. In addition, an aqueous solution of copper sulfate (copper sulfate aqueous solution) (10.0 ppm, 100 ppm) was prepared. An aqueous solution of SL-Na was added to the copper sulfate aqueous solution (10.0 ppm, 100 ppm) in an amount ranging from 0.5 to 2 equivalents, and the mixture was stirred at room temperature for 15 hours to prepare a mixture of copper sulfate and SL-Na (CuSO4 + SL-Na aqueous solution).

[0096] (2) Antimicrobial activity evaluation Using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus (all Gram-negative bacteria) as test bacteria, the antimicrobial activity evaluation method 1 described above (test bacteria suspension method: reaction time 10 minutes and 30 minutes) was carried out to determine the antimicrobial activity (logarithmic decrease value) of the copper sulfate aqueous solution (CuSO4 aqueous solution) and the mixture of SL-Na and copper sulfate (CuSO4 + SL-Na aqueous solution) (Initial bacterial count (Log[CFU / mL]): approximately 7). Figure 2 shows the results of determining the antimicrobial activity (logarithmic decrease value) against the test bacteria (Escherichia coli, Pseudomonas aeruginosa) for CuSO4 aqueous solution (10.0 ppm, 62.7 μM), CuSO4 (62.7 μM) + SL-Na (62.7 μM: 1 eq.) aqueous solution, and CuSO4 (62.7 μM) + SL-Na (125 μM: 2.0 eq.) aqueous solution. Figure 3 also shows the results of determining the antibacterial activity (logarithmic reduction value) against the test bacteria (Escherichia coli, Staphylococcus aureus) for CuSO4 aqueous solution (100 ppm, 627 μM), CuSO4 (627 μM) + SL-Na (314 μM: 0.5 eq.) aqueous solution, CuSO4 (627 μM) + SL-Na (627 μM: 1.0 eq.) aqueous solution, and CuSO4 (627 μM) + SL-Na (1.25 mM: 2.0 eq.) aqueous solution.

[0097] As shown in Figures 2 and 3, the antibacterial effect of the CuSO4 + SL-Na aqueous solution was higher than that of the CuSO4 aqueous solution alone, and it was confirmed that mixing the CuSO4 aqueous solution with the SL-Na aqueous solution further enhanced the antibacterial effect against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). From the results of Experimental Example 1, it is thought that this strong antibacterial effect against Gram-negative bacteria is due to the formation of SL and complexes (SL-copper complexes) in the aqueous solution.

[0098] Experimental Example 3: Evaluation of a Mixture of SL-Na and Copper Sulfate (UV-Vis Spectrum) Using the method described in Experimental Example 1, an aqueous solution of copper sulfate (6.27 mM) and an aqueous solution of SL-Na (6.27 mM) were prepared separately. These were then mixed at various ratios (CuSO4:SL-Na = 20:80 to 80:20 [molar ratio]) to prepare a mixture of SL-Na and copper sulfate (CuSO4 + SL-Na aqueous solution) with a total volume of 1.0 mL. The prepared aqueous solutions of copper sulfate, SL-Na, and CuSO4 + SL-Na at each ratio were subjected to a UV-Vis absorption analyzer to measure the UV-Vis spectra. The results are shown in Figure 4(a). As shown in Figure 4(a), the UV-Vis spectrum of copper sulfate aqueous solution (H) showed a maximum peak around 800 nm, while the UV-Vis spectra of CuSO4 + SL-Na aqueous solutions (B-G) showed a maximum peak at 680-685 nm, indicating a peak shift. This peak originates from the electron transition of the d orbital, suggesting that a complex (Cu-Cu bond) is formed in the CuSO4 + SL-Na aqueous solution. The absorbance at 680 nm in the CuSO4 + SL-Na aqueous solution was measured. 2＋ Figure 4(b) shows a graph plotted against concentration (mM). As shown in Figure 4(b), Cu 2＋ The highest absorbance was observed when the concentration was 312 μM (0.312 mL) and the SL concentration was 688 μM (0.688 mL). This suggests that copper(II) ions and SL form a stable complex in a CuSO4 + SL-Na aqueous solution with a molar ratio of 1:2.

[0099] Experimental Example 4: Evaluation of Antimicrobial Activity (SL-Copper Complex) (1) Preparation of Test Samples Copper sulfate (1.25 g, 5.00 mM) was dissolved in 10 mL of distilled water to prepare a 500 mM copper sulfate aqueous solution (CuSO4 aqueous solution). Acidic SL (1.25 g, 20.0 mM) was dissolved in 50 mL of distilled water, and then calcium hydroxide (741 mg, 10.0 mM) was added to prepare a 100 mM SL2-Ca aqueous solution. 1.0 mL of CuSO4 aqueous solution and 5.0 mL of SL2-Ca aqueous solution were mixed and then diluted with 4.0 mL of distilled water. The precipitated white solid (CaSO4・2H2O) was removed by centrifugation and filter filtration, and the filtrate was collected (Cu concentration 50 mM). This was serially diluted with distilled water to obtain the test sample (evaluation sample).

[0100] (2) Antimicrobial activity evaluation Using Escherichia coli as the test organism, the antimicrobial activity evaluation method 1 described above (test organism suspension method: reaction time of 10 minutes and 30 minutes) was performed to determine the antimicrobial activity (logarithmic decrease value) (Initial bacterial count (Log[CFU / mL]) of E. coli: approximately 7) for the sample to be evaluated. For comparison, the antimicrobial activity of a copper sulfate aqueous solution (comparative sample) adjusted to the same copper concentration was also evaluated in the same manner. The results are shown in Table 5.

[0101]

[0102] As shown in Table 5, it was confirmed that the filtrate (test sample) recovered by the above method exhibited a higher antibacterial effect against E. coli than copper sulfate containing the same concentration of copper.

[0103] As a result of the above method, it is thought that a complex of SL and copper is formed in the filtrate in the form of a hydrate (Cu2(SL)4・2H2O), as shown in the reaction equation below. [Reaction equation] (i) 2SL + Ca(OH)2 → SL2-Ca + 2H2O (ii) 2SL2-Ca + 2[CuSO4・5H2O] → Cu2(SL)4・2H2O + 2[CaSO4・2H2O]↓ + 4H2O (SL: acidic SL)

[0104] Experimental Example 5: Evaluation of Antimicrobial Activity 1 (SL-Copper Complex) (1) Preparation of Test Samples To acid-type SL (2.0 g, 3.21 mM), copper(II) acetate monohydrate (321 mg, 1.61 mM) was added, and then 20 mL of distilled water was added and stirred at 80°C for 7 hours, adding distilled water as needed. After the reaction, the obtained solution was freeze-dried to remove water, and Cu2(SL)4・2H2O was recovered as a pale blue solid. This was diluted with distilled water to various concentrations and used as the test samples (evaluation samples). In addition, as a comparative sample, an aqueous solution of copper sulfate with the same copper concentration as the evaluation samples was prepared.

[0105] (2) Antimicrobial activity evaluation: Using Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa as test bacteria, the aforementioned test samples (evaluation sample, comparison sample) were subjected to the antimicrobial activity evaluation method 1 described above (reaction time 10 minutes and 30 minutes) to determine the antimicrobial activity (logarithmic decrease value) against each test bacterium (Initial bacterial count (Log[CFU / mL]) of E. coli, S. aureus, P. aeruginosa: approximately 7). The results are shown in Table 6.

[0106]

[0107] As shown in Table 6, it was confirmed that the SL-copper composite (Cu2(SL)4・2H2O) (evaluated sample) exhibited a higher antibacterial effect than copper sulfate (comparative sample) containing the same concentration of copper.

[0108] In the aforementioned sample under evaluation, it is believed that a complex of SL and copper is formed in the hydrated state (Cu2(SL)4・2H2O), as shown in the reaction equation below. [Reaction equation] 4SL + 2[Cu(OAc)2・H2O] → Cu2(SL)4・2H2O + 4AcOH↑ (SL: acidic SL, Ac: acetyl group (CH3CO-))

[0109] Experimental Example 6: Evaluation of Antimicrobial Activity 2 (SL-Copper Composite) Test samples (evaluation sample, comparison sample) with copper concentrations of 100 μM and 1000 μM were prepared using the method described in Experimental Example 5 (1). The aforementioned test samples, held at 15, 20, 30, or 40°C, were subjected to the antimicrobial activity evaluation method 1 (reaction times of 10 minutes and 30 minutes) using E. coli as the test bacterium, and the antimicrobial activity (logarithmic reduction value) (Initial bacterial count (Log[CFU / mL]) of E. coli: approximately 7) of the test samples against E. coli was determined. The results are shown in Table 7.

[0110]

[0111] As shown in Table 7, it was confirmed that the SL-copper composite (Cu2(SL)4・2H2O) (evaluated sample) exhibited a higher antibacterial effect than copper sulfate (comparative sample) containing the same concentration of copper at all operating temperatures. Furthermore, it was confirmed that the antibacterial effect increased with increasing operating temperature.

[0112] Experimental Example 7: Evaluation of Antimicrobial Activity 3 (SL-Copper Complex) Test samples (evaluation sample, comparison sample) with copper concentrations of 0.25, 0.5, 1.0, 2.0, or 4.0 mM, prepared by the method described in Experimental Example 5 (1), were subjected to the aforementioned antimicrobial activity evaluation method 2 using Escherichia coli as the test bacterium, and the MIC value against Escherichia coli was determined for each test sample. The results are shown in Table 8.

[0113]

[0114] As shown in Table 8, the SL-copper composite (Cu2(SL)4・2H2O) (evaluation sample) had a lower MIC value than copper sulfate (comparison sample) containing the same concentration of copper, confirming that it exhibited a higher antibacterial effect.

[0115] Experimental Example 8: Evaluation of Antimicrobial Activity 4 (SL-Copper Complex) Each test sample (evaluation sample, comparison sample) with a copper concentration of 100 μM or 1000 μM, prepared by the method described in Experimental Example 5 (1), was subjected to the aforementioned antimicrobial activity evaluation method 3 (chemical substance loading reaction time of 10 minutes) using E. coli, and the antimicrobial activity (logarithmic decrease value) against E. coli in the presence of the chemical substance was determined (Initial bacterial count (Log[CFU / mL]) of E. coli: approximately 7). The results are shown in Table 9.

[0116]

[0117] As shown in Table 9, the SL-copper composite (Cu2(SL)4・2H2O) (evaluated sample) was confirmed to exhibit a higher antibacterial effect than copper sulfate (comparative sample) containing the same concentration of copper, in the presence of any chemical substance. Furthermore, as shown in Table 9, even under conditions where no inhibition by chemical substances was observed in the SL-copper composite (Cu2(SL)4・2H2O) (evaluated sample), inhibition was observed in the copper sulfate aqueous solution (comparative sample). From this, it was confirmed that the SL-copper composite (Cu2(SL)4・2H2O) is less susceptible to inhibition by chemical substances compared to copper sulfate.

[0118] Experimental Example 9: Evaluation of Antiviral Activity (SL-Copper Complex) For each test sample (evaluation sample, comparison sample) with copper concentrations of 100 μM and 1000 μM, prepared by the method described in Experimental Example 5 (1), the antiviral activity (logarithmic reduction value) against the test viruses (herpesvirus, influenza virus) was determined using the antiviral activity evaluation method described above (Initial viral infectivity titer (Log[TCID]). 50 [ / mL]) of Herpes virus and Influenza virus: approximately 6). The results are shown in Table 10.

[0119]

[0120] As shown in Table 10, the SL-copper complex (Cu2(SL)4・2H2O) (evaluated sample) was confirmed to exhibit a higher antiviral effect than copper sulfate (comparative sample) containing the same concentration of copper.

[0121] Experimental Example 10: Evaluation of Antimicrobial Activity 5 (SL-Copper Complex + Ethanol) (1) Preparation of Test Samples The SL-copper complex (Cu2(SL)4・2H2O), ethanol, and distilled water prepared by the method described in (1) of Experimental Example 5 were mixed to obtain the copper and ethanol concentrations shown in Table 11 to prepare the test samples.

[0122] (2) Antimicrobial activity evaluation Using Escherichia coli and Staphylococcus aureus as test bacteria, the aforementioned antimicrobial activity evaluation method 1 (reaction time of 10 minutes and 30 minutes) was performed on the test samples kept at 20°C to determine the antimicrobial activity (logarithmic decrease value) for each test bacterium (Initial bacterial count (Log[CFU / mL]) of E. coli, S. aureus: approximately 7). The results are shown in Table 11.

[0123]

[0124] As shown in Table 11, adding ethanol to the SL-copper complex (Cu2(SL)4・2H2O) resulted in an antibacterial effect equivalent to or better than that of the SL-copper complex (Cu2(SL)4・2H2O) alone. In particular, it was confirmed that the antibacterial effect was significantly enhanced when 20% ethanol was added against E. coli and when 30% ethanol was added against Staphylococcus aureus, compared to the SL-copper complex (Cu2(SL)4・2H2O) alone.

[0125] Experimental Example 11: Evaluation of Antimicrobial Activity 6 (SL-Copper Complex + Surfactant) (1) Preparation of Test Samples The SL-copper complex (Cu2(SL)4・2H2O), surfactant (APG or LADO), and distilled water prepared by the method described in Experimental Example 5 (1) were mixed to obtain the copper concentration and surfactant concentration shown in Table 12 to prepare the test sample.

[0126] (2) Antimicrobial activity evaluation Using Escherichia coli and Staphylococcus aureus as test bacteria, the aforementioned antimicrobial activity evaluation method 1 (reaction time of 10 minutes and 30 minutes) was performed on the test samples kept at 20°C to determine the antimicrobial activity (logarithmic decrease value) against the test bacteria (Initial bacterial count (Log[CFU / mL]) of E. coli, S. aureus: approximately 7). The results are shown in Table 12.

[0127]

[0128] As shown in Table 12, adding a surfactant (APG or LADO) to the SL-copper complex (Cu2(SL)4・2H2O) yielded effects equivalent to or better than those of the SL-copper complex (Cu2(SL)4・2H2O) alone. In particular, it was confirmed that adding LADO significantly increased the effectiveness against E. coli, and adding either APG or LADO significantly increased the effectiveness against Staphylococcus aureus, compared to the SL-copper complex (Cu2(SL)4・2H2O) alone.

[0129] Experimental Example 12: Evaluation of Antimicrobial Activity 7 (SL-Copper Complex + Ammonia) (1) Preparation of Test Samples Using the SL-copper complex (Cu2(SL)4・2H2O), ammonia, and distilled water prepared by the method described in Experimental Example 5 (1), the samples were mixed to obtain the copper concentration and ammonia concentration (1 to 20 equivalents relative to the copper concentration) shown in Table 13 to prepare the test samples.

[0130] (2) Antimicrobial activity evaluation Using Escherichia coli as the test bacterium, the above-described antimicrobial activity evaluation method 1 (reaction times of 1 minute, 5 minutes, 10 minutes, and 30 minutes) was performed on the test sample held at 20°C to determine the antimicrobial activity (logarithmic decrease value) against the test bacterium (Initial bacterial count (Log[CFU / mL]) of E. coli: approximately 7). The results are shown in Table 13.

[0131]

[0132] As shown in Table 13, adding ammonia to the SL-copper complex (Cu2(SL)4・2H2O) yielded a greater effect compared to the SL-copper complex (Cu2(SL)4・2H2O) alone. In particular, at a copper concentration of 10 ppm, the effect was significantly enhanced when adding 34.0, 85.0, and 170 ppb (2.0, 5.0, and 10 equivalents relative to the copper concentration), and at a copper concentration of 100 ppm, the effect was significantly enhanced when adding 170 and 340 ppb (1.0 and 2.0 equivalents relative to the copper concentration), compared to the SL-copper complex (Cu2(SL)4・2H2O) alone.

[0133] Experimental Example 13: Evaluation of the antibacterial activity of a mixture of SL-Na and silver nitrate. The same experiment as in Experimental Example 2 was conducted using silver nitrate instead of copper sulfate. (1) Preparation of test samples. Aqueous solutions of silver nitrate (silver nitrate aqueous solution) (0.3 ppm, 1 ppm) were prepared. In addition, SL-Na aqueous solution was added to the silver nitrate aqueous solution in a range of 0.5 to 2 equivalents, and after mixing, it was allowed to stand for 1 hour to prepare a mixture of silver nitrate and SL-Na (AgNO3 + SL-Na).

[0134] (2) Antimicrobial activity evaluation: Using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus as test bacteria, the antimicrobial activity evaluation method 1 described above (reaction time 1 to 30 minutes) was carried out to determine the antimicrobial activity (logarithmic decrease value) of the copper sulfate aqueous solution (AgNO3 solution) and the mixture of SL-Na and silver nitrate (AgNO3 + SL-Na aqueous solution) (Initial bacterial count (Log[CFU / mL]): approximately 7).

[0135] Figure 5 shows the results of evaluating the antibacterial activity of AgNO3 aqueous solutions (0.3 ppm, 1.77 μM) and AgNO3 (1.77 μM) + SL-Na (1.77 μM: 1.0 eq.) aqueous solutions against the test bacteria (Escherichia coli). Figure 6 shows the results of evaluating the antibacterial activity of AgNO3 aqueous solutions (1 ppm, 5.89 μM), AgNO3 (5.89 μM) + SL-Na (2.95 μM: 0.5 eq.) aqueous solutions, AgNO3 (5.89 μM) + SL-Na (5.89 μM: 1.0 eq.) aqueous solutions and AgNO3 (5.89 μM) + SL-Na (11.8 μM: 2.0 eq.) aqueous solutions against the test bacteria (Pseudomonas aeruginosa, Staphylococcus aureus).

[0136] As shown in Figures 5 and 6, the antibacterial effect of the AgNO3 + SL-Na aqueous solution was higher than that of the silver nitrate aqueous solution alone, and it was confirmed that mixing the silver nitrate aqueous solution with the SL-Na aqueous solution further enhanced the antibacterial effect against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). From the results of Experimental Examples 1 to 5, it was considered possible that a complex was formed between silver ions and SL in the aqueous solution when the silver nitrate aqueous solution and the SL-Na aqueous solution were mixed. From this, it is thought that the strong antibacterial effect against Gram-negative bacteria is due to the complex of SL and silver (SL-silver complex) formed in the aqueous solution.

[0137] Experimental Example 14: Evaluation of Physical Properties of SL-Copper Composite (Cu2(SL)4・2H2O) (1) Evaluation of Surface Activity The static surface tension of the SL-copper composite (Cu2(SL)4・2H2O) recovered in Experimental Example 5 was measured using the method described above. As comparative samples, the static surface tension of acid-type SL and SL-Na aqueous solution was measured in the same manner. The results for acid-type SL are shown in Figure 7(a), and the results for the SL-copper composite are shown in Figure 7(b). The critical micelle concentration (CMC) calculated from the measurement results is shown in Table 14.

[0138]

[0139] As shown in these results, it was confirmed that the interfacial micelle concentration (CMC) decreases when acidic SL is combined with copper (Cu2(SL)4・2H2O). In other words, Cu2(SL)4・2H2O exhibits surfactant activity at lower concentrations than acidic SL. Furthermore, from the results of the aforementioned experimental examples, including Experimental Example 5, it was confirmed that Cu2(SL)4・2H2O possesses antibacterial activity in addition to surfactant activity.

[0140] (2) Evaluation of foaming properties and foam stability The foaming properties and foam stability of the SL-copper composite (Cu2(SL)4・2H2O) recovered in Experimental Example 5 were measured using the method described above. As a comparison sample, an aqueous solution of acid-type SL, which is known to be a low-foaming surfactant, was used. The results are shown in Figures 8(a) and (b). From Figures 8(a) and (b), the foaming properties and foam stability of the SL-copper composite were about the same as those of acid-type SL. From this, it can be concluded that the SL-copper composite is a low-foaming surfactant, similar to acid-type SL.

[0141] Production Example 1: Production of SL-Copper Composite (1) Production Method 2.0 g (3.21 mmol) of acid-type SL was mixed with 321 mg (1.61 mmol) of copper acetate, and 20 mL of distilled water was added to this mixture. The mixture was heated at 80°C for 7 hours, with water added as needed while stirring. During this time, the acetic acid produced was evaporated. The resulting solution was then freeze-dried to obtain 2.12 g (0.80 mmol, yield 99%) of a pale blue solid.

[0142] As shown in the equation below, it is thought that a complex (hydrate) of SL and copper (SL-copper complex) is formed by the above reaction. [Reaction Equation] 4SL + 2[Cu(OAc)2・H2O] → Cu2(SL)4・2H2O + 4AcOH (SL: acidic SL, Ac: acetyl group (CH3CO-))

[0143] (2) Evaluation (a) UV-Vis Spectrum Measurement The pale blue solid (test sample) obtained in the above reaction was dissolved in distilled water and then diluted, and the UV-Vis spectrum (A) was measured using a UV-Vis absorption analyzer. As a control, the UV-Vis spectrum (B) of copper acetate was measured in the same manner. The results are shown in Figure 9. As shown in Figure 9, while copper acetate has a maximum peak around 800 nm, the test sample has a maximum peak at 680-685 nm, showing a peak shift.

[0144] (b) FT-IR spectral analysis The pale blue solid (test sample) obtained in the above reaction was recovered by freeze-drying the solution to remove water. The recovered pale blue solid was subjected to FT-IR spectral analysis in its solid state. As a control, acidic SL and copper acetate were also subjected to FT-IR spectral analysis in the same manner. The results are shown in Figure 10.

[0145] As shown in Figure 10, the test sample showed different peaks from those of acid-type SL and copper acetate. Specifically, when comparing the spectra of the test sample and acid-type SL, at 3400 cm⁻¹ -1 Nearby (broad peak originating from the OH bond of the OH group in the sophorose moiety), 1000 cm -1 Nearby (peaks originating from the OH group CO bond of the sophorose moiety) and 2800-2900 cm -1 While the peak (derived from the carbon chain -CH2-) is common, the acidic SL peak is 1700 cm⁻¹. -1 A peak originating from a carboxyl group in the vicinity is present at 1600 cm⁻¹ in the test sample. -1 It had shifted to the vicinity. This 1600 cm -1The same peaks were detected in the spectrum of copper acetate. This clearly indicates that a change occurred in the carboxyl group of the acidic SL in the solid obtained by the reaction. Furthermore, comparison with the spectrum of copper acetate suggests that copper bonded to the carboxyl group of the acidic SL, forming a SL-copper complex. This result supports the formation of the SL-copper complex according to the reaction equation.

[0146] Production Example 2: Production of SL-Copper Composite (1) Production Method 15.6 g (25.0 mmol) of acid-type SL was mixed with 926 mg (12.5 mmol) of calcium hydroxide and stirred at room temperature for 30 minutes. After stirring, 16.25 g (12.5 mmol) of copper sulfate was added and stirred at room temperature for 10 minutes. The precipitate (white solid) was removed by centrifugation and filtration of the supernatant, and the recovered filtrate was freeze-dried to obtain 19 g (quant.) of pale blue solid.

[0147] As shown in the equation below, it is thought that a complex (hydrate) of SL and copper (SL-copper complex) is formed by the above reaction. [Reaction equation] (i) 2SL + Ca(OH) 2 → SL2-Ca + 2H2O (ii) 2SL2-Ca + 2[CuSO4・5H2O] → Cu2(SL)4・2H2O + 2[CaSO4・2H2O] + 4H2O (SL: acid type SL)

[0148] (2) Evaluation (a) UV-Vis Spectrum Measurement The pale blue solid (test sample) obtained in the above reaction was dissolved in distilled water and then diluted, and the UV-Vis spectrum was measured using a UV-Vis absorption analyzer. The results are shown in Figure 11. As shown in Figure 11, copper sulfate (B) has a maximum peak around 800 nm, while the test sample (A) has a maximum peak at 680-685 nm, showing a peak shift.

[0149] (b) FT-IR spectral analysis The pale blue solid obtained in the above reaction (test sample) and the precipitate removed during the reaction were subjected to FT-IR spectral analysis while still in solid form. As a control, acidic SL, calcium salt of SL synthesized in the past (SL2-Ca), and calcium sulfate dihydrate (CaSO4・2H2O, standard) were also subjected to similar FT-IR spectral analysis. The results are shown in Figure 12.

[0150] As shown in Figure 12, the test sample showed a different peak from SL2-Ca, and the peak of the precipitate coincided with the peak of calcium sulfate dihydrate. Specifically, when comparing the spectra of the test sample and acid-type SL, at 3400 cm⁻¹ -1 Nearby (broad peak originating from the OH bond of the OH group in the sophorose moiety), 1000 cm -1 Nearby (peaks originating from the OH group CO bond of the sophorose moiety) and 2800-2900 cm -1 While the peak (derived from the carbon chain -CH2-) is common, the acidic SL peak is 1700 cm⁻¹. -1 A peak originating from a carboxyl group in the vicinity is present at 1600 cm⁻¹ in the test sample. -1 The peak was shifted to the vicinity, and its peak position coincided with that of the solid (SL-copper composite) obtained in Production Example 1. For SL2-Ca, the peak originating from the carboxyl group was the same as the peak of acidic SL (1700 cm). -1 The peak position was shifted from the vicinity, but it differed from that of the test sample. From this, it is clear that a change occurred in the carboxyl group of the acidic SL in the solid obtained by the above reaction. Furthermore, by comparing it with the spectrum of the solid (SL-copper composite) obtained in Production Example 1, it was considered that copper was similarly bonded to the carboxyl group of the acidic SL, and a composite of SL and copper was formed. This result supports the formation of the SL-copper composite according to the above reaction equation.

Claims

1. An antimicrobial substance consisting of a complex of an acid-type sophorolipid and a group 11 element.

2. An antimicrobial substance according to claim 1, characterized by having surfactant properties.

3. The antimicrobial substance according to claim 1 or 2, wherein the microorganism is a bacterium, a virus, or a fungus.

4. An antimicrobial agent containing an effective amount of the antimicrobial substance described in claim 1 having antimicrobial activity.

5. A surfactant containing an effective amount of the antimicrobial substance described in claim 1, having surfactant properties.

6. A composition containing the antimicrobial substance described in claim 1, the antimicrobial agent described in claim 4, or the surfactant described in claim 5.

7. An antimicrobial material treated with the antimicrobial substance described in claim 1, or the antimicrobial agent described in claim 4.

8. An antimicrobial processed product comprising the antimicrobial material described in claim 7.

9. A method for producing a composite of an acid-type sophorolipid and a group 11 element, comprising the following steps: (1) a step of reacting an acid-type sophorolipid and a salt of a group 11 element by heating in the presence of water; (2) a step of removing the acid from the reaction solution.

10. A method for producing a composite of an acid-type sophorolipid and a group 11 element, comprising the following steps: (i) reacting an acid-type sophorolipid with an alkali metal salt or an alkaline earth metal salt in the presence of water to form an alkali metal or alkaline earth metal salt of the acid-type sophorolipid; (ii) reacting the alkali metal or alkaline earth metal salt of the acid-type sophorolipid with an acid acid of a group 11 element in the presence of water; (iii) removing the alkali metal or alkaline earth metal salt of the acid from the reaction solution.

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