JP4573611B2 - Aureobasidium culture - Google Patents

Description

  The present invention relates to an aureobasidium culture obtained from an aureobasidium culture medium containing β-glucan. The aureobasidium culture of the present invention has a bioregulatory function and a physiologically active function, and is suitably used for foods, pharmaceuticals, cosmetics, feeds, chemical products and the like.

  β-glucan is contained in large amounts in cell walls of yeast, basidiomycetes, fungi, lactic acid bacteria, and other microorganisms. In addition, cells that secrete β-glucan outside the cells are also known. The cell walls and extracts of these microorganisms and basidiomycetes, and β-glucan secreted and produced outside the cells have physiological functions such as antitumor activity and immune enhancement, and therefore are being applied to food and cosmetics. In particular, β-glucans derived from basidiomycetes contained in the fruiting bodies and mycelium of basidiomycetes have been used as pharmaceuticals. However, the amount of β-glucan contained in basidiomycetes varies greatly depending on the growth / cultivation conditions, and it is necessary to separate β-glucan by extraction, and the resulting β-glucan is complex and has a wide variety of molecular species. The quality is unstable, such as a mixture of high and low molecular weight substances. As a strain that secretes and produces β-glucan outside the fungus body, there is a strain of the genus Aureobasidium (Aureobasidium sp.), And its culture solution (Aureobasidium culture solution) is used as a food or drink or food additive. I came. The β-glucan contained therein has a β-1,3-bonded glucose residue as a main chain, and this glucose residue has a β-1,6-bonded glucose residue as a side chain (branched chain). 1,6-β-D-glucan is well known (see Non-Patent Document 1 and Non-Patent Document 2).

However, the β-glucan content of the aureobasidium culture solution obtained by culturing the microorganism of the genus Aureobasidium is about 0.1 to 0.2% by mass, which is very inefficient. In addition, these microorganisms belonging to the genus Aureobasidium are called black yeasts by common names, and the cells or culture solution are colored black by the melanin pigment produced by the cells, and the resulting β-glucan is also colored. The β-glucan that is produced has a significantly deteriorated quality. In addition, the viscosity of the culture solution is high and coagulation is difficult to handle, and due to changes over time, further coloring occurs, water solubility changes, thickening, odor generation, etc., causing problems Yes.
Acta Chemica Scandinavia 17, 1351-1356 (1963) Agric. Biol. Chem. 47 (6), 1167-1172 (1983)

  The object of the present invention is to contain β-glucan, which has excellent physiological functions such as antitumor activity and immune enhancement, at a high concentration, excellent in color tone, taste and solubility in water, stable over time, and easy to handle. It provides an Aureobasidium culture with excellent quality.

The present invention removes the components from the Aureobasidium culture solution so that the content of the cells and the components having a weight average molecular weight of 50 or more and 200 or less is less than 30% by mass, and then the Aureobasidium culture solution. And is a powdered aureobasidium culture obtained by performing a homogenization process before, during or after the concentration, and has a whiteness (W value) of 70 measured by a color difference meter. The above- mentioned problems are solved by providing a powdered aureobasidium culture having a yellowness (YI value) of 30 or less .

  According to the present invention, β-glucan excellent in physiological functions such as antitumor activity and immune enhancing action is contained at a high concentration, excellent in color tone, taste, solubility in water, not changed over time, easy to handle, quality Can provide an excellent Aureobasidium culture.

Hereinafter, the aureobasidium culture of the present invention will be described in detail.
The aureobasidium culture of the present invention is an aureobasidium culture having a water content of 99% by mass or less, obtained from an aureobasidium culture solution in which a strain of the genus Aureobasidium is cultured. The aureobasidium culture of the present invention preferably has a water content of 97% by mass or less from the viewpoint of the stability of the culture (such as no change over time in odor, coloring, solubility in water, etc.) More preferably, it is 95 mass% or less.

  The aureobasidium culture solution contains β-D-glucan having 1,3,1,6-glucose bond (also referred to as 1,3,1,6-β-D-glucan) as a physiologically functional component. The aureobasidium culture of the present invention may contain 0.1% by mass or more of 1,3,1,6-β-D-glucan in order to fully exhibit its physiological functionality. The content is preferably 0.5% by mass or more, more preferably 1% by mass or more, and most preferably 2% by mass or more. 1,3,1,6-β-D-glucan may contain a 1,4-glucose bond in the molecule.

The aureobasidium culture of the present invention is preferably a concentrate obtained by removing water from the aureobasidium culture solution and concentrating 1,3,1,6-β-D-glucan. Aureobasidium culture solution, when left as it is, causes various problems such as coloring, generation of odor and increase in odor, decrease in solubility in water, generation of insoluble matter, etc. By using a concentrate, these problems can be preferably improved.
Although it is a preferable degree of concentration, it is preferable to reduce the water content. However, the water content is preferably ½ or less, more preferably 1% of the water content in the original aureobasidium culture solution. It is preferable to concentrate to / 3 or less. It is also preferable to concentrate to a powder state. Moreover, it is preferable to concentrate so that the content of 1,3,1,6-β-D-glucan is 1% by mass or more in the aureobasidium culture, more preferably 2% by mass or more. is there.

Moreover, it is preferable to remove the cells as much as possible before concentrating the aureobasidium culture solution, and after concentrating, it is preferable to leave it for one day or more before use.
In addition, sugars, amino acids, and other components having a weight average molecular weight of 50 or more and 200 or less in the aureobasidium culture solution tend to be colored, so that they are reduced or removed before concentration. Preferably it is. Concentrate the aureobasidium culture solution in which the content of the component having a weight average molecular weight of 50 or more and 200 or less in the aureobasidium culture solution is less than 30% by mass, preferably less than 20% by mass, more preferably 10% by mass or less. Is preferred. In addition, when the concentrated culture has a sugar, amino acid, or other component having a weight average molecular weight of 50 to 200 in an amount of 30% by mass or more, coloring or odor tends to occur during storage. The content of the component having a weight average molecular weight of 50 or more and 200 or less is less than 30% by mass, preferably less than 20% by mass, and more preferably 10% by mass or less.

In addition, the content of 1,3,1,6-β-D-glucan in the aureobasidium culture solution before concentration is preferably 0.1% by mass or more.
The weight average molecular weight of 1,3,1,6-β-D-glucan in the aureobasidium culture solution before concentration is 5000 to 800,000 (500 to 500,000 when the culture is used for cosmetics). ) To 30,000 to 400,000 (10,000 to 300,000 when the culture is used for cosmetics) is more preferable, and the molecular weight may be reduced using an enzyme or the like before or during concentration. The weight average molecular weight of 1,3,1,6-β-D-glucan in the obtained aureobasidium culture is preferably 5,000 to 500,000 (500 to 500,000 when the culture is used for cosmetics). 30,000 to 400,000 (10,000 to 300,000 when the culture is used for cosmetics) is preferable.

The method for concentrating the aureobasidium culture solution is not particularly limited as long as it is a method for removing water or powdering. For example, spray drying, freeze drying, freeze vacuum drying, evaporator, heating operation (boiling, drum drying) ), Solvent precipitation with a solvent such as alcohol.
The form of the aureobasidium culture of the present invention may be liquid, gel, jelly, solid, or powder, but is preferably in powder form.

  In order to obtain an aureobasidium culture, the aureobasidium culture solution is preferably subjected to homogenization (also referred to as homogenization) by physical stirring or the like before or after concentration. Examples of the homogenization treatment include, for example, treatment using a pressure homogenizer, treatment using a high-pressure homogenizer, ultrasonic treatment using an ultrasonic crusher or the like, treatment using a mill mixer, treatment using a homogenizer, and the like.

  In order to obtain an aureobasidium culture, it is also preferable that the aureobasidium culture solution is treated with activated carbon and / or a resin before and after cell separation or during cell separation. Alternatively, the aureobasidium culture solution may be treated with activated carbon and / or resin before, during or after concentration. Any activated carbon having any particle size can be used, and pulverized, granulated, pelletized, fibrous, etc. can be used. Moreover, although what was dried may be sufficient, the activated carbon activated with water vapor | steam is preferable from the point of efficiency. Moreover, you may use the impregnated activated carbon which gave special treatment to activated carbon and improved adsorption | suction ability, such as a bad smell and a metal vapor | steam. In the treatment with resin, anion exchange resins, cation exchange resins, nonionic (nonpolar) resins, hydrophobic resins, chelating resins, etc. can be used singly or in combination. In view of efficiency, it is more preferable to use a type of resin that has both adsorption characteristics of activated carbon and ion exchange resin. The activated carbon and resin are preferably food-grade in terms of safety. In the treatment of activated carbon or resin, it may be added to the culture solution or culture as it is, or a column or filter filled with activated carbon or resin may be prepared and passed through the culture solution or culture. The resin or activated carbon used for the treatment may be regenerated with acid or alkali and used repeatedly.

  Further, in order to obtain an aureobasidium culture, it is preferable that the aureobasidium culture solution is subjected to filtration treatment with celite and / or cellulose powder before or after cell separation or during cell separation. Alternatively, the aureobasidium culture solution may be filtered with celite and / or cellulose powder before, during or after concentration. Conventionally known celite and cellulose powders can be used, but food grades are particularly preferred from the viewpoint of safety.

  The powder, which is a preferred form of the aureobasidium culture of the present invention, does not change over time such as coloring, odor, solubility in water, etc., and is white and has excellent handling properties. The quality change and quality deterioration of the powder are closely related to the measured values of the whiteness (W value) and yellowness (YI value) of the powder by a color difference meter. The powder excellent in the quality of the present invention is a powder passing through 16 meshes, and the whiteness (W value) is preferably 70 or more, more preferably 80 or more, and further preferably 85 or more. The yellowness (YI value) is preferably 30 or less, more preferably 20 or less, and even more preferably 15 or less. In particular, whiteness (W value) is 70 or more and yellowness (YI value) is 30 or less, preferably W value is 80 or more and YI value is 20 or less, more preferably W value is 85 or more and YI value is 15 or less. In addition to being visually white and easy to handle, generation of odor and coloring during storage is suppressed, resulting in a quality with excellent long-term stability, which is most preferable. The powdery Aureobasidium culture of the present invention having such W value and YI value is easy to be blended with other foods, pharmaceuticals, cosmetics, chemical products and the like, and looks good when blended. Of course, the appearance of the powder itself is good, and there is no resistance to ingestion, which is preferable. The W value and the YI value can be measured using a color difference meter, and any device and any manufacturer can be used.

  In the aureobasidium culture of the present invention, the absorbance at 660 nm (liquid layer length 10 mm) of an aqueous solution (water-diluted solution) having a solid concentration of 1% by mass is 1.0 or less from the viewpoint of solubility in water. Is preferably 0.5 or less, and most preferably 0.2 or less. In the present invention, the absorbance is also referred to as turbidity.

Next, the aureobasidium culture solution used for obtaining the aureobasidium culture of the present invention will be described.
The aureobasidium culture solution is obtained by culturing a microorganism belonging to the genus Aureobasidium, and by culturing the microorganism, 1,3,1,6-β- Any strain that produces D-glucan can be used, and preferred examples thereof are strains of Aureobasidium pullulans, specifically IFO4464, IFO4466, IFO6353, IFO7757, ATCC9348, AT CC3092, ATCC42023, ATCC433023, etc. can be used, and in particular, Aureobasidium pullulans ADK-34 strain (a strain deposited as FERM BP-8391) is used. It is preferable from the content of 6-β-D-glucan, the problem of change over time, the ease of obtaining a concentrate, and the like. In addition, the microorganisms separated in the environment (for example, food, soil, indoors, etc.) can be used. Moreover, the stock strain or isolate isolate | separated by single bacteria, Furthermore, the mutant strain which performed mutation operation in them by a conventional method can be used. Examples of the mutation operation include UV treatment, chemical treatment with nitrosoguanidine, ethidium bromide, ethyl methanesulfonate, sodium nitrite, and the like.

The medium for culturing these microorganisms belonging to the genus Aureobasidium may be a medium containing nutrient sources (carbon source, nitrogen source, inorganic salts) that can be normally used by microorganisms belonging to the genus Aureobasidium, and further if necessary. A normal medium containing an organic nutrient source can be used, and various synthetic media, semi-synthetic media, natural media, and the like can be used.
As the carbon source, saccharides are preferable. Examples include monosaccharides such as glucose, sucrose, fructose, mannose, galactose, xylose and arabinose, disaccharides such as maltose, lactose and trehalose, oligosaccharides such as fructooligosaccharide and xylooligosaccharide, dextrin and starch Polysaccharides can be used alone or in combination. In addition, alcohols such as glycerol and ethylene glycol, sugar alcohols such as mannitol, sorbitol, and erythritol, organic acids, and the like are appropriately used. In addition, beet juice, sugarcane juice, fruit juices including citrus fruits, or those obtained by adding sugar to these juices can be used. The main carbon source is preferably 6 monosaccharides such as glucose and fructose, 2 monosaccharides such as sucrose and lactose, polysaccharides such as starch and dextrin, and hydrolysates of these carbohydrates. These carbon sources may be added at any time during the culture. For example, sucrose is added to the medium in a concentration range of 3 to 500 g / l, preferably 5 to 300 g / l, more preferably 10 to 200 g / l. By appropriately feeding as described above, the production rate and production amount of β-glucan can be relatively increased.

Nitrogen sources include peptone, meat extract, soy flour, casein, amino acids, malt extract, corn steep liquor, casein digest, yeast extract, organic nitrogen sources such as urea, sodium nitrate, ammonium sulfate, ammonia gas, aqueous ammonia, etc. Inorganic nitrogen sources and others can be used alone or in combination.
As inorganic salts, for example, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, and cobalt chloride, heavy metal salts, vitamins, and the like can be added and used as necessary. . In addition, when foaming occurs during culture, various known antifoaming agents can be appropriately added to the medium.

  The culture conditions for the strain are not particularly limited, and can be appropriately selected within a range in which the strain can grow well. Usually, it is good to culture at pH 5.0 to 8.5 and 20 ° C. to 35 ° C. for about 2 to 8 days. The culture conditions can be appropriately changed according to the type and characteristics of the microorganism strain used, external conditions, etc., and the optimum conditions can be selected. It is preferable to add 1 to 5% (v / v) of the main culture solution as a seed culture solution in the case of flask culture. This does not apply if culture is possible.

  Culturing is performed under aerobic conditions such as aeration and shaking. The culture time is preferably carried out to the desired 1,3,1,6-β-D-glucan production concentration, and usually 2 to 5 days. Alternatively, saccharides or medium components that are β-glucan substrates may be continuously added to continuously produce 1,3,1,6-β-D-glucan. For example, when sucrose is used as the main raw material for the production of 1,3,1,6-β-D-glucan, it is added to the culture solution as a powder or as a liquid sugar dissolved in water at a high concentration. The added amount is preferably 0.1 to 500 g per liter of the culture solution. When the addition amount is more than 500 g / l, the production rate is remarkably slow, which is not preferable. After the addition of the substrate, the substrate is preferably treated at 25 to 35 ° C. for 1 to 7 days, particularly about 2 to 5 days by shaking or aeration and stirring, and the reaction is allowed to proceed under aerobic conditions. 1,3,1,6-β-D-glucan can be produced from a certain sucrose.

  Alternatively, microbial cells obtained by culturing a strain of the genus Aureobasidium may be isolated, and a microbial strain or a strain extract may be used as a catalyst. In this case, the cultured cells are used as is, or a homogenized cell disruption solution, or the cultured cells or cell disruption solution is immobilized in an alginate gel, ion exchange resin, ceramic, chitosan, or the like. What is necessary is just to add to the saccharide solution or culture medium which is a substrate as an immobilized microbial cell. Examples of the solution that can be used for preparing suspensions of these cultured cells, cultured cell preparations, or treated cultured cells include the above-mentioned medium, Tris-acetic acid, Tris-hydrochloric acid, sodium succinate, and sodium citrate. , Single or mixed buffer solutions such as sodium phosphate and potassium phosphate. The pH of the buffer is preferably 3.5 to 9.0, more preferably 5.0 to 8.0, and still more preferably 5.2 to 7.8. The amount of cultured cells in the suspension is not limited, but is preferably about 0.1 to 5% in terms of wet volume ratio.

  The addition of saccharides or a medium as a substrate to the suspension can be carried out at any concentration and in any volume, but the amount of substrate added per time is preferably within a range where the activity of the cells can be maintained. For example, 0.1 to 500 g per liter of suspension may be divided into one time or several times, or may be continuously added at about 0.5 to 500 g / day. The solution in which the bacterial cells and the substrate are allowed to act can be incubated at an arbitrary time and temperature to produce 1,3,1,6-β-D-glucan, but under the same conditions as the culture of the strain. It is preferable to do.

After culturing as described above, β-glucan secreted and produced outside the cells is separated and collected from the culture solution according to a conventional method. Specifically, solids such as bacterial cells are separated and removed from the culture solution by centrifugation or filtration, or impurities and salts are obtained with activated carbon / ion exchange resin (cation exchange resin, anion exchange resin) or hydrophobic resin. For example, various known purification means can be selected and combined. For example, in microbial cell separation, the culture solution may be directly or concentrated, or diluted with an appropriate solvent such as water, and centrifugation, celite filtration, filtration with cellulose powder, etc. may be performed alone or in combination. it can. In addition, for example, adsorption / elution to activated carbon, ion exchange resin, hydrophobic resin, solvent precipitation using ethanol, methanol, ethyl acetate, n-butanol, etc., column method using silica gel or thin layer chromatography, reverse Separation and purification can be performed by using high-performance liquid chromatography for fractionation using a phase column alone or in combination as appropriate, and optionally repeatedly.

Bacteria may be sterilized before and after the β-glucan secreted outside the cells is separated by the above method. The sterilization of the cells may be any temperature as long as the cells are killed, but it is 50 ° C. or higher, preferably 60 ° C., more preferably 80 ° C. or higher. Furthermore, the temperature can be further increased, and for example, secreted β-glucan prepared in the microbial cells at 90 ° C. or higher or 121 ° C. under pressure can be extracted with hot water. The sterilization and hot water extraction time can be set arbitrarily, but it is 10 minutes or more and 8 hours or less, preferably 15 minutes or more and 6 hours or less, and more preferably 30 minutes or more and 2 hours or less to prevent impurities from being mixed. , 3,1,6-β-D-glucan is preferable because it does not deteriorate.
Β-glucan components other than 1,3,1,6-β-D-glucan may be produced in the aureobasidium culture solution obtained by the method as described above. You may refine | purify.

Next, the use of the aureobasidium culture of the present invention will be described.
The aureobasidium culture of the present invention has excellent bioregulatory functionality and physiological activity functions, for example, lipid metabolism improving action, intestinal regulation action, blood sugar level rise inhibiting action, cholesterol lowering action, blood sugar level lowering action, antitumor action , Immunopotentiating action, immunostimulating action, intestinal immunity enhancing action, skin immunity enhancing action, skin damage repairing action, lifestyle-related disease prevention / amelioration action, etc., excellent solubility in water, Since it has a thickening property and has no change over time such as odor, coloring, and poor solubility in water, it can be preferably used for foods, pharmaceuticals, cosmetics, feeds, chemical products and the like. When used in these applications, the aureobasidium culture of the present invention may be used alone, or the aureobasidium culture of the present invention may be used in combination with other components. Moreover, it is also preferable to use it as a composition (also referred to as a β-glucan complex) with 1,3,1,4-β-D-glucan or β-glucan derived from a grass family plant.

  Examples of foods that can be used or blended with the aureobasidium culture of the present invention include oil and fat foods, bakery products, confectionery, noodles, processed rice products, processed wheat products, processed corn products, beans (soybeans, red beans, etc.) ) Processed products, cereals (buckwheat, sea bream, awa, millet), agricultural products (potato, sweet potato, taro, troroimo, etc.), dairy products, soups, beverages, seasonings, processed meat products, processed fish products Cooking, side dishes, health foods, low calorie foods, foods for allergic patients, foods for infants, foods for the elderly, beauty foods, medicinal foods, etc.Frozen foods, retort foods, instant foods, canned foods, etc. Can be given.

Examples of the oil and fat food include margarine, shortening, whipped cream, cream, salad oil, custard cream, dip cream, fat spread, mayonnaise, tartar sauce, dressing, and oil frying.
Examples of the bakery product include bread, white bread, confectionery bread, side dish bread, French bread, croissant, pie, castella, sponge cake, butter cake, shoe confectionery, waffle, steamed bread, fermented confectionery and the like.
Examples of the confectionery include snacks, donuts, biscuits, crackers, buns, Japanese confectionery, yokan, midway, uiro, dumplings, daifuku mochi, candy, gum, chocolate, rice cake, ice cream, soft cream, sorbet, popsicle , Lact ice, ice confectionery, jelly, pudding, dessert, topping and the like.

Examples of the above processed rice products include cooked rice (including frozen cooked rice, sterile cooked rice, etc.), rice balls, rice ball rice cakes, rolled rice cakes, chili rice cakes, rice cakes, pilaf, tea pickles, doria, rice noodles, arabe, rice crackers, etc. can give.
Examples of the processed wheat products include cereal, udon, pizza, pasta, hoto, Chinese soba, fried noodles, champon, okonomiyaki, monjayaki, piroxy, buns, cup noodles and their ingredients.
Examples of the corn processed product include corn flakes, corn snacks, popcorn and the like.
Examples of the processed soybean product include tofu, soy milk, soy milk beverage, yuba, deep-fried oil, deep-fried chicken, ganmodoki, red bean paste, miso, various bean dishes, and the like.
Examples of processed cereals (buckwheat, lobster, abalone, millet) and processed agricultural products (potato, sweet potato, taro, troroimo, etc.) include buckwheat, potato snacks, potato chips, sweet potatoes, potato fries, various potato dishes, etc. Can be given.

Examples of the dairy products include milk, processed milk, yogurt, whey drink, lactic acid bacteria drink, butter, cheese, coffee whitener and the like.
Examples of the soup include potage soup, consomme soup, stew, miso soup, soup, soup, curry and the like, and these may be added to these ingredients. Or you may add directly in these like croutons.
Examples of the beverages include soft drinks, carbonated drinks, colas, juices, fruit juices, vegetable juices, tomato juices, shakes, sake, beer, sparkling wine, western liquor, wine, fruit liquors, cocktails, tea, tea, coffee , Cafe au lait, oolong tea, green juice, mineral water, water and the like.
Examples of the above-mentioned seasonings include soy sauce, fish sauce (squid kana soy sauce, sardine soy sauce, salt soup, nanpura, etc.), miso, jam, sauce, Worcester sauce, tomato sauce, tomato ketchup, tomato paste, tomato puree, chili sauce, sauce , Pepper, pepper, garlic, ginger, vinegar, chili oil, tabasco, salt, various spices.
Examples of the processed meat products include hamburger, sausage, salami sausage, ham, meatball, meat dumpling, meat bun, dumpling, shumai, various meat dishes, and the like.

Examples of the processed fishery products include kamaboko, deep-fried sweet potato, tsumire, and kneaded products.
Examples of the health food or medicinal food include supplements, tablets, drinks, sports drinks and the like.

  Examples of pharmaceuticals that can use or be mixed with the aureobasidium culture of the present invention include lipid metabolism improving action, intestinal regulating action, blood sugar level raising inhibiting action, cholesterol lowering action, blood sugar level lowering action, antitumor action, immune enhancement Action, immunostimulatory action, intestinal immunity enhancing action, skin immunity enhancing action, lifestyle-related disease prevention / amelioration action, eyeball lens fluid replacement, articular cartilage fluid improvement action, etc., cancer prevention or treatment drug, infection prevention or Examples thereof include therapeutic agents for skin, ointments, ointments, patches, nasal preparations, ear drops, eye drops, suppositories, vaginal preparations and the like. Furthermore, it is also useful as an ointment base, tablet, granule, excipient for powder formulation, internal use, and liquid dispersant.

  Examples of feeds that can use or blend the aureobasidium culture of the present invention include livestock feeds, pet feeds, seafood feeds, farmed fish feeds, etc. It can also be used as a preventive agent, a therapeutic agent or preventive agent for pets, a therapeutic agent or prophylactic agent for seafood, a therapeutic or preventive agent for cultured fish, an infectious disease preventive agent, and the like.

  When the aureobasidium culture of the present invention is used for foods, pharmaceuticals, feeds, and the like, other substances that impart some physiological activity to the body may be further blended. For example, cholesterol elevation inhibitor, blood pressure elevation suppressor, blood cholesterol regulation function agent, blood glucose level elevation inhibitor, gut microbiota ameliorator, intestinal regulator, immune enhancer, anticancer agent, antiallergy Agents, digestion and absorption regulating agents, anti-aging agents, antioxidants, blood circulation promoters, amino acids, peptides, lipids, polysaccharides, oligosaccharides, proteins, carbohydrates, dietary fiber, enzyme components, etc. .

  Specific examples of these other substances that give physiological activity include polyunsaturated fatty acids (EPA, DHA) that optimize blood lipid concentration, plant sterols that regulate serum cholesterol, and esterified products thereof, diacylglycerol, Unsaturated fatty acids such as γ-linolenic acid, α-linolenic acid, linoleic acid, conjugated linoleic acid, beet fiber, corn fiber, psyllium seed coat, tea polyphenol, lecithin, bonito peptide, sardine peptide, casein deca peptide effective for blood pressure lowering, soybean Foods and pharmaceuticals that contain lactic acid bacteria, gluconic acid, oligosaccharides, various dietary fibers, etc. that improve the intestinal environment and work for intestinal regulation, such as isolated proteins. As other ingredients known to have health functions, chlorella, spirulina, propolis, chitin, chitosan, deoxyribonucleic acid, ribonucleic acid, ganoderma, agaricus, ginkgo biloba extract, citrus fruit , Turmeric, garcinia, apple fiber, gymnema, collagen, blueberry, aloe, saw palmetto, capsanthin, lutein, β-cryptoxanthin, renin, taurine, casein, collagen, glucosamine, casein phosphopeptide (CPP), milk basic protein (MBP) , Lactoferrin, glutathione, theanine, gaba, aspartame, xylitol, recardent, alginic acid, sodium alginate, fucoidan, chondroitin, hyaluronic acid, cellulose, pectin, indigestible Xistrin, glucomannan, inulin, fructan, cyclofructan, difructose, levan, mucin, flavonoid, polyphenol, catechin, tannin, anthocyanin, rutin, quercetin, soy isoflavone, soy saponin, soy globulin, chlorogenic acid, citric acid, lactic acid, Malic acid, capsaicin, sesame lignan, allicin, caffeine, chlorophyll, nattokinase, β-lactoglobulin, plant fermentation enzyme, mevalonic acid, chlorophyll, royal jelly, ginseng, prunes, chamomile, thyme, sage, peppermint, lemon balm, mallow, Oregano, Catnip tea, Yarrow, Hypiscus, Echinesia and other herbs, vitamins, calcium-containing compounds such as calcium fortifiers, iron-containing compounds and other iron fortification , Mineral reinforcing agent containing essential minerals, more animals and plants extract, seaweed extract, bioactive ingredient natural ingredients such as crude drugs components and the like.

  As vitamins that can be added, those listed as fortifiers in food additives can be used, such as vitamin A or β-carotene, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, pantothenic acid. , Niacin, biotin, vitamin C, vitamin D, vitamin E, vitamin K, derivatives thereof, oil-coated vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, pantothenic acid, niacin, biotin, vitamin C, coenzyme Q10 (Vitamin Q) etc. Specific examples of vitamin derivatives include thiamine hydrochloride, thiamine sulfate, dibenzoylthiamine, dibenzoylthiamine hydrochloride, thiamine nitrate, thiamine lauryl sulfate, bisbenchamine, etc. as vitamin B1, and riboflavin butyrate as vitamin B2. Riboflavin 5′-phosphate sodium, pyridoxine hydrochloride as vitamin B6, L-ascorbic acid stearate, sodium L-ascorbate and the like as vitamin C.

  As calcium-containing compounds that can be blended, those listed as calcium fortifiers in food additives can be used, such as calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, acidic calcium pyrophosphate, calcium hydroxide, Calcium carbonate, calcium lactate, calcium sulfate, primary calcium phosphate, dibasic calcium phosphate, tertiary calcium phosphate, calcium pantothenate, calcium pyrohydrogen phosphate, coral calcium, dolomite, eggshell calcium, beef bone powder calcium, scallop shell calcium, milk calcium, etc. Can be given.

As iron-containing compounds that can be blended, those listed as iron enhancers in food additives can be used, such as ferric chloride, iron citrate, ammonium iron citrate, sodium iron citrate succinate, lactic acid Examples thereof include iron, ferrous pyrophosphate, ferric pyrophosphate, ferrous sulfate, ferrous gluconate, heme iron, lever powder, and oil-coated iron.
Moreover, you may make it contain an essential mineral, For example, zinc, potassium, magnesium, manganese, phosphorus, sodium, selenium, iodine, molybdenum etc. are mention | raise | lifted as an essential mineral. Examples of sources for supplying such essential minerals include wheat extract, wheat germ, young wheat leaf, chlorella, oyster meat extract, seawater concentrate, nuts, fish meal, liver powder, brown rice powder, and brewer's yeast.

  Specific examples of physiologically active ingredients of naturally derived ingredients such as animal and plant extract ingredients, seaweed extract ingredients, and herbal medicine ingredients that can be blended include Ashitaba extract, Avocado extract, Achacha extract, Altea extract, Arnica extract, Aloe extract, Apricot extract, Apricot kernel extract, Ginkgo biloba extract, Fennel extract, Turmeric extract, Oolong tea extract, Ages extract, Echinacea leaf extract, Ogon extract, Prunus extract, Auren extract, Barley extract, Hypericum extract, Odrianthus extract, Dutch mustard extract, Orange extract, Seawater dried Products, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, chamomile extract, carrot extract, kawara mugi extract, licorice extract, oil-soluble licorice extract, calcade extract, oyster extract, kiyu Extract, Kina extract, Cucumber extract, Guanosine, Gardenia extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Clematis extract, Chlorella extract, Mulberry extract, Gentian extract, Black tea extract, Yeast extract, Burdock extract, Fermented rice bran extract, Rice germ Oil, Comfrey extract, Collagen, Cowberry extract, Saishin extract, Psycho extract, Saitai extract, Salvia extract, Salmon extract, Sasa extract, Hawthorn extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, Perilla extract, Linden extract, Shimotake Extract, Peonies Extract, Ginger Root Extract, Birch Extract, Horsetail Extract, Kizuta Extract, Hawthorn Extract, Elderflower Extract, Achillea millefolium extract, mint extract, sage extract, mallow extract, nematode extract, senbu extract, soybean extract, taisu extract, thyme extract, tea extract, clove extract, chigaya extract, chimpi extract, toki extract, tokisenka extract, tonin extract, spruce Extract, Dokudami Extract, Tomato Extract, Natto Extract, Carrot Extract, Garlic Extract, Novara Extract, Hibiscus Extract, Bacmond Extract, Parsley Extract, Honey, Hamamelis Extract, Parietalia Extract, Hikikoshi Extract, Bisabolol, Biwa Extract, Fukitane Popo Extract, Fukinoto Extract, Bukuryo Extract, Butcher Bloom Extract, Grape Extract, Placenta Extract, Propolis, Loofah Extract, Safflower Extract, Peppermint Extract, Body Extract, Button Extract, Hop Extract, Pine Extract, Maronier Extract, Citrus Extract, Mukuroji Extract, Melissa Extract, Peach Extract, Cornflower Extract, Eucalyptus Extract, Yukinoshita Extract, Yuzu Extract, Yakuinin Extract, Mugwort Extract, Lavender Extract, Examples include apple extract, lettuce extract, lemon extract, lotus extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract and the like.

In addition to the above components, sweeteners, colorants, seasonings, bittering agents, toughening agents, surfactants, solubilizers, thickeners, pastes, excipients commonly used in foods and pharmaceuticals, etc. , Preservatives, fragrances, antibacterial agents, bactericides, antioxidants, salts, solvents, chelating agents, neutralizing agents, acidulants, pH adjusters, preservatives, buffering agents, fats and oils, processed oils, starches, flours, Ingredients such as buckwheat flour, flour, eggs, dairy products, and dairy products can be used.
Examples of excipients that can be preferably used include agar, gelatin, guar gum, starch, dextrin, cellulose, chocolate, cocoa butter, coconut oil, palm kernel oil, hardened oil and other oils, various proteins, wheat flour, rice flour , Soy flour, soybean flour, flour such as wheat germ, soy protein, skim milk powder, casein, albumin, globulin, cheese and other dairy products and processed milk products, egg white, egg yolk and other processed egg products, sugar, glucose, maltose And sugars such as lactose, salts such as sodium chloride and calcium carbonate, etc., and a mixture or composite material thereof may be used.

  Next, examples of cosmetics that can be used or blended with the aureobasidium culture of the present invention include skin lotion, emulsion, skin milk, cream, ointment, external preparation, lotion, calamine lotion, sunscreen agent, suntan agent. , After-shave lotion, pre-shave lotion, makeup base, pack, cleansing, face wash, anti-acne cosmetic, essence and other basic cosmetics; foundation, white powder, eye shadow, eyeliner, eyebrow, teak, lipstick, nail color Make-up cosmetics such as shampoo, rinse, conditioner, hair color, hair tonic, set agent, hair styling, hair restorer, body powder, deodorant, hair remover, massage cosmetics, body cosmetics, skin cosmetics, Esthetic cosmetics, hay fever and Improvement agents such as ghee, antiallergic agents, rough skin prevention agents, skin disease improvement agents, skin damage repair agents, wrinkle protection agents, cold compresses, hot compresses, poultices, patches, scrub cosmetics, soaps, tissues Examples include paper, body shampoo, hand soap, perfume, toothpaste, oral care products, mouthwash, gum massage cream, and bath preparation.

  When blended in the above cosmetics, other substances that give some physiological activity to the skin may be blended. For example, whitening ingredients, anti-inflammatory agents, anti-aging agents, slimming agents, tanning agents, antioxidants, hair growth agents, hair growth agents, blood circulation promoters, moisturizing ingredients other than polyhydric alcohols or sugars, drying agents, cooling agents , Warming agents, amino acids, wound healing promoters, irritation relieving agents, analgesics, cell activators, antiallergic agents, rough skin prevention agents, skin damage repair agents, enzyme components, and the like may be blended.

  Specific examples of other substances that give physiological activity to the skin include, as biologically active ingredients of naturally derived ingredients such as animal and plant extract ingredients, seaweed extract ingredients, herbal medicine ingredients, for example, Ashitaba extract, Avocado extract, Achacha extract, Altea extract, Arnica extract, Aloe extract, Apricot extract, Apricot kernel extract, Ginkgo biloba extract, Fennel extract, Turmeric extract, Oolong tea extract, Age extract, Echinacea leaf extract, Ogon extract, Oat extract, Auren extract, Barley extract, Hypericum extract, Steller's root extract, Dutch mustard extract, orange extract, dried seawater, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, chamomile extract, carrot extract, Chinese mugwort extract, licorice extract, oil-soluble licorice ex , Kalkade extract, Oyster extract, Kiwi extract, Kina extract, Cucumber extract, Guanosine, Gardenia extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Clematis extract, Chlorella extract, Mulberry extract, Gentianana extract, Black tea extract, Yeast extract, Burdock Extract, Fermented rice bran extract, Rice germ oil, Comfrey extract, Collagen, Cowberry extract, Saishin extract, Psycho extract, Saitai extract, Salvia extract, Soap extract, Sasa extract, Hawthorn extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, perilla extract, linden extract, citrus extract, peony extract, shrimp root extract, birch extract, horsetail extract, kizuta extract Hawthorn extract, elderberry extract, yarrow extract, mint extract, sage extract, mallow extract, sensu extract, senbu extract, soybean extract, taisu extract, thyme extract, tea extract, clove extract, chimp extract, toki extract, tow Calendula extract, Spruce extract, Spruce extract, Dokudami extract, Tomato extract, Natto extract, Carrot extract, Garlic extract, Novara extract, Hibiscus extract, Bacmond extract, Parsley extract, Honey, Hamamelis extract, Parietalia extract, Hikikoshi extract, Bisabolol, Biwa extract, Japanese dandelion extract, Japanese quince extract, Bukuryo extract, Butcher bloom extract, Grape extract, Placentae Kiss, Propolis, Loofah extract, Safflower extract, Peppermint extract, Bodaiju extract, Button extract, Hop extract, Pine extract, Maronnier extract, Citrus extract, Mulberry extract, Melissa extract, Peach extract, Cornflower extract, Eucalyptus extract, Yukinosita extract, Yuzu extract, Yokuinin Examples include extract, mugwort extract, lavender extract, apple extract, lettuce extract, lemon extract, lotus extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract and the like.

  Specific examples of preferable physiologically active components such as other naturally-derived components include deoxyribonucleic acid, raffinose, mucopolysaccharide, hyaluronic acid or a salt thereof such as sodium hyaluronate, chondroitin sulfate sodium, collagen, elastin, chitin, chitosan Biopolymers such as hydrolyzed eggshell membranes; amino acid derivatives such as amino acids, sarcosine and N-methyl-L-serine; polyhydric alcohols or sugars such as ethyl glucose, sodium lactate, urea, sodium pyrrolidonecarboxylate, betaine, whey Other moisturizing ingredients; oily ingredients such as sphingolipids, ceramides, cholesterol, cholesterol derivatives, phospholipids; anti-inflammatory agents such as ε-aminocaproic acid, glycyrrhizic acid, β-glycyrrhetinic acid, lysozyme chloride, guaiazulene, hydrocortisone; Vitamins such as vitamin A, B2, B6, D, calcium pantothenate, biotin, nicotinamide, vitamin E; active ingredients such as allantoin, diisopropylamine dichloroacetic acid, 4-aminomethylcyclohexanecarboxylic acid; carotenoids, flavonoids, tannins Antioxidants such as lignan and saponin; cell activators such as α-hydroxy acid, β-hydroxy acid and mevalonic acid; blood circulation promoters such as γ-oryzanol; wound healing agents such as retinol and retinol derivatives; Whitening agents such as arbutin, kojic acid, placenta extract, sulfur, ellagic acid, linoleic acid, tranexamic acid, glutathione; cephalanthin, licorice extract, red pepper tincture, hinokitiol, garlic iodide extract, pyridoxine hydrochloride, nicotinic acid, nicoti Acid derivatives, calcium pantothenate, D-pantothenyl alcohol, acetyl pantothenyl ethyl ether, biotin, allantoin, isopropylmethylphenol, estradiol, ethinylesteradiol, capronium chloride, benzalkonium chloride, diphenhydramine hydrochloride, takanal, camphor, salicylic acid, Nonyl acid vanillyl amide, nonanoic acid vanillyl amide, piroctone olamine, glyceryl pentadecanoate, l-menthol, mononitroguaiacol, resorcin, γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormone, cantalistin tincture, cyclosporine, zinc pyrithione , Hydrocortisone, minoxidil, polyoxyethylene sorbitan monostearate, mint oil, sasani Examples include hair restorers such as shiki extract and herbs such as fucoidan and echinesia.

  Examples of the ascorbic acids include ascorbic acid, ascorbic acid sulfate, ascorbic acid phosphate, ascorbic acid higher fatty acid ester, and salts thereof. These salts are sodium salt, potassium salt, magnesium salt, calcium salt, barium salt, ammonium salt, monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt, triisopropanolamine. Examples include salt. Examples of the ascorbic acid sulfate include ascorbic acid-2-sulfate and ascorbic acid-3-sulfate, and examples of the ascorbic acid phosphate include ascorbic acid-2-phosphate and ascorbic acid. -3-phosphate esters, which are known substances and are described in JP-B-44-31237 and JP-B-54-21415. Examples of higher fatty acid esters of ascorbic acid include ascorbic acid-2-palmitic acid monoester, ascorbic acid-2,6-palmitic acid diester, ascorbic acid-2-stearic acid ester, and the like.

  In addition to the above components, oils, powders (pigments, dyes, resins), fluorine compounds, resins, surfactants, stickers, preservatives, fragrances, antibacterial agents, and bactericides that are commonly used in cosmetics Ingredients such as salts, solvents, chelating agents, neutralizing agents, pH adjusting agents and insect repellents can be used.

  Examples of the above powders include dyes such as red 104, red 201, yellow 4, blue 1, black 401, rake dyes such as yellow 4 Al lake, yellow 203 Ba rake; nylon powder , Silk powder, urethane powder, Teflon (registered trademark) powder, silicone powder, polymethyl methacrylate powder, cellulose powder, silicone elastomer spherical powder, polyethylene powder, etc .; yellow iron oxide, red iron oxide, black iron oxide Colored pigments such as chromium oxide, carbon black, ultramarine, and bitumen; white pigments such as zinc oxide, titanium oxide, and cerium oxide; extender pigments such as talc, mica, sericite, kaolin, and plate-like barium sulfate; Pearl pigment; barium sulfate, calcium carbonate, magnesium carbonate, aluminum silicate, silicate mug Metal salts such as Siumu; silica, alumina such as inorganic powder; bentonite, smectite, boron nitride and the like. These powders have a spherical shape, a rod shape, a needle shape, a plate shape, an indefinite shape, a flake shape, a spindle shape, and the like.

These powders are conventionally known surface treatments such as fluorine compound treatment, silicone treatment, silicone resin treatment, pendant treatment, silane coupling agent treatment, titanium coupling agent treatment, oil agent treatment, N-acylated lysine treatment, The surface treatment may or may not be performed in advance by polyacrylic acid treatment, metal soap treatment, amino acid treatment, inorganic compound treatment, plasma treatment, mechanochemical treatment, or the like.
Among these powders, silicone elastomer spherical powder, polyethylene powder, polypropylene powder, Teflon (registered trademark) powder, silicone rubber, urethane powder, polyalkylsilsesquioxane, nylon, silica beads, alumina beads, apatite, allyl Spherical powders (including hollow resin powders) such as fluorinated acrylic beads are preferably blended because they retain the physiologically active component and are excellent in sustained release effect.

  Examples of the oil agent include volatile and non-volatile oil agents and solvents and resins that are usually used in skin cosmetics. The oil agent may be a liquid, paste, or solid at room temperature, but a liquid that is excellent in handling is preferable. Examples of oil agents include, for example, higher alcohols such as cetyl alcohol, isostearyl alcohol, lauryl alcohol, hexadecyl alcohol, octyldodecanol; fatty acids such as isostearic acid, undecylenic acid, oleic acid; glycerin, sorbitol, ethylene glycol, propylene Polyols and sugars such as glycol and polyethylene glycol; myristyl myristate, hexyl laurate, decyl oleate, isopropyl myristate, hexyl decyl dimethyloctanoate, glyceryl monostearate, diethyl phthalate, ethylene glycol monostearate, oxy Esters such as octyl stearate; Hydrocarbons such as liquid paraffin, petroleum jelly and squalane; Waxes such as lanolin, reduced lanolin and carnauba wax Mink oil, cacao butter, coconut oil, palm kernel oil, camellia oil, sesame oil, castor oil, oils and fats, such as olive oil, ethylene-alpha-olefin co-oligomer, and the like.

  Examples of other forms of oil include, for example, dimethylpolysiloxane, methylhydrogenpolysiloxane, methylphenylpolysiloxane, polyether-modified organopolysiloxane, fluoroalkyl / polyoxyalkylene co-modified organopolysiloxane, alkyl-modified Silicone compounds such as organopolysiloxane, terminal-modified organopolysiloxane, fluorine-modified organopolysiloxane, amodimethicone, amino-modified organopolysiloxane, silicone gel, acrylic silicone, trimethylsiloxysilicate, silicone RTV rubber; perfluoropolyether, fluorinated Fluorine compounds such as pitch, fluorocarbon and fluoroalcohol are listed.

Examples of the solvent include purified water, cyclic silicone, ethanol, light liquid isoparaffin, lower alcohol, ethers, LPG, fluorocarbon, N-methylpyrrolidone, fluoroalcohol, volatile linear silicone, and next-generation fluorocarbon. .
As the surfactant, for example, an anionic surfactant, a cationic surfactant, a nonionic surfactant, or a betaine surfactant can be used.

  Examples of adhesives and resins include sodium polyacrylate, cellulose ether, calcium alginate, carboxyvinyl polymer, ethylene / acrylic acid copolymer, vinyl pyrrolidone polymer, vinyl alcohol / vinyl pyrrolidone copolymer, nitrogen substituted acrylamide type Polymer, polyacrylamide, cationic polymer such as cationized gar gum, dimethylacrylammonium polymer, acrylic acid / methacrylic acid acrylic copolymer, POE / POP copolymer, polyvinyl alcohol, pullulan, agar, gelatin, tamarind seed polysaccharide, Xanthan gum, carrageenan, high methoxyl pectin, low methoxyl pectin, gar gum, gum arabic, crystalline cellulose, arabinogalactan, karaya gum, gum tragacanth, algin , Albumin, casein, curdlan, gellan gum, dextran, cellulose, polyethyleneimine, highly polymerized polyethylene glycol, cationized silicone polymer, synthetic latex, and the like.

It is also preferable to impart a UV protection effect to cosmetics. In this case, it is preferable to blend an ultraviolet protective agent (also referred to as an ultraviolet absorber) as shown below.
Examples of the ultraviolet protective agent (including organic and inorganic types, which may be any of UV-A and B) include fine particle titanium oxide and fine particle zinc oxide. Examples of the organic ultraviolet protective agent include 2-methoxyhexyl paramethoxycinnamate, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfate, and 2,2′-dihydroxy-4- Methoxybenzophenone, p-methoxyhydrocinnamic acid diethanolamine salt, paraaminobenzoic acid (hereinafter abbreviated as PABA), ethyldihydroxypropyl PABA, glyceryl PABA, homomenthyl salicylate, methyl-O-aminobenzoate, 2-ethylhexyl-2-cyano- 3,3-diphenyl acrylate, octyl dimethyl PABA, octyl methoxycinnamate, octyl salicylate, 2-phenyl-benzimidazole-5-sulfate, triethanolamine salicylate, 3- (4-methylbenzili Den) camphor, 2,4-dihydroxybenzophenine, 2,2 ', 4,4'-tetrahydroxybenzophenone, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone, 2-hydroxy-4-N- Octoxybenzophenone, 4-isopropyldibenzoylmethane, butylmethoxydibenzoylmethane, 4-tert-butyl-4'-methoxydibenzoylmethane, 4- (3,4-dimethoxyphenylmethylene) -2,5-dioxo-1 -Imidazolidinepropionate 2-ethylhexyl, their polymer derivatives, silane derivatives and the like. Furthermore, what sealed these in the polymer may be used.

  Next, examples of chemicals that can be used or blended with the aureobasidium culture of the present invention include medical detergents, garment detergents, contact lens preservatives, contact lens cleaning liquids, dry eye prevention agents, disinfectants, Examples include soaps, hygiene products, medical products, toothpaste products, toiletry products, diaper products, feminine sanitary products, thickeners, and viscosity modifiers.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Analysis example-1 (water content)
For the measurement of the water content, 20 g of the culture solution and the concentrated solution were accurately weighed into a mass-measured eggplant-shaped flask and dried with an evaporator to calculate the solid content mass. The powder was measured for loss on drying using an infrared moisture meter (FD-230: manufactured by Kett Science Laboratory), and the moisture content was calculated.

Analysis example-2 (calculation of the molecular weight of β-glucan, the ratio of components having a molecular weight of 200 or less)

The weight average molecular weight of β-glucan and the ratio of components having a weight average molecular weight of 200 or less were analyzed by HPLC analysis. That is, each culture solution was prepared by concentrating or adding water so as to have a solid content of 1%, and then adding twice the amount of distilled water and mixing well to prepare a sample for HPLC. The powder was dissolved in distilled water so as to be 10 mg / ml, and further doubled amount of distilled water was added and mixed well to prepare a sample for HPLC. For separation, Shodex packed columns KS-804 and KS-802 (manufactured by Showa Denko), which are HPLC gel filtration columns, were used at a flow rate of 0.5 ml / min. The temperature was 80 ° C., and a molecular weight measuring device TriSEC Model 302 manufactured by Viscotech was used for detection, and three points of differential pressure viscosity, differential refraction, and light scattering were measured, and the molecular weight was calculated from the three pieces of information. First, a solution (1.5 mg / ml, 2.5 mg / ml, 5.0 mg / ml) with an accurate β-glucan concentration was prepared, and the refractive index specific to the sample was calculated from the sample concentration and the differential refractive area value. did. In the measurement of the differential pressure viscosity, pullulan was used as a standard material for the intrinsic viscosity. The approximate molecular weight is calculated from the differential refraction detection area value and the light scattering detection area value measured for the purified β-glucan solution (1.5 mg / ml), and the molecular weight is corrected from the calculated β-glucan intrinsic viscosity. Of molecular weight. The results are shown in Table 1.
A peak area value with a molecular weight of less than 200 with respect to the total peak area value was calculated from the area value of each peak obtained by RI measurement.

Analysis Example 3 (Measurement of whiteness and yellowness of powder)
The powdered sample was measured for whiteness and yellowness with a color difference meter. For the measurement, a colorimetric color difference meter (ZE2000: manufactured by Nippon Denshoku Industries Co., Ltd.) is used. First, measurement correction is performed with a standard white tile, and 2 g of each sample is accurately taken into a cell and measured. XYZ values and Lab values were calculated according to the color specification method, and whiteness (W value) and yellowness (YI value) were calculated from these values.

Analysis Example 4 (Measurement of turbidity of 660 nm)
Absorbance (turbidity) at 660 nm was measured by adding distilled water or concentrating with an evaporator so that the solution had a solid concentration of 1%, and dissolving the powder in distilled water so that the solid concentration was 1%. Then, 1 ml was added to a 10 mm square quartz cell and measured with a spectrophotometer (manufactured by Hitachi, Ltd .: U-3300).

Analysis Example-5 (Detection of 1,3,1,6-β-D-glucan)
The content of 1,3,1,6-β glucan contained in each sample is determined by measuring the amount of polysaccharide having β bonds in the solid content, and then measuring the residual glucose having 1,3,1,6 bonds in the polysaccharide. This was done by checking the group. That is, the culture solution or the concentrate was first calculated in accordance with Analysis Example-1 and then freeze-dried to obtain a powder. Add 5 mg of powder sample to 1 ml of 20 mM citrate buffer (pH 5.0) and mix well. Add 1 μL of pullulanase (Wako Pure Chemical Industries) and 1 mg of α-amylase derived from human saliva (Sigma). The mixture was reacted at 40 ° C. for 16 hours, twice as much ethanol was added, and allowed to stand at −20 ° C. for 1 hour. Then, the polysaccharide was precipitated by centrifugation, freeze-dried, and the mass of the precipitate was measured. The precipitate was dissolved in distilled water to 2 mg / ml, and the amount of polysaccharide was measured by the phenol sulfuric acid method. In the determination of polysaccharides by the phenol-sulfuric acid method, a pullulan calibration curve was used. The amount of β-glucan in the powder sample and the amount of β-glucan in the culture solution or concentrate were calculated from the amount of polysaccharide after the enzyme treatment. Next, 1,3,1,6 glucose residues in the enzyme-treated product were confirmed.

  Analysis was performed by the methylation method. To 3 mg of the enzyme-treated product, 2 ml of DMSO was added and dissolved, 0.5 ml of a carbanion reagent (adjusted with DMSO and sodium hydride) was added, and the mixture was reacted at room temperature for 4 hours. While cooling, 1.5 ml of methyl iodide was added and allowed to react for 1.5 hours. The reaction solution was dialyzed against running water for 3 hours and overnight with 200-fold distilled water, and then concentrated to dryness with a vacuum evaporator. After repeating this methylation operation three times, 0.1 ml of 72% by weight sulfuric acid was added, and after reaction with ice cooling, the mixture was heated at 100 ° C. for 4 hours. The mixture was neutralized and centrifuged to recover the supernatant, desalted and concentrated with a vacuum evaporator, followed by acid hydrolysis. Next, alditolization was performed. That is, 1 ml of distilled water and 1 drop of 2N aqueous ammonia were added to the concentrated sample, 3 mg of sodium borohydride was added, and the mixture was reacted at room temperature for 2 hours. The mixture was neutralized with an ion exchange resin and concentrated with a vacuum evaporator. Further, 1 ml of methanol was added, and the mixture was concentrated with a vacuum evaporator. Next, 0.1 ml of acetic anhydride and 0.1 ml of pyridine were added to the concentrated sample and reacted at 100 ° C. for 2 hours. 1 ml of distilled water was added and the mixture was concentrated with a vacuum evaporator. This concentration operation was repeated three times, 20 μl of acetone was added to the dried sample, and measurement was performed by gas chromatography. Gas chromatography uses capillary column TC-WAX (manufactured by GL Sciences), carrier gas He110kPa, column temperature 230 ° C, inlet temperature 250 ° C, detection method performs hydrogen flame ionization detection, 5890A Gas Chromatograph (manufactured by HEWLETT PACKARD) Was used. In this analysis, inositol was used as an internal standard, and 2,3,4,6 methyl 1 acetyl glucose, 2,4,6 methyl 1,3 acetyl glucose, , 4 methyl 1,3,6 acetylglucose, it was confirmed that 2,4 methyl 1,3,6 acetylglucose was obtained from each sample.

Production Example 1 (Production of Aureobasidium Culture Solution)
Aureobasidium pullulans (Aureobasidium pullulans) ADK-34 strain (FERM BP-8391) was inoculated into 120 ml of YM medium (manufactured by Difco) to obtain a preculture solution (cultured at 26 ° C., 180 rpm, 3 days). Sucrose 90 g, yeast extract _{15g, K 2 HPO 4 1.0g,} KCl 0.5g, MgSO 4 · 7H 2 O 0.5g, FeSO 4 · 7H 2 O 0.01g, 5L jar containing the distilled water 3L The mentor was inoculated with 100 ml of the preculture solution and cultured at 26 ° C. for 72 hours to obtain 3 L (pH 4.5) of the culture solution (Aureobasidium culture solution-1). The obtained Aureobasidium culture solution-1 was sterilized by heating at 80 ° C. for 30 minutes, added with an equal amount of distilled water, mixed well, then centrifuged at 8000 rpm for 30 minutes, and the culture supernatant ( About 6 L of Aureobasidium broth-1A) was obtained. The water content of the obtained Aureobasidium culture solution-1A was 99.5% by mass, and the content of 1,3,1,6-β-D-glucan was 0.43% by mass. The absorbance at 660 nm was 0.881. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 350,000.

Production Example 2 (Production of aureobasidium culture medium: high-pressure homogenizer treatment-dilution-preparation of centrifugal supernatant)
Aureobasidium culture solution-1 obtained in Production Example 1 was sterilized by heating at 80 ° C. for 30 minutes, added with an equal amount of distilled water, mixed well, and then mixed with a pressure homogenizer (manufactured by SMT) at 400 atm. The mixture was treated for 30 minutes and centrifuged at 8000 rpm for 30 minutes to obtain about 5 L of a culture supernatant (Aureobasidium culture solution-1B). The water content of the obtained Aureobasidium culture solution-1B was 99.41% by mass, and the content of 1,3,1,6-β-D-glucan was 0.53% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 330,000. The absorbance at 660 nm was 0.433.

Production Example 3 (Production of aureobasidium culture solution: activated carbon treatment-dilution-centrifugal supernatant)
Aureobasidium culture solution-1 obtained in Production Example 1 was sterilized by heating at 80 ° C. for 30 minutes, 200 g of activated carbon (Ajinomoto Fine Techno Co., Ltd .: Hokuetsu ZN) was added, and the mixture was stirred at room temperature for 2 hours. Distilled water was added and mixed well, and then activated carbon and bacterial cells were removed by centrifugation to obtain about 6 L of a culture supernatant (Aureobasidium culture solution-1C). The water content of the obtained Aureobasidium culture solution-1C was 99.45% by mass, and the content of 1,3,1,6-β-D-glucan was 0.49% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 340,000. The absorbance at 660 nm was 0.436.

Production Example 4 (Production of Aureobasidium Culture Solution: High Pressure Homogenizer Treatment-Celite Filtration Separation-Preparation of Ion Exchange Resin Treatment Supernatant)
Aureobasidium culture solution-1 obtained in Production Example 1 was sterilized by heating at 80 ° C. for 30 minutes, treated with a high-pressure homogenizer (manufactured by SMT) for 10 minutes at 200 atmospheres, and Celite (Tokyo Kogyo Trading Company: Cell Pure) was used. The mixture was added and mixed, and filtered with a filter press (manufactured by Azumi Filter Paper Co., Ltd .: AF-58). 100 g of ion exchange resin (Ajinomoto Fine Techno Co., Ltd .: Hokuetsu KS) was added to 2 L of the obtained supernatant solution, stirred for 2 hours at room temperature, the resin was removed, and the culture supernatant solution (Aureobasidium culture solution-1D) ) The water content of the obtained aureobasidium culture solution-1D was 98.72% by mass, and the content of 1,3,1,6-β-D-glucan was 1.22% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 310,000. The absorbance at 660 nm was 0.088.

Production Example 5 (Production of aureobasidium culture solution: sonication-dilution-preparation of cellulose powder filtration separation)
Aureobasidium broth-1 was sterilized by heating at 80 ° C. for 30 minutes, 100 ml of the broth was treated with an ultrasonic crusher (manufactured by Tommy Seiko Co., Ltd.) for 20 minutes, an equal amount of water was added, and cellulose powder (Japan) 20 g of Paper Chemical Co., Ltd. (KC Flock / W / 50) was added and mixed, and 170 ml of a culture supernatant (Aureobasidium culture solution-1E) was obtained by suction filtration. The water content of the obtained Aureobasidium culture 1E was 99.51% by mass, and the content of 1,3,1,6-β-D-glucan was 0.43% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 300,000. The absorbance at 660 nm was 0.036.

Production Example 6 (Production of aureobasidium culture solution)
This was carried out in the same manner as in Production Example 1 except that IFO6353 strain was used instead of Aureobasidium pullulans ADK-34 strain (FERM BP-8391) to obtain 3 L (pH 4.5) of culture solution (Aureo) Vasidium culture solution-2).
Aureobasidium culture fluid-2 was sterilized by heating at 80 ° C. for 30 minutes, added with an equal amount of distilled water, mixed well, and then centrifuged at 8000 rpm for 30 minutes to obtain a culture supernatant (Aureobasidium). Culture solution-2A) About 6 L was obtained. The water content of the obtained Aureobasidium culture solution-2A was 99.44% by mass, and the content of 1,3,1,6-β-D-glucan was 0.05% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 1.1 million. The absorbance at 660 nm was 1.066.

Production Example 7 (Manufacture of aureobasidium culture solution: pressure homogenizer treatment-celite filtration separation-preparation of ion exchange resin / activated carbon treatment supernatant)
3 L of Aureobasidium broth-2 was sterilized by heating at 80 ° C. for 30 minutes, treated with a pressure homogenizer (manufactured by SMT) for 40 minutes at 800 atmospheres, and 100 g of Celite (manufactured by Tokyo Kogyo Trading Co., Ltd .: SSC) The mixture was added and mixed, and filtered with a filter press (manufactured by Azumi Filter Paper Co., Ltd .: AF-58). 300 g each of ion exchange resin (Ajinomoto Fine Techno Co., Ltd .: Hokuetsu KS) and activated carbon (Ajinomoto Fine Techno Co., Ltd .: Hokuetsu ZN) were added to the resulting supernatant and stirred at room temperature for 2 hours, after which the resin and activated carbon were removed. As a result, a culture supernatant (Aureobasidium broth-2B) was obtained. The water content of the obtained Aureobasidium culture solution-2B was 98.89% by mass, and the content of 1,3,1,6-β-D-glucan was 0.11% by mass. The weight average molecular weight of 1,3,1,6-β-D-glucan was about 1.05 million. The absorbance at 660 nm was 0.566.

Example 1 (Production of aureobasidium culture α: ethanol precipitation (eta precipitation) purified product)
An equal amount of ethanol was added to 200 ml of the culture supernatant (Aureobasidium broth-1A to 1E, -2A to 2B) obtained in Production Examples 1 to 7, and the resulting precipitate was separated, and 20 ml of ethanol. Was further washed and dried with a freeze dryer to obtain a white solid. This was pulverized by a mill and passed through a 16 mesh sieve to obtain powdered aureobasidium cultures-1Aα to 1Eα and −2Aα to 2Bα.

Example 2 (Production of aureobasidium culture β: preparation of concentrate)
While applying 200 ml of the culture supernatant (Aureobasidium broth-1A-1E, -2A-2B) obtained in Production Examples 1-7 at 50 ° C. using an evaporator, the water content was increased to 98% by mass. Water was distilled off and concentrated to obtain liquid aureobasidium cultures-1Aβ-1Eβ and -2Aβ-2Bβ.

Example 3 (Production of Aureobasidium culture γ: Preparation of concentrate)
While heating 200 ml of the culture supernatant (Aureobasidium broth-1A-1E, -2A-2B) obtained in Production Examples 1-7, water was distilled off to 97 mass% and concentrated. Liquid Aureobasidium cultures-1Aγ-1Eγ, -2Aγ-2Bγ were obtained.

Example 4 (Production of Aureobasidium culture δ: Preparation of spray-dried product)
To 1000 ml of the culture supernatant obtained in Production Examples 1 to 7 (Aureobasidium broth-1A to 1E, -2A to 2B), an equal amount of distilled water is added and spray-dried (manufactured by Tokyo Rika). As a result, white powdered Aureobasidium cultures -1Aδ to 1Eδ and -2Aδ to 2Bδ were obtained.

Example 5 (Production of Aureobasidium culture ε: Preparation of freeze-dried product)
1000 ml of the culture supernatant (Aureobasidium broth-1A-1E, -2A-2B) obtained in Production Examples 1-7 was concentrated to 400 ml, freeze-dried, and white solid Aureova Cidium cultures-1Aε-1Eε, -2Aε-2Bε were obtained.

Example 6 (Aureobasidium culture ζ: Cellobiose as additive)
20 g of cellobiose (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 100 ml of Aureobasidium culture solution-1D, mixed and dissolved, and then lyophilized. The obtained powder was pulverized in a mortar and passed through a 16 mesh sieve to obtain 18 g of white powder Aureobasidium culture-1Dζ.

Example 7 (Production of Aureobasidium culture η: Preparation of concentrate)
200 ml of the culture supernatant obtained in Production Examples 1 to 7 (Aureobasidium cultures-1A to 1E, -2A to 2B) was heated to 50 ° C. using an evaporator until the water content reached 55% by mass. Water was distilled off and concentrated to obtain liquid aureobasidium cultures -1Aη to 1Eη and -2Aη to 2Bη.

Test evaluation-1 (quality analysis)
Moisture content of aureobasidium cultures obtained in Examples 1 to 7, mass average molecular weight of β-glucan, ratio of components having a mass average molecular weight of 200 or less, measurement results of whiteness / yellowness of powdered samples, turbidity The measurement results of 660 nm and the measurement results of the content of 1,3,1,6-β-D-glucan are summarized in Table 1.

Test evaluation-2 (daily test)
The color, odor, and water solubility of each sample were examined over time. In the test, the sample was placed in a sealed container, stored at room temperature (25 ° C.), and the change in each state was examined according to the following evaluation criteria after 1 day, 3 days, 7 days, 10 days, and 30 days. As comparative examples, Aureobasidium broth-1A and Aureobasidium broth-2A were evaluated.

<Evaluation of coloring>
Both the liquid and the powder were visually evaluated for coloring. The results are shown in Table 2.
Evaluation criteria ○: No change Δ: Slightly colored ×: Colored (black, brown or yellow colored)

<Evaluation of odor>
The presence or absence of odor was confirmed. The results are shown in Table 2.
Evaluation criteria ○: No change △: Slight odor.
X: Odor occurred.

<Solubility in water>
The change in the state of the 1% by mass aqueous solution of the aureobasidium culture obtained in Examples 1 to 7 was visually confirmed and evaluated. The change in the state as it was was confirmed visually. As a comparative example, changes in the state of Aureobasidium culture solution-1A and Aureobasidium culture solution-2A were confirmed by visual observation and evaluated. The results are shown in Table 2.
Evaluation criteria ○: No change Δ: Turbidity or small amount of precipitation occurred.
X: Precipitation is recognized.

Claims (5)

After removing the components from the Aureobasidium culture solution so that the content of the cells and the components having a weight average molecular weight of 50 to 200 is less than 30% by mass, the Aureobasidium culture solution is concentrated, A powdered aureobasidium culture obtained by performing a homogenization process before, during or after the concentration, and having a whiteness (W value) of 70 or more obtained by measurement with a color difference meter, and / or Or the powdered aureobasidium culture whose yellowness (YI value) is 30 or less . Aureobasidium 660nm absorbance of water dilutions of the culture (solid content concentration of 1 mass%) of (length 10mm liquid layer), 1.0 or less claim 1 Symbol placement Aureobasidium culture. 1,3,1,6- content of beta-D-glucan having a glucose binding (1,3,1,6-β-D- glucan) is, according to claim 1 or 2 is at least 0.1 wt% Aureobasidium culture as described. Aureobasidium culture, Aureobasidium culture according to any one of claims 1 to 3, which was obtained through the step of treating the activated carbon and / or ion exchange resin. Aureobasidium culture, celite and / or Aureobasidium culture according to any one of claims 1 to 4 by the cellulose powder is obtained through the step of filtering process.