WO2006088102A1 - Emulsifying agent derived from red yeast - Google Patents

Abstract

A novel emulsifying agent that is highly safe, exhibiting alone high emulsifying capability and emulsification stability; a water-soluble composition containing a lipid-soluble substance obtained by the use of the emulsifying agent; and processes for producing them. There is provided an emulsifying agent comprising as an active ingredient a glycoprotein complex contained in the culture fluid obtained by culturing red yeast in a culture fluid. Further, there is provided a water-soluble composition comprising the emulsifying agent and a lipid-soluble substance. Still further, there are provided processes for producing the emulsifying agent and the water-soluble composition.

Description

 Specification

 Emulsifier derived from red yeast

 Technical field

 [0001] The present invention relates to a novel emulsifier and a water-soluble composition. More specifically, the present invention is obtained from red yeast used as an auxiliary agent in the production of an emulsion that is a mixture of water and fat. The present invention relates to an emulsifier containing a sugar protein complex as a component, a water-soluble composition obtained by using the emulsifier, and a production method thereof.

 Background art

 [0002] Emulsions in which oil droplets are dispersed in water or in which water droplets are uniformly dispersed constitute foods such as mayonnaise and dressing, and industrial products such as inks and paints. Oil and oil are difficult to mix, so an auxiliary agent is used to emulsify the oil and promote its stability.

 [0003] Conventionally, as such industrial emulsifiers, alkyl sulfates obtained by organic synthesis, polyoxyethylene-based low molecular weight synthetic emulsifiers, and the like have been used, but these are subject to degradation by microorganisms. It has been pointed out that when it is released into the environment, it may accumulate in the environment without undergoing biodegradation, causing pollution. In addition, paints that have been used by dissolving in conventional organic solvents have similar environmental pollution concerns, and from the viewpoint of work safety, the solvent used is converted to water. It is hoped for.

 [0004] In order to cope with this, for example, when using paint or the like, it was necessary to apply a certain viscosity to the paint or the like in order to prevent dripping at the application part after application. The emulsifiers used in the past had insufficient viscosity, and it was necessary to add a thickener separately, which resulted in a larger composition and had problems in production and economy.

[0005] Further, as emulsifiers applied to mammals, synthetic emulsifiers such as sugar esters also exist, but it is desired to use natural products for safety, for example, as natural emulsifiers, Proteins such as casein, lipids such as lecithin, or plant polysaccharides such as gum arabic are used. However, although these have high emulsifiability, they have a drawback that the aqueous phase and the oil phase are separated when left for a long time due to the low viscosity of the solution. In order to solve this problem, the ability to add a large amount of these emulsifiers or the stability by using in combination with a thickener such as xanthan is necessary, which has economic and manufacturing problems. Another problem is that gum arabic is derived from plants and its production is influenced by the climate and it is difficult to supply it quickly and stably.

 [0006] Other patent specifications that describe the use of polyalcohols in emulsions containing carotenoids or vitamins include alcohols such as ethanol (see Patent Document 1), nonionic emulsifiers such as polyglycerin. It is necessary to additionally use fatty acid esters (see patent document 2) or both (see patent document 3).

 [0007] However, alcohols and nonionic emulsifiers are preferred for application to many formulations, particularly mammals, or generally should be approved for use in food. Furthermore, a method for producing a carotenoid emulsion based on glycerin or other polyhydric alcohol and used for filling soft gelatin capsules is described (see Patent Document 4). The emulsifiers used in the examples of the document are likewise nonionic emulsifiers and a relatively low content of active substance is regarded as another drawback.

 [0008] Coenzyme Q10, a type of coenzyme Q known as ubidecarenone or coenzyme Q10, is known as a fat-soluble substance, and there is a technology for maintaining its homogenization and soluble state. Has been developed. For example, a fat emulsion treated with a high pressure homogenizer of the Manton-Gorin type using a nonionic emulsion such as polyethylene glycol and hydrogenated castor oil polyoxyethylene (20) -ether is disclosed (Patent Document). Five). In addition, an emulsion for intravenous injection that has been treated with a vegetable oil such as soybean oil or a phospholipid emulsifier such as phosphatidylcholine to have a particle size of 0.5 to 300 m is disclosed (Patent Document 6).

 However, in the former method, the fat emulsion has a problem that the particle size is large and the transparency is inferior. Furthermore, the latter intravenous emulsifier has the problem of poor storage stability when the concentration of Coenzyme Q10 is low and high. The water-soluble composition containing Coenzyme Q10 is required to be emulsifiable without the need for oil components, and to be free of special conditions and complicated processes during production.

[0009] In addition, in a food manufacturing process, when trying to add a fat-soluble natural product to food that is heat-treated in the manufacturing process, such as canned food, the emulsion is destroyed by heating, and the fat-soluble natural product is displayed. There was a problem that tallying occurred as a phenomenon that surfaced. In order to clear this problem, heat-resistant emulsification is attempted using sucrose condensed ricinoleic acid ester and alcohol (Patent Document 7). However, in the above prior art, if the heating conditions are severe, such as retorting of canned coffee (generally 125 ° C, 20 minutes), the emulsification is destroyed and taring occurs. Therefore, there is a demand for an emulsifier that is more resistant to heat.

 [0010] Emulsifiers used in cosmetics are used to blend UV diffusing agents and UV absorbing agents for absorbing UV rays into the skin, and to uniformly disperse UV diffusing agents and UV absorbing agents in cosmetics, etc. Added to. In this case, many emulsifiers are used, and it has been pointed out that when applied directly to the skin and used for a long period of time, it may cause various obstacles. There was a need for an emulsifier.

 [0011] Bathing agents include those containing inorganic salts derived from hot springs as the main component, or those consisting of alkali carbonates and organic acids that generate carbon dioxide in bath water. It is mainly blended. In recent years, interest in the skin care effect of bathing has increased, and many proposals have been made for bathing agents that have been given skin care effects. Skin bathing effects can be imparted by bath additives, such as those containing a moisturizing component such as polyhydric alcohol, polysaccharides and milk ingredients, those containing plant extracts that are considered to have skin care effects, and those containing oil. Etc. However, water-soluble moisturizing ingredients are diluted in bath water and are therefore less effective on the skin. In addition, the use of such a large amount that the effect can be expected is a problem such as causing skin stickiness after hot water force S and not being economical. On the other hand, those that use oil are effective means that achieve effects and real feelings when used in relatively small amounts, and a type in which oil is emulsified in a bath with an emulsifier is usually used. However, there is a limit to the amount of oil that can be added, and if it is added too much, the fluidity of the preparation deteriorates and hinders formulation. Therefore, a smaller amount of an effective emulsifier is required. In addition, since a large amount of water is used at the time of bathing and a large amount of emulsifier is required, it is anticipated that there will be problems with safety, and naturally derived emulsifiers have been demanded.

For these reasons, naturally-derived emulsifiers have been conventionally demanded, and the following are reported as emulsifiers from biological materials. The cell wall of Saccharomyces cerevisiae contains a substance that is a fusion of mannose and protein. It is known (see Non-Patent Document 1). In this case, the preparation of this substance requires destruction of the cells, which is very laborious and has not been put into practical use. In addition, Can dida lipolytics is known to produce substances that have an emulsifying action in the medium! /, And (see Non-Patent Document 2), but it is possible to use poorly soluble carbon sources such as hexadecane during culture. It is necessary, and it has been said that a soluble carbon source such as glucose has a low emulsifying action.

 [0013] Emulsifiers are used in various production processes as well as so-called detergents as indispensable substances in modern life, and are included in various daily products and foods. In particular, saponin, known as a natural emulsifier, has been used for a long time, and the use of natural emulsifiers is limited in its surface activity and productivity. Synthetic emulsifiers are generally used as emulsifiers, such as cost and production volume. However, biotoxicity, environmental persistence, V, and other problems have been pointed out.

 [0014] On the other hand, various emulsifier-like substances derived from microorganisms have been reported, and these are called biosurfactants, but their biotoxicity and environmental residues are low in micelle formation critical concentration (CMC) compared to synthetic detergents. It has the advantage of not having sex. It can also be mass-produced by culture! /

 There are various microorganisms in nature, some of them have unknown functions, and biosurfactant producing bacteria have been reported. Several biosurfactants, including rhamnolipid (see Non-Patent Document 3) and sopho mouth lipid (see Non-Patent Document 4), have already been put into practical use with increased productivity. Among these, those derived from yeast are sophorolipid, which is a glycolipid type for the classification of surfatatant, and this production fungus, Candida bombicola, is a red yeast used in the present invention. Different in taxonomy, the biosurfactants they produce are also different from those of the present invention.

 Patent Document 1: Japanese Patent Laid-Open No. 47-25220

 Patent Document 2: Japanese Patent Publication No. 61-260860

 Patent Document 3: Japanese Patent Publication No. 60-000419

 Patent Document 4: Japanese Patent Publication No. 58-128141

Patent Document 5: Japanese Patent Laid-Open No. 60-199814 Patent Document 6: Japanese Patent Laid-Open No. 61-56124

 Patent Document 7: Japanese Patent Laid-Open No. 4-299940

 Non-Patent Document 1: D. R. Cameron et al., Applied and Environmental Microbiology, June 1988, p. 1420—1425

 Non-Patent Document 2: M. C. Cirigliano et al., Applied and Environmental Mi crobiology, Oct. 1984, p. 747—750

 Non-Patent Document 3: M. Benincasa et al., 2002. J. Food Eng. 54: 283-28 8

 Non-Patent Document 4: M. Deshpande and L. Daniels, 1995. Bioresour. Techno 1. 53: 143- 150

 Disclosure of the invention

 Problems to be solved by the invention

[0015] An object of the present invention is to solve the above-mentioned problems and to provide high safety, high safety by itself, a novel emulsifier exhibiting high emulsifiability and emulsification stability, and fat solubility obtained by using the emulsifier. It is to provide a water-soluble composition containing a substance and a method for producing them.

 Means for solving the problem

 [0016] As a result of intensive studies to solve the above-mentioned problems, the present inventors have added a glycoprotein complex in a culture solution of red yeast to a solution of a fat-soluble substance, thereby homogenizing the fat-soluble substance. Thus, the emulsion can be stably emulsified, and it has been found that the emulsification is stable for a long time even after the emulsion is heated, and the present invention has been completed based on this finding.

 That is, the present invention relates to an emulsifier comprising, as an active ingredient, a sugar protein complex in a culture solution obtained by culturing red yeast in a culture solution.

 The present invention also relates to a water-soluble composition comprising the above emulsifier and a fat-soluble substance.

 Further, the present invention relates to a method for producing an emulsifier, characterized by culturing red yeast in a culture solution, and separating and recovering a fraction containing a glycoprotein complex in the obtained culture solution. About.

Further, the present invention provides a water-soluble composition characterized by mixing the emulsifier and a fat-soluble substance. The present invention relates to a method for manufacturing a product.

 [0018] The present invention is described in detail below.

 First, the emulsifier of the present invention comprises a sugar protein complex in a culture solution obtained by culturing red yeast in a culture solution as an active ingredient.

 [0019] The red yeast used in the present invention is not particularly limited in terms of its genus and species as long as it is an enzyme mother capable of producing a glycoprotein complex, but its culture is easy and nutritional. Because it can grow from inexpensive materials as a source, Rhodotorula genus, Xanthophyllomyces genus, Rhodospo ridium genus, Candida genus, Saccharomyces genus etc. It is done.

 [0020] Among these red yeasts, Rho dotorula mucilagmosa KUGPP-1, Rhodotorula mucilaginosa NBR C0003, Xanthophyllomyces dendrorhous NBRC10129, Rhodosporidiu m toruloides NBRC0388 and the like are more preferably used because they produce a large amount of an emulsifying substance. It is done.

 The red yeast can be used alone or in combination of two or more.

 Rhodotorula mucilaginosa KUGPP-1 was separated from Antarctica and was deposited with an accession number of FERM BP-10500 on February 2, 2006 (the original deposit on February 8, 2005 was transferred to an international deposit) In addition, it is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located in 1st, 1st, 1st, Tsukuba, Higashi, Ibaraki, Japan.

 The other red yeast can be obtained from NBRC (Biotechnology Division, Biotechnology Headquarters, National Institute of Technology and Evaluation).

 [0021] The red yeast is easily cultivated and has properties suitable for culturing in large quantities to produce a glycoprotein complex that is an aggregation inhibitor (emulsifying component). That is, the red yeast is a fraction (glycoprotein complex) that grows well in a medium consisting only of inorganic salts containing glucose as the only carbon source and can be precipitated and concentrated with acetone or the like in the culture medium. Have a strong emulsifying activity.

[0022] Regarding the method for cultivating red yeast, the culture is carried out in a medium usually used for culturing microorganisms. It ’s fine.

 Glucose is preferably used as a carbon source necessary for the growth of red yeast contained in the medium. The concentration is preferably about 0.1 to 5% by volume, more preferably about 0.5 to 1.5% by volume.

 In this case, alcohols such as ethanol and oils such as corn oil and soybean oil can be used as the carbon source instead of glucose.

[0023] As the nitrogen source, an ammonium salt is preferably used, and ammonium phosphate is particularly preferable from the viewpoint of pH buffering ability and replenishment of phosphorus content.

 In addition, since the pH of the medium decreases as the culture progresses, it is possible to carry out the culture by adding alkali dropwise, preferably adjusting the pH to 6 to 8, more preferably pH 6.5 to 7.5. I like it.

 Any alkali such as sodium hydroxide, potassium hydroxide, ammonia, and an amine compound can be used as the alkali used in this case, but ammonia is preferably used in terms of supplying a nitrogen source for cell growth. .

 [0024] In addition, a yeast extract may be added to the medium to supplement vitamins and the like.

 Also, since the glucose of the carbon source gradually decreases as the cell grows, the force that needs to be gradually added can be added at this time. A mixture of ammonia and ethanol can also be added. As an additional method, any method such as a method of intermittently adding a certain amount or a method of adding continuously can be adopted.

 [0025] As the culture temperature, the surface force of the cell growth rate is usually a force of 0 to 50 ° C, more preferably around 25 to 35 ° C.

 The culture time is not particularly limited, but is preferably 12 to 70 hours, more preferably 24 to 48 hours.

 For the culture, either agitation culture or stationary culture can be employed, and either aeration culture or closed culture may be used, but in order to accelerate the growth of microorganisms, agitation culture is preferably performed under aeration.

[0026] From the culture medium cultured in this manner, the fraction containing the glycoprotein complex, Sarakoko, is separated and recovered. Centrifugation of red yeast cells prior to separation and collection It can also be removed in advance from the culture medium by an operation such as separation, or the culture supernatant can be used.

 Glycoprotein complexes can be separated and recovered by methods using quaternary amines such as cetyl pyridinium hydrochloride and cetyltributyl odor, lower alcohols such as methanol, ethanol and isopropanol, and acetone. The method of adding to the supernatant can be used, and the former method is preferably used for obtaining a higher purity product, and the latter method is preferably used for obtaining a sample more rapidly. In addition, both methods can be used in combination, and either order may be used.

 The glycoprotein complex separated and recovered by the above method is used as a powder dried by a known method, or again dissolved in water and used as an aqueous solution.

 It can also be separated and recovered by fractionation based on affinity such as ion exchange column and affinity column, and also fractionation based on molecular weight such as ultrafiltration and gel filtration column.

 [0027] The glycoprotein complex contained as an active ingredient in the emulsifier of the present invention is a high-molecular sugar protein complex secreted from cells of red yeast into the culture medium by culturing the red yeast. It is.

 Since the glycoprotein complex is produced in the culture solution, the action as an emulsifier is remarkable, and the glycoprotein complex is recovered from the culture solution after the red yeast is produced. Separation from red yeast cells is easy.

 As the glycoprotein complex, one that is secreted by red yeast cells into the culture medium by culturing the red yeast can be used. In addition, it can be used even if it stays in the cell wall or cells before being secreted.

[0028] The glycoprotein complex is a complex having a sugar and a protein, and refers to a glycoprotein in which a sugar and a protein are combined, or a group of substances in which lipids or the like are added to the glycoprotein. It is not limited. Sugar refers to one or more oligosaccharides, and also includes a repeating structure of a specific sugar. The protein part has two or more amino acid forces.

The glycoprotein complex can be used alone or in combination of two or more. [0029] The glycoprotein complex preferably has an average molecular weight of 30,000 or more. That is, the molecular weight shown by gel filtration of the fraction containing the glycoprotein complex is preferably 30,000 or more.

 The average molecular weight can be determined by a known method such as laser scatterometer or gel filtration. In the present invention, it was determined by gel filtration as described in the examples below. That is, the average molecular weight can be measured using the obtained glycoprotein complex as a gel filtration carrier (Sephacryl S-400HR, φlcm X length 100 cm) and using dextran as a molecular weight marker.

[0030] The emulsifier of the present invention contains the glycoprotein complex produced by the red yeast as an active ingredient, but the culture solution of the red yeast that produced the glycoprotein complex is used as it is or the glycoprotein complex. Purified (= separated / recovered) body can be used. Especially when an emulsifier is used in a small amount, the concentration of the glycoprotein complex in the emulsifier can be increased by purification.

 Furthermore, in order to use the glycoprotein complex that remains in the cell walls, it is possible to use the cells as they are or to use a disrupted solution of the cells.

[0031] As described above, the other components in the emulsifier include red yeast culture fluid components other than the glycoprotein complex, and various additives may be added to enhance the effect as an emulsifier. An agent may be used.

 In order to maintain the dosage form of the emulsifier and to prevent the effect of the glycoprotein complex from degrading due to degradation, etc., as a additive, a stabilizer is used or the actual use is facilitated. In addition, a liquid such as water can be used. Further, as an additive, for example, an antioxidant, an antiseptic, a cosmetic active agent, a humidifier, a sphingolipid, a fat-soluble polymer and the like may be contained.

 Furthermore, in addition to the emulsifier of the present invention, an existing emulsifier can be used in combination.

 What is necessary is just to determine suitably the addition amount of the said additive and the existing emulsifier according to the use.

[0032] The dosage form of the emulsifier may be either liquid or solid, but is in contact with a fat-soluble substance. In order to mix the emulsifier and the fat-soluble substance uniformly, it is preferable to use liquid form.

[0033] The content of the glycoprotein complex in the emulsifier is not particularly limited, but 0.0001 to 10% by weight of the total emulsifier is preferable, and 0.001 to 1% by weight is more preferable. That's right.

 [0034] The method for producing an emulsifier of the present invention is characterized in that red yeast is cultured in a culture solution, and a fraction containing the glycoprotein complex in the obtained culture solution is separated and recovered. Specifically, it is as described above.

 In addition, it is preferable to obtain a fraction having a molecular weight of 30,000 or more shown by gel filtration by separating and collecting a fraction containing the glycoprotein complex.

[0035] Next, the present invention relates to a water-soluble composition containing a fat-soluble substance obtained by contacting an oil-soluble substance with an emulsifier containing a glycoprotein complex produced by the red yeast! Explain in an instant.

 That is, the water-soluble composition of the present invention contains the emulsifier and the fat-soluble substance.

 [0036] The fat-soluble substance used in the present invention is not particularly limited as long as it is physiologically acceptable. For example, fat-soluble drugs such as Coenzyme Q10; fat-soluble vitamins A, D, E, K, and Vitamins such as derivatives thereof; essential oil (for example, pine oil, lime oil, yuzu oil), vegetable oil (for example, soybean oil, rapeseed oil, bean flower oil, corn oil, sesame oil, cottonseed oil, olive oil, palm oil, sunflower oil Oils such as beef tallow, lard, etc .; fat-soluble pigments (eg, Anato, Tucon, Benikouji, chlorophyll, etc.); perfumes (eg, orange oil); carotenoids (eg, canthaxanthin Astaxanthin, zeaxanthin, lycopene, apocarotinanol, β-strength rotin, etc.).

 These fat-soluble substances can be used alone or in combination of two or more.

 [0037] The water-soluble composition of the present invention can contain various components such as pigments, seasonings, antibacterial agents, and the like, in addition to the additives exemplified in the above-mentioned emulsifiers, foods, cosmetics, bathing agents. Various performances as industrial products including the above can be imparted.

In addition, water; a solvent such as alcohol such as ethanol is added to the water-soluble composition. Can also be preferred.

 [0038] The content of the glycoprotein complex in the water-soluble composition is not particularly limited, but 0.0001% to 10% by weight of the whole water-soluble composition is preferable. 0.001% to 1% by weight is more preferred.

 The content of the fat-soluble substance is not particularly limited, but 0.001 to 30% by weight of the entire water-soluble composition is preferable, and 0.001 to 10% by weight is more preferable.

 [0039] The method for producing a water-soluble composition of the present invention is characterized by mixing the emulsifier and the fat-soluble substance.

 [0040] In the method for producing the water-soluble composition, an emulsifier and a fat-soluble substance are brought into contact with each other, and both are mixed. The mixing method is not particularly limited as long as both can be sufficiently brought into contact with each other, such as shaking and stirring. However, in order to quickly and sufficiently mix a material having a relatively high viscosity such as a fat-soluble substance, stirring is performed. In addition, it is preferable to vigorously stir the mixture. As a method for mixing such an emulsifier and a fat-soluble substance, known methods such as a method using a milling blender and a juicer, a method using a Manton-Gorin homogenizer, and a method using ultrasonic waves can be used.

[0041] In addition, it is necessary to consider the processing temperature, processing time, etc. under the conditions in which the emulsifier and the fat-soluble substance are brought into contact with each other and further mixed, but the type of the fat-soluble substance to be used and the fat obtained Depending on the use of the water-soluble composition containing a soluble substance, it may be carried out under appropriate conditions.

 For example, heat may be generated during mixing, so when using a material that does not have enough heat resistance, it is necessary to mix carefully so as not to reach a high temperature. Specifically, for the application to foods, etc., and the case where volatile solvents such as paints are included, the latter does not cause microbial growth. It should be done in a short time so that the solvent will volatilize!

[0042] In addition, when adding an emulsifier to a fat-soluble substance, both may be mixed after adding them at the same time 1S Add both of them gradually and mix them, or gradually add one to the other. Any form such as mixing can be adopted.

The invention's effect [0043] According to the present invention, a novel emulsifier having high safety and high emulsifiability and emulsification stability can be obtained, and a fat-soluble substance stably emulsified by using the emulsifier. It is possible to obtain a water-soluble composition containing benzene, and the contribution to the industry is very large.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0044] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

 Example 1: Preparation of crude emulsifier derived from yeast culture solution

 Prepare 10 ml of YM medium (0.03 g yeast extract, 0.03 g molasses extract, 0.05 g peptone, 0 lg darcose, pH 6.0) and sterilize using autoclave. It was. This was inoculated with the red fermentation mother Rhodotorula mucilaginosa KUGPP-1 (FERM BP-10500) deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology. A culture solution was obtained. This preculture was inoculated into 500 ml of the same medium and cultured at 30 ° C. for 48 hours, and then about 500 ml of supernatant from which the cells had been removed was collected by centrifugation. The supernatant was concentrated at about 30 m using an ultrafiltration membrane with a cutoff value of MW 100,000. Thereafter, dialysis was performed against buffer A [20 mM potassium phosphate buffer (pH 7.0)], and this was used as a crude emulsifier (sample A).

 The activity of this emulsifier was determined by adding sample 100 1 to 1.8 ml of Buffer A and dissolving it in dimethyl sulfoxide to 200 μg Zml (Wax Junyaku Co., Ltd.). ) Was added and mixed well, and the index was that it did not aggregate after standing at about 26 ° C for 15 minutes. In other words, if it does not aggregate after standing at about 26 ° C for 15 minutes, it has emulsification activity.

[0046] About four times the amount of acetone was added to Sample A, and the precipitate was collected by centrifugation. This precipitate is suspended in about 5 ml of buffer B [20 mM Tris-HCl buffer (pH 8.0)], dialyzed, and then anion-exchanged resin DEAE—TOYOPEARL650M (Tosoh) pre-equilibrated with buffer B. Load a column (φ1.6 cm x 10 cm) packed with a single company, and elute with a linear concentration gradient of sodium chloride from 0 to 1.0 M (total elution volume 360 ml) to obtain a sodium chloride concentration. About 20 ml of the emulsifying active fraction eluted between 300 mM and 350 mM. This fraction Was concentrated to about 1.5 ml with an ultrafiltration membrane, and this was used as sample B.

 This was subjected to gel filtration using a column (φ 1 cm x 100 cm) packed with Sephacryl S-400HR (Amersham) equilibrated with buffer C [50 mM potassium phosphate buffer (pH 7.0)]. Then, about 5 ml of an active fraction (emulsifying agent) was obtained, and this was designated as sample C (which was almost a single purified product from the peak during gel filtration).

 Using sample C, the amount of sugar (converted to glucose) was determined using the phenol-sulfuric acid method, and the amount of protein (converted to BSA) was determined using the BCA kit (PIERCE). The total amount of sugar was 2.6 mg. The total protein amount was 0.3 mg. From this, it was found that this emulsifier contains sugar and protein in a ratio of about 9: 1.

[0047] Example 2: Evaluation of emulsifying action

 Add 100 1 of sample A prepared in Example 1 to 1.8 ml of Buffer A [20 mM phosphate buffer (PH 7.0)], and add 200 gZml in dimethyl sulfoxide. 1001 of wastaxanthin (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 1 was added and mixed well. As a control, a culture solution of Saccharomyces cerevisiae NBRC223 prepared according to Example 1 was used.

 When observed by standing at 25 ° C. for up to 24 hours, in the control, agastaxanthin aggregates started to precipitate after about 1 hour, and after 24 hours, all had precipitated. However, when sample A was added, there was no agglomeration even after 24 hours. Further, even when this mixture of sample A and wastaxanthin was treated at 100 ° C. for 1 hour, the aggregates did not precipitate. Furthermore, even when the mixture of sample A and ataxanthin was allowed to stand at room temperature (25 ° C) for 1 week, no aggregates were observed o

 [0048] Example 3: Purification of emulsifying agent

Sample A prepared in Example 1 is filled with 30 ml of DEAE—TOYOPEARL650M (manufactured by Tosohichi Co., Ltd.), which is an anion exchange resin that has been equilibrated in advance with buffer B [20 mM Tris-HCl buffer (pH 8.0)]. Column (φ1.6 cm x 10 cm), eluting with sodium chloride linear gradient method (total elution volume 760 ml) to 1.0 M and 0 force, and sodium chloride concentration from 300 mM to 400 mM About 20 ml of the emulsifying active fraction eluted during the period was recovered. This fraction Was concentrated to about 2 ml using a centrifugal ultrafiltration membrane Centrivep-10 (Amicon). This was subjected to gel filtration using a column (φ1.6 cm × 100 cm) packed with Superose 12 (manufactured by Pharmacia) equilibrated with buffer C [50 mM potassium phosphate buffer (pH 7.0)] About 2 ml of active fraction (emulsifying agent) was obtained.

 Next, ProteinaseK (manufactured by SIGMA) was prepared to 200 UZml.

9 (final Proteinase K concentration 20UZml) and kept at 25 ° C for 2 hours.

 When this sample was used to evaluate the emulsifying action in the same manner as in Example 2, the emulsifying action was not observed. In addition, the activity of the fraction during this purification process was consistent with the strength of sugar absorption. From these facts, the emulsifying agent was considered to be a glycoprotein.

 [0049] Example 4: Measurement of molecular weight of emulsifying agent

 1.5 ml of the sample C purified in Example 1 was applied to a gel filtration carrier (Sephacryl S-400 HR, φ 1 cm X length 100 cm), and molecular weight was measured using dextran as a molecular weight marker. As a result, it was estimated that this emulsifying agent had a molecular weight of about 730,000.

 [0050] Example 5: Emulsifying action of β-carotene, vitamins (Α, Ε, Κ) and Coenzyme Q10

 Emulsifying agent 100 1 prepared in Example 3 was added to 0.9 ml of 10 mM potassium phosphate buffer (pH 7.0), 10 mg of carotene (manufactured by Nacalai Testa), vitamin A (Nacalai Tester) ), Vitamin E (manufactured by Nacalai Testa), vitamin K1 (manufactured by Nacalai Testa), and Coenzyme Q10, and mixed well. As a control, a culture solution of Sacc haromyces cerevisiae NBRC223 prepared according to Example 1 was used.

 When observed by standing at 25 ° C for up to 24 hours, in the control, aggregates of each sample started to precipitate immediately after the addition, and after 1 hour, all of them had precipitated. Coarse force When the above crude emulsifier was added, no aggregation was observed even after 24 hours.

[0051] Example 6: Emulsification of other red yeast cultures

Rhodotorula mucilagino, a red yeast, in 100 ml of YM medium in a test tube sa NBRC0003, Xanthophyllomyces dendrorhous NBRC10129, Rhodos poridium toruloides NBRC0388, respectively, and cultured at 30 ° C, 120rpm for 48 hours. About 100 ml of supernatant was collected. This fraction was concentrated to about 10 ml using a centrifugal ultrafiltration membrane Centrivep-10 (Amicon), and this was used as a crude emulsifier.

[0052] In the same manner as in Example 2, the emulsifying effect on wastaxanthin and in the same manner as in Example 5, the emulsifying effect on j8-carotene, vitamins (A, E, K), and Coenzyme Q10 were evaluated. As a result, an emulsifying action was observed in all strains.

 Moreover, when the crude emulsifier derived from red yeast was filtered using a centrifugal ultrafiltration membrane Centribrep-30 (manufactured by Amicon), the filtrate (that is, the molecular weight of less than 30,000) was emulsified. Therefore, it was estimated that more than 30,000 substances have an emulsifying effect.

 [0053] Example 7: pH stability

 Sample B prepared in Example 1 was mixed with 50 mM sodium acetate buffer at pH 5 and 6, 50 mM potassium phosphate buffer at pH 6, 7 and 8, 50 mM Tris buffer at pH 8 and 9, 5 OmM sodium carbonate at pH 9 and 10. After dialyzing with buffer solution, add 100 1 of sample B to a final concentration of 10 / z gZml, 1. Carry it with 8 ml of each pH buffer solution, and add 200 μl to dimethylsulfoxide. Axaxanthin (manufactured by Wako Pure Chemical Industries) dissolved in gZml was added at 100 / zl and mixed well. Measure the absolute value (I) of absorbance at 440nm immediately after mixing and the absolute value (に お け る) of absorbance at 440nm after standing at room temperature (about 26 ° C) for 15 minutes to calculate (I)-(Π) (ΔΑ), which is shown in FIG. In addition, astaxanthin aggregation was observed.

 440

 As a result, the absolute value of absorbance at 440 nm, which is the maximum absorption wavelength of wastaxanthin, decreases. If the above (I) one (Π) was within the range of 0 to 0.1, it was determined that the emulsification was stable.

 As a result, it has been found that the emulsifier of the present invention is effective over a wide pH range.

[0054] Example 8: Strength of emulsifying action (using aastaxanthin)

Add 100 C of Sample C prepared in Example 1 to a final concentration (sugar equivalent) of 1, 5, and 10 gZml, respectively, and add it to 1.8 ml of Buffer A. Dimethyl sul 100 1 power of axaxanthin (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in loxide / ml in hydroxide! ] Well mixed. At the same time, a commercially available biosurfactant, Surfatatin Na (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 1, 5 and 10 gZml. Table 1 shows the results of measuring the absolute value of absorbance at 440 nm after standing at 26 ° C for 24 hours.

 As a result, the emulsifier of sample C stably emulsified the astaxanthin even at a low concentration, whereas surfatatin Na aggregated the atastaxanthin at a low concentration (the absolute value of the absorbance of astaxanthin decreased) . That is, it became clear that the emulsifier of the present invention exhibits a higher emulsifying action than is surfatatin Na against wastaxanthin.

[0055] [Table 1]

 [0056] Example 9: Effect of suppressing dye fading

 Add 100 1 of sample C prepared in Example 1 to 1. 8 ml of buffer A [20 mM phosphate buffer (pH 7.0)] (final concentration in terms of sugar: 10 μg / ml) To this, 100 1 wasastaxanthin dissolved in dimethyl sulfoxide so as to be 1 mg / ml was added and mixed well. In addition, Surfactatin Na (manufactured by Wako Pure Chemical Industries, Ltd.), a commercially available biosurfactant, was added to the same concentration, and each was stored at 26 ° C for 1 month, and then the spectrum of these solutions at 200 nm to 700 nm was The results of measurement (U-2000A, manufactured by Hitachi, Ltd.) are shown in FIG. At this time, sample C containing 100 1 of buffer A instead of surfatatin Na was used as a control, and the spectrum was measured immediately after the addition of astaxanthin and used as a comparative control. Data 1 is for control (immediately after addition), data 2 is for sample C (after storage at 26 ° C for 1 month), and data 3 is for surfatatin Na (after storage at 26 ° C for 1 month) .

As a result, in the presence of the whey agent of the present invention, the absorption spectrum of istaxanthin is unchanged and stable. In the presence of surfatatin Na, the absorption spectrum is changed because astaxanthin aggregates. The color tone changed over time. That is, the present invention It was found that the emulsifier retained the color immediately after addition even after being left for 1 month.

Example 10: Sugar composition analysis

 Concentrated sulfuric acid was added to Sample C prepared in Example 1 to a final concentration of 2N, and treated at 100 ° C. for 4 hours. After neutralization with sodium hydroxide sodium hydroxide, a sample of 20 gZml (glucose equivalent sugar concentration) was used for analysis with a sugar analysis column (TSKgel Suger AXG, manufactured by Tosoh Corporation). The results are shown in Fig. 3.

 At the same time, when compared with the monosaccharide standard, these three peaks coincided with the peaks of mannose, galactose, and glucose in order from the one with the highest retention time.

 Example 11: Particle diameter

1. Add 100 1 of the astaxanthin solution dissolved in 12.5 gZml of dimethyl sulfoxide to 8 ml of buffer A, and add the sample C prepared in Example 1 to the final concentration Α: 0 125 ^ ₈ / πι1, Β: 100 1 added to make 0.25 g / ml and mixed well. Surfactin Na (manufactured by Wako Pure Chemical Industries, Ltd.) was also carried out at the same concentration. After storage for 24 hours at 26 ° C, the particle size of astaxanthin in the solution was measured using a dynamic light scattering particle size distribution device (LB-550, manufactured by HORIBA, Ltd.), and the average particle size was measured. The results for (nm) are shown in Table 2. From this result, it was found that when the emulsifier of the present invention was used, a fat-soluble substance, axaxanthin having a smaller particle size, was formed as compared with using a commercially available biosurfactant.

 [0059] [Table 2]

 Industrial applicability

[0060] According to the present invention, it is possible to obtain a novel emulsifier having high safety and exhibiting high emulsifiability and emulsification stability alone, and by using the emulsifier, a fat-soluble substance stably emulsified. It is possible to obtain a water-soluble composition containing benzene, and the contribution to the industry is very large. Brief Description of Drawings

FIG. 1 is a graph showing the pH stability of an emulsifier.

 FIG. 2 is a graph showing the effect of suppressing dye fading.

FIG. 3 is a graph of sugar composition analysis.

Claims

The scope of the claims  [I] An emulsifier comprising, as an active ingredient, a glycoprotein complex in a culture solution obtained by culturing red yeast in a culture solution.  [2] The emulsifier according to claim 1, wherein the emulsifier is at least one selected from the genera Rhodotorula, Xanthophyllomyces and Rhodosporidium.  [3] The emulsifier according to claim 1 or 2, wherein the emulsifier is at least one selected from the red yeast power Rhodotorula mucilaginosa KUGPP-1, Rhodotorula mucilaginosa NBRC0003, Xanthophyllomyces dendrorhous NBRC10129, and Rhodosporidium toruloides NBRC0388.  [4] The emulsifier according to any one of claims 1 to 3, wherein the glycoprotein complex has a molecular weight of 30,000 or more as indicated by gel filtration.  [5] A water-soluble composition comprising the emulsifier according to any one of claims 1 to 4 and a fat-soluble substance.  6. The water-soluble composition according to claim 5, wherein the fat-soluble substance is one or more selected from fat-soluble drugs, vitamins, fats and oils, fat-soluble pigments, fragrances and carotenoids.  [7] The fat-soluble drug is Coenzyme Q10, the vitamins are one or more selected from vitamins A, D, E, K, and derivatives thereof, and the fat is selected from essential oils, vegetable oils and animal oils 7. The water-soluble composition according to claim 6, wherein the carotenoid is at least one selected from canthaxanthin, austaxanthin, zeaxanthin, lycopene, apocarotinal and j8-power rotin power.  [8] A method for producing an emulsifier, comprising culturing red yeast in a culture solution, and separating and collecting a fraction containing the glycoprotein complex in the obtained culture solution.  [9] The fraction according to claim 8, wherein a fraction having a molecular weight of 30,000 or more shown by gel filtration is obtained by separating and collecting a fraction containing the glycoprotein complex. Production method.  [10] A method for producing a water-soluble composition, comprising mixing the emulsifier according to any one of claims 1 to 4 and a fat-soluble substance. [II] The fat-soluble substance contains fat-soluble drugs, vitamins, fats and oils, fat-soluble pigments, fragrances, and carotenoids. 11. The production method according to claim 10, wherein the production method is at least one selected from the group. The fat-soluble drug is Coenzyme Q10, the vitamins are one or more selected from vitamins A, D, E, K, and their derivatives, and the fat is selected from essential oils, vegetable oils and animal oils 12. The production method according to claim 11, wherein the carotenoid is at least one selected from canthaxanthin, austaxanthin, zeaxanthin, lycopene, apocarotinal and j8-power rotin power.