JP2014124163A - Method of producing royal jelly having tyrosinase inhibitory action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new technique which can obtain a royal jelly having tyrosinase inhibitory action.SOLUTION: There is provided a method of producing a royal jelly having tyrosinase inhibitory action which includes the steps of causing a protease derived from Bacillus subtilis to act on the royal jelly and pancreatin to act thereon. There is also provided a royal jelly composition containing the royal jelly obtained by the production method.

Description

  The present invention relates to a method for producing a royal jelly having a tyrosinase inhibitory action, and a royal jelly composition containing the royal jelly obtained by the method.

  Royal jelly is a milky-white paste-like substance secreted by the pharyngeal gland of young worker bees. In addition, it contains trace components such as vitamins, minerals and carbohydrates. Examples of vitamins include vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, vitamin A, vitamin C, and vitamin E that are effective in promoting growth and preventing aging. Minerals include potassium, magnesium, calcium, copper, iron, phosphorus and the like. Examples of carbohydrates include glucose and fructose. Furthermore, acetylcholine-like substances, organic acids (such as 10-hydroxydecenoic acid), fats, and protein-like antibacterial substances such as leuallysin are included.

  As described above, royal jelly contains various nutritional components and has many known effects such as antibacterial action, immune enhancement action, anti-inflammatory action, anti-aging action, prevention / treatment action for menopause, and anticancer action. ing.

  Tyrosinase is an oxidoreductase involved in melanin synthesis and catalyzes a reaction that introduces a hydroxyl group into tyrosine to produce dopa.

  Melanin is a pigment that exists in the skin, and is produced by stimulation of ultraviolet rays and the like in melanocytes, causing stains and freckles. Specifically, free radicals (active oxygen) are generated by ultraviolet rays, and melanocytes (pigment cells) in the epidermis are activated by the stimulation. In the activated melanocytes, tyrosinase is activated, and as a result, tyrosine in the melanocytes is converted into dopa by tyrosinase, and melanin is generated by further reaction. Therefore, a substance having a tyrosinase inhibitory action is expected to have a melanin production inhibitory effect and thus a whitening effect.

  As a tyrosinase inhibitor derived from food, for example, kojic acid is disclosed (Patent Document 1).

Japanese Patent Publication No.61-10447

  An object of the present invention is to provide a new technique capable of obtaining a royal jelly particularly having a tyrosinase inhibitory action.

  As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by causing a protease derived from Bacillus subtilis and pancreatin to act on royal jelly, thereby completing the present invention. .

That is, the present invention
1. A method for producing a royal jelly having a tyrosinase inhibitory action, characterized by comprising a step of allowing a proteolytic enzyme derived from Bacillus subtilis to act on royal jelly and a step of causing pancreatin to act, and
2. A royal jelly composition containing the royal jelly obtained by the production method,
Consists of.

  According to the present invention, a royal jelly having a tyrosinase inhibitory action can be produced by a safe and simple method, and a royal jelly composition can be provided using this royal jelly having a tyrosinase inhibitory action.

  In the present invention, royal jelly having a tyrosinase inhibitory action is produced by allowing a proteolytic enzyme derived from Bacillus subtilis and pancreatin to act on the royal jelly.

  Examples of the country of origin of royal jelly include Japan, China, Taiwan, Thailand, Brazil, European countries, Oceania countries, the United States, etc. Royal jelly of any country of origin may be used. Further, a plurality of royal jelly of the country of origin may be appropriately mixed and used. The royal jelly is preferably a liquid, and when lyophilized royal jelly is used, it can be used after being dissolved in purified water, tap water, or an appropriate buffer. The frozen royal jelly can be used after being thawed.

  Further, the royal jelly may be one to which processing such as heating, centrifugation, alcohol extraction, filtration, freeze drying or hot air drying, and various nutrients are added.

  The proteolytic enzyme derived from Bacillus subtilis that acts on royal jelly is obtained by culturing Bacillus subtilis, and may be a culture solution or a purified product. The proteolytic enzyme derived from Bacillus subtilis may be obtained by gene recombination, and may be further modified with, for example, sugar or polyethylene glycol.

  The solid content concentration of the royal jelly after mixing with a proteolytic enzyme derived from Bacillus subtilis can be 0.1 to 30 W / W%, preferably 3 to 10 W / W%. Here, for example, 10 W / W% means that 10 g of a dried product is obtained when 100 g of royal jelly is dried by freeze drying or the like.

  The treatment conditions of the royal jelly with a proteolytic enzyme derived from Bacillus subtilis, such as the treatment time, pH, and temperature, are not particularly limited as long as the target tyrosinase inhibitor is produced, and the stability of the royal jelly and It can be appropriately set in consideration of the stability and reactivity of the proteolytic enzyme derived from Bacillus subtilis.

  The time for allowing the protease derived from Bacillus subtilis to act on the royal jelly is not particularly limited as long as the target tyrosinase inhibitor is produced, but it is preferably several hours to 1 day, preferably 3 hours to 10 hours. Even long-term reactions had little effect on the production of tyrosinase inhibitors.

  The pH at which Bacillus subtilis-derived proteolytic enzyme is allowed to act on royal jelly is not particularly limited as long as the target tyrosinase inhibitor is produced, but pH 3 to 11, preferably pH 5 to 11 is suitable.

  The temperature at which the Bacillus subtilis-derived proteolytic enzyme is allowed to act on the royal jelly is not particularly limited as long as the target tyrosinase inhibitor is produced, but is 30 ° C to 80 ° C, preferably 40 ° C to 60 ° C. Is appropriate.

  While the proteolytic enzyme derived from Bacillus subtilis is reacted with the royal jelly, it may be left still, and may be shaken or stirred.

  The pancreatin that acts on royal jelly is preferably produced from digestive fluids of animals, particularly pigs. Pancreatin is a mixture of various enzymes contained in pancreatic juice, and mainly produced from porcine pancreas is used as a digestive enzyme agent. In the present invention, commercially available pancreatin can also be used as a digestive enzyme agent.

  The solid content concentration of the royal jelly after mixing with pancreatin can be 0.1 to 30 W / W%, preferably 3 to 10 W / W%. Here, for example, 10 W / W% means that 10 g of a dried product is obtained when 100 g of royal jelly is dried by freeze drying or the like.

  The treatment conditions of royal jelly with pancreatin, for example, conditions such as treatment time, pH, and temperature are not particularly limited as long as the target tyrosinase inhibitor is produced. Stability of royal jelly and protein derived from Bacillus subtilis It can be set as appropriate in consideration of the stability and reactivity of the decomposing enzyme.

  The time for which pancreatin is allowed to act on royal jelly is not particularly limited as long as the target tyrosinase inhibitor is produced, but it is several hours to 1 day, preferably 3 hours to 20 hours, more preferably 6 hours to 18 hours. Time is preferred. Even long-term reactions had little effect on the production of tyrosinase inhibitors.

  The pH at which pancreatin is allowed to act on royal jelly is not particularly limited as long as the target tyrosinase-inhibiting substance is produced, but pH 3-11, preferably pH 5-11 is suitable.

  The temperature at which pancreatin is allowed to act on royal jelly is not particularly limited as long as the target tyrosinase inhibitor is produced, but it is suitably 30 ° C to 80 ° C, preferably 40 ° C to 60 ° C.

  While pancreatin is allowed to react with the royal jelly, the pan creatine may be left still, and may be shaken or stirred.

  The treatment of royal jelly with an enzyme may be carried out by allowing pancreatin to act on royal jelly after allowing a protease from Bacillus subtilis to act on the royal jelly, or proteolysis from Bacillus subtilis after causing pancreatin to act on the royal jelly. You may implement by making an enzyme act. Preferably, it is carried out by allowing pancreatin to act after allowing a proteolytic enzyme derived from Bacillus subtilis to act on royal jelly. That is, the method for producing a royal jelly having a tyrosinase inhibitory action according to the present invention comprises a step of causing a royal jelly to act on a proteolytic enzyme derived from Bacillus subtilis and a step of causing pancreatin to act. Preferably, the royal jelly is made to have a Bacillus subtilis. The method includes a step of causing a proteolytic enzyme derived from the agent to act and a step of causing pancreatin to act after the step.

  The production of royal jelly having tyrosinase inhibitory action according to the present invention generally includes a step of inactivating these enzymes after treatment of the royal jelly with a proteolytic enzyme derived from Bacillus subtilis and pancreatin. The method for inactivating the proteolytic enzyme and pancreatin derived from Bacillus subtilis may be to inactivate the proteolytic enzyme and pancreatin derived from Bacillus subtilis to the extent that there is no problem as a food. Examples of the deactivation method include a method of deactivation by heating, a method of deactivation using a drug, and a method of removing a proteolytic enzyme derived from Bacillus subtilis and pancreatin by filtration, and these methods can be used alone. It is also possible to combine two or more types. Preferably, the proteolytic enzyme derived from Bacillus subtilis and pancreatin are inactivated by heating. It is appropriate to heat at a heating temperature of 60 ° C. or higher, preferably 80 ° C. or higher. Furthermore, when a food sterilization step is required, it may be combined with a step of inactivating the Bacillus subtilis-derived proteolytic enzyme and pancreatin.

  Further, when a Bacillus subtilis-derived proteolytic enzyme and pancreatin are allowed to act on royal jelly, a Bacillus subtilis-derived proteolytic enzyme, a pancreatin stabilizer or a reaction accelerator may be added.

  A royal jelly produced by the action of a proteolytic enzyme derived from Bacillus subtilis and pancreatin is a royal jelly having a tyrosinase inhibitory action.

  The tyrosinase-inhibiting substance may be purified by treating the royal jelly having a tyrosinase-inhibiting action according to the present invention with various types of chromatography. Examples of the purification method include gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, normal phase chromatography, ultrafiltration, electrophoresis and the like. These can be used for purification alone or in combination.

  Gel filtration chromatography includes a carrier for gel filtration chromatography that can separate proteins having various molecular weights, and a carrier for gel filtration chromatography that can separate proteins having a molecular weight of about 10,000 or less is preferable. Examples of ion exchange groups used in ion exchange chromatography include anion exchangers and cation exchangers. Examples of the anion exchanger include a diethylaminoethyl group (DEAE group) and a quaternary aminoethyl group (QAE group). Examples of the cation exchanger include a carboxymethyl group (CM group) and a sulfopropyl group (SP group). Examples of the carrier used in the hydrophobic chromatography include a carrier to which a butyl group (Butyl group), an ethyl group (Ethyl group), and a phenyl group (Phenyl group) are bonded. Examples of the carrier used for the reverse phase chromatography include octadecyl group (C18), and a carrier in which C30, C8, C4, etc. having different alkyl group length from octadecyl group are bonded. Examples of the carrier used for normal phase chromatography include silica gel, as well as a carrier bonded with a cyanopropyl group, a functional group having a diol structure, an aminopropyl group, a polyamine, and the like.

  A royal jelly composition can be provided by adding various components to the royal jelly having an inhibitory action on tyrosinase or a purified product thereof. Examples of the various components include foods, sugars, lipids, emulsifiers, thickeners, seasonings, fragrances, sourness adjusters, preservatives, fruit juices, fragrances, various nutritional components, and the like, and do not impair the effects of the present invention. Can be used in Moreover, various components may be used independently and may be used in mixture of 2 or more types. Examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose and the like. Examples of emulsifiers include sucrose fatty acid esters, glycerin fatty acid esters, and lecithin. Examples of the thickener include carrageenan, gum arabic, xanthan gum, guar gum, pectin, locust bean gum thickener starch, gellan gum and the like. Examples of the sourness adjuster include citric acid, lactic acid, malic acid, fumaric acid, gluconic acid, tartaric acid and the like. Examples of the preservative include benzoic acid and its salt, sorbic acid and its salt, paraben, sodium sulfite, pectin degradation product, glycine and the like. Examples of the fruit juice include tomato juice, plum juice, apple juice, lemon juice, orange juice, and berry juice. Examples of the fragrances include spices such as herbs and spices, fruit fragrances, and fragrances such as vanilla. In addition, other preferable nutrients include vitamins such as vitamin D and minerals such as calcium, magnesium, iron, manganese, and zinc.

  The royal jelly composition according to the present invention can also be used as food. Specific examples of such foods include, for example, beverages, confectionery, candy, gum, bread, livestock meat products, dairy products, retort foods, instant foods, frozen foods, jelly-like foods, beekeeping products, pickles, seasonings And so on. These foods are also useful as so-called health foods, functional foods, foods for specified health use, functional nutritional foods, dietary supplements, supplements and the like. Moreover, granule, powder, tablet, capsule, chewable, drink, jelly, paste, grain etc. can be mentioned as the shape as those foods.

  Moreover, the royal jelly which has a tyrosinase inhibitory action based on this invention, Preferably the refined product can be manufactured as a pharmaceutical composition containing the support | carrier (pharmaceutical carrier) accept | permitted for a pharmaceutical as needed. Such a pharmaceutical composition can be used as a whitening agent. Examples of the pharmaceutical carrier include fillers, fillers, binders, moistening agents, disintegrating agents, lubricants, diluents and excipients that are usually used depending on the form of use of the preparation. These are appropriately selected and used depending on the administration form of the resulting preparation. More specifically, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, Examples include gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. These are used singly or in combination of two or more according to the dosage form of the intended drug. In addition, stabilizers, bactericides, buffers, isotonic agents, chelating agents, surfactants, pH adjusters, and the like can be used as appropriate. Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. The L-amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like. The saccharide is not particularly limited, for example, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, dextran, hydroxypropyl starch, chondroitin sulfate, Any of polysaccharides such as hyaluronic acid and their derivatives may be used. The cellulose derivative is not particularly limited, and may be any of methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose sodium and the like. There is no particular limitation on the surfactant, and either an ionic surfactant or a nonionic surfactant can be used. Examples of the surfactant include polyoxyethylene glycol sorbitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, and fatty acid glyceride. Buffers include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or their corresponding salts (for example, alkali metal salts such as sodium salts, potassium salts, calcium salts, magnesium salts thereof, An alkaline earth metal salt). Examples of the isotonic agent include sodium chloride, potassium chloride, saccharides and glycerin. Examples of the chelating agent include sodium edetate and citric acid.

  The royal jelly having a tyrosinase inhibitory action according to the present invention exhibits a melanin production inhibitory action and thus a whitening action in humans and non-human animals. Therefore, the royal jelly composition containing the royal jelly having the tyrosinase inhibitory action according to the present invention is useful as a cosmetic or food that can be expected to have a whitening effect.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

  100 g of raw royal jelly was suspended in water, the pH was adjusted to 7.0 using 2N sodium hydroxide solution, and 1 L was prepared to prepare a raw royal jelly solution. 1 g of proteolytic enzyme derived from Bacillus subtilis was added to this solution, and the enzyme treatment was performed at 45 ° C. for 6 hours. Next, the pH of this treatment solution was adjusted to 8 using a 20% sodium hydroxide solution, 1 g of pancreatin was added, and the enzyme treatment was performed at 45 ° C. for 6 hours. The solution after completion of the enzyme treatment is adjusted to pH 5.5 using 20% sodium hydroxide solution or 10% citric acid solution, heated at 80 ° C. for 10 minutes to deactivate the enzyme, and further filtered. The insoluble residue was removed and an enzyme-treated royal jelly solution was obtained.

  The enzyme-treated royal jelly solution and fresh royal jelly solution obtained in Example 1 were examined for tyrosinase inhibitory activity. Specifically, 0.1 mL of the sample solution and 1.7 mL of 5 mM L-dopa solution are mixed and incubated at 37 ° C. for 5 minutes, 0.2 mL of tyrosinase solution is added, and then incubated at 37 ° C. for 5 minutes. Absorbance at 475 nm was measured to measure tyrosinase inhibitory activity. As a result, when the raw royal jelly solution was 2.5 mg / mL, the inhibition rate of tyrosinase activity was 9%, whereas when the enzyme-treated royal jelly solution was 2.5 mg / mL, the inhibition rate of tyrosinase activity was 28%. The enzyme-treated royal jelly solution was found to have higher tyrosinase inhibitory activity than the fresh royal jelly.

  As described above, it was found that a royal jelly having a high tyrosinase inhibitory activity can be produced by allowing a proteolytic enzyme derived from Bacillus subtilis and pancreatin to act on the royal jelly.

  The royal jelly having a tyrosinase inhibitory action produced according to the present invention can be suitably used as a royal jelly composition for foods and drinks, and particularly suitable for health foods and beauty supplements.

Claims (2)

A method for producing a royal jelly having a tyrosinase inhibitory activity, comprising a step of allowing a protease derived from Bacillus subtilis to act on royal jelly, and a step of causing pancreatin to act; The royal jelly composition containing the royal jelly obtained by the said manufacturing method.