JP6129503B2 - Method for producing royal jelly having α-glucosidase inhibitory action - Google Patents

Description

  The present invention relates to a method for producing a royal jelly having an α-glucosidase inhibitory action, a royal jelly composition containing the royal jelly obtained by the method, and a hypoglycemic agent.

  Royal jelly is a milky white paste-like substance secreted by the pharyngeal glands of young worker bees, and its components are abundant in amino acids including essential amino acids and are composed of high-quality proteins. 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.

  It has been reported that an α-glucosidase inhibitor inhibits α-glucosidase localized in the microvilli of the small intestine and suppresses a rapid increase in blood glucose level after meal and a subsequent increase in insulin value (Non-patent Document 1). In order to suppress the metabolism of carbohydrates (particularly starch-derived oligosaccharides, sucrose, etc.) in humans and non-human animals, for example, it exhibits an effect of suppressing blood sugar elevation, and hyperglycemic symptoms and obesity derived from hyperglycemia It is useful for improving various diseases such as diabetes. In addition, foods produced by adding an α-glucosidase inhibitor are suitable for dietary abnormal patient foods, and are also suitable for healthy people as metabolic metabolic preventive foods.

  As an α-glucosidase inhibitor derived from food, for example, an enzyme hydrolyzate of animal protein or vegetable protein (Patent Document 1) and tea polyphenol (Patent Document 2) are disclosed.

JP 09-065836 A Japanese Patent Laid-Open No. 05-017364

Tattersall R.D. , “Alpha-glucosidase inhibition as an adjustment to the treatment of type 1 diabetics.”, Diabetic Medicine, 1993, Vol. 10, No. 8, p. 688-983 Miwa I. "Inhibition of intestinal alpha-glucosides and sugar absorption by flavones", Chemical & Pharmaceutical Bulletin, 1986, Vol. 34, No. 2, p. 838-844

  An object of the present invention is to provide a new technique capable of obtaining a royal jelly having an α-glucosidase 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 an α-glucosidase inhibitory action, 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,
2. 2. a royal jelly composition containing the royal jelly obtained by the production method; A hypoglycemic agent containing royal jelly obtained by the production method,
Consists of.

  According to the present invention, a royal jelly having an α-glucosidase inhibitory action can be produced by a safer and simpler method, and a royal jelly composition and a hypoglycemic agent are provided using this royal jelly having an α-glucosidase inhibitory action. be able to.

  In the present invention, royal jelly having an α-glucosidase inhibitory action is produced by allowing a protease derived from Bacillus subtilis and pancreatin to act on 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 processing conditions of royal jelly with a proteolytic enzyme derived from Bacillus subtilis, for example, conditions such as processing time, pH, and temperature are not particularly limited as long as the target α-glucosidase inhibitor is produced. It is possible to set appropriately depending on the stability and reactivity of the protease and the protease derived from Bacillus subtilis.

  The time for allowing the Bacillus subtilis-derived proteolytic enzyme to act on the royal jelly is not particularly limited as long as the target -glucosidase inhibitor is produced, but it is preferably several hours to 1 day, preferably 3 hours to 10 hours. . Even a long reaction had little effect on the production of α-glucosidase inhibitors.

  The pH at the time of allowing the protease derived from Bacillus subtilis to act on the royal jelly is not particularly limited as long as the target α-glucosidase inhibitor is produced, but pH 3-11, preferably pH 5-11 is suitable. is there.

  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 α-glucosidase inhibitor is produced, but is 30 ° C. to 80 ° C., preferably 40 ° C. to 60 ° C. ° C is suitable.

  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 α-glucosidase inhibitor is produced, and the stability of royal jelly and the origin of Bacillus subtilis It is possible to set appropriately in consideration of the stability and reactivity of the proteolytic enzyme.

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

  The pH at which pancreatin is allowed to act on royal jelly is not particularly limited as long as the target α-glucosidase inhibitor is produced, but pH 3 to 11, preferably pH 5 to 11, is appropriate.

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

  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 an α-glucosidase inhibitory action according to the present invention is characterized by comprising a step of allowing a protease deriving from Bacillus subtilis to act on royal jelly and a step of causing pancreatin to act. The method includes a step of allowing a protease derived from Bacillus subtilis to act, and a step of causing pancreatin to act after the step.

  The production of a royal jelly having an α-glucosidase 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.

  The royal jelly produced by the action of a proteolytic enzyme derived from Bacillus subtilis and pancreatin is a royal jelly having an α-glucosidase inhibitory action.

  You may refine | purify an alpha-glucosidase inhibitory substance by processing the royal jelly which has the alpha-glucosidase inhibitory action which concerns on this invention using various 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.

  By adding various components to the royal jelly having an α-glucosidase inhibitory action according to the present invention or a purified product thereof, it can be provided as a royal jelly composition. 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 the scope of the present invention is not impaired 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 the alpha-glucosidase inhibitory action which concerns 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 hypoglycemic 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, polyvinyl pyrrolidone, 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.

  A “hypoglycemic agent” refers to a drug that improves a hyperglycemic state and lowers blood sugar. The “hypoglycemic agent” preferably means an agent that suppresses a rapid increase in blood glucose level after a meal, that is, a drug having an inhibitory effect on increase in blood glucose, and can also be referred to as an agent for suppressing increase in blood glucose.

  The royal jelly having an α-glucosidase inhibitory action according to the present invention suppresses metabolism of carbohydrates (particularly starch-derived oligosaccharides, sucrose, etc.) in humans and non-human animals, and as a result, suppresses blood sugar elevation. Indicates. Therefore, the royal jelly composition and the hypoglycemic agent containing the royal jelly having an α-glucosidase inhibitory action according to the present invention are useful for the improvement and prevention of hyperglycemia symptoms and various diseases such as obesity and diabetes derived from hyperglycemia. is there. In addition, the food produced by adding the royal jelly having an α-glucosidase inhibitory action according to the present invention is suitable as a hypoglycemic food for a patient with metabolic abnormality and also suitable for a healthy person as a metabolic abnormality preventing food. ing.

  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 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 raw royal jelly solution obtained in Example 1 were examined for α-glucosidase inhibitory activity according to the method described in a previous report (Non-Patent Document 2). As a result, as shown in Table 1, it was revealed that the enzyme-treated royal jelly solution had higher α-glucosidase inhibitory activity because the 50% inhibitory concentration (IC50) was lower than that of the fresh royal jelly solution.

(Table 1)
───────────────────────────────
α-Glucosidase inhibitory activity
IC50 (mg / mL)
───────────────────────────────
Enzyme-treated royal jelly solution 94
Raw royal jelly solution 125
───────────────────────────────

  As described above, it was found that a royal jelly having a high α-glucosidase 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 an α-glucosidase inhibitory activity produced by the present invention can be suitably used for food and drink as a royal jelly composition or a blood glucose level lowering agent, and can be particularly suitably used for health foods.

Claims (1)

An α-glucosidase-inhibiting food composition comprising, as an active ingredient, an enzyme-treated royal jelly obtained by a process of causing a protease that is derived from Bacillus subtilis and pancreatin to act.