JP2010030975A - Bee larva solubilized product, preparation method thereof, and pharmaceutical product, cosmetic, or food and drink containing the bee larva solubilized product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bee larva solubilized product that has increased water solubility, and a pharmaceutical product, a cosmetic, or food and drink containing the bee larva solubilized product. <P>SOLUTION: The bee larva solubilized product is obtained by subjecting bee larvae to a combination of protease treatment and an acidification treatment. Examples of a protease used for the protease treatment are a neutral protease derived from Bacillus subtilis, a neutral protease derived from Aspergillus oryzae, and an alkaline protease derived from Bacillus licheniformis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is a solubilized bee treatment product obtained by subjecting a bee, or an insoluble component obtained by subjecting a bee to extraction with an extraction solvent whose main component is water, a protease treatment and an acidification treatment, The present invention relates to a method for producing the same, as well as a pharmaceutical, a cosmetic or a food or drink containing the solubilized bee treatment product.

Bees are larvae and moths of bees such as bees, wasps, wasps, wasps, etc., and are used as food in some areas. In addition, it is known that bees have abundant nutrients such as proteins, fats and carbohydrates, and have various physiological effects such as blood flow improving effects and nourishing tonic effects. It is also treated as a food material. For example, Patent Document 1 discloses a bee powder obtained by processing a queen bee larva into a powder form. The honeybee powder of Patent Document 1 is prepared by heat-treating a queen bee larvae and drying it until the water content becomes 10% or less, and then pulverizing the obtained dried larvae.
JP 2001-204421 A

  However, the bee powder of Patent Document 1 has a problem that it is not easily dissolved when dissolved in water as it is. For this reason, for example, when applied as a beverage, the bee powder cannot be blended at a high concentration, and application to an aqueous solution, such as a beverage, has been extremely difficult. The present invention has been made in view of such conventional circumstances, and the object thereof is to contain a treated product of a solubilized bee that has improved solubility in water, a method for producing the same, and a treated product of the solubilized bee. It is to provide pharmaceuticals, cosmetics or food and drinks.

  In order to achieve the above-mentioned object, the solubilized bee treatment product according to claim 1 is an insoluble component obtained by extracting a bee child or a bee child with an extraction solvent whose main component is water. Is obtained by combining protease treatment and acidification treatment.

  The solubilized bee treatment product according to claim 2 is a bee child or an insoluble component obtained by extracting the bee child with an extraction solvent whose main component is water, protease treatment, cellulase treatment and acidity. It is characterized by being obtained by combining the processing.

  The treated product of the solubilized bee according to claim 3 is the invention according to claim 1 or claim 2, wherein the protease treatment is neutral protease derived from Bacillus subtilis, neutral derived from Aspergillus oryzae. The treatment is performed using at least one selected from protease and alkaline protease derived from Bacillus licheniformis.

The solubilized bee treatment product according to claim 4 is the invention according to any one of claims 1 to 3, wherein the solubilized bee treatment product has reduced allergenicity. Features.
The method for producing a solubilized bee-treated product according to claim 5 is characterized in that a bee or an insoluble component obtained by extracting bees with an extraction solvent whose main component is water is treated with protease and acidic. It is characterized by processing in combination.

The method for producing a solubilized bee treatment product according to claim 6, wherein the bee child or an insoluble component obtained by extracting the bee child with an extraction solvent whose main component is water is treated with protease, cellulase The treatment is characterized by a combination of treatment and acidification treatment.

  The method for producing the solubilized bee treatment product according to claim 7 is the invention according to claim 5 or claim 6, wherein the protease treatment is derived from a neutral protease derived from Bacillus subtilis, Aspergillus oryzae. It is characterized by being treated with at least one selected from neutral proteases of Bacteria and alkaline proteases derived from Bacillus licheniformis.

The pharmaceutical product, cosmetic product, or food or beverage product according to claim 8 contains the processed bee candy according to any one of claims 1 to 4.
The food and drink according to claim 9 is the allergy-reducing food and drink according to the invention according to claim 8.

  ADVANTAGE OF THE INVENTION According to this invention, the solubilized bee treatment product which improved the solubility with respect to water, its manufacturing method, and the pharmaceutical, cosmetics, or food / beverage products containing the solubilized bee treatment product can be provided.

Hereinafter, the embodiment which materialized the solubilized bee treatment thing of the present invention is described. In the following, the solubilized bee treatment product is simply referred to as a bee treatment product.
Examples of the bee pup used in the present embodiment include at least one selected from bee larvae and bee wings. These bee larvae and moths may be either male or female, and may be any larvae and moths of bees such as bees, wasps, wasps and wasps. Furthermore, the place of production of bees used in the present embodiment may be any of Asian countries such as China, Taiwan and Japan, European countries, North American countries, and South American countries.

  The processed bee child of this embodiment is a bee child (including a bee powder obtained by processing a bee child into a powder form, including a raw bee child), or an extract whose main component is water. An insoluble component obtained by extraction with a solvent (that is, an insoluble fraction (extraction residue) in the extraction solvent whose main component is water) is used as a raw material. As a method for pulverizing a bee puppet, any conventionally used method may be adopted, and for example, a method disclosed in Patent Document 1 may be mentioned. Moreover, when using a raw bee child as a raw material, it is preferable to pre-process in paste form, such as by grinding. Moreover, when obtaining the insoluble fraction with respect to the extraction solvent whose main component of a bee is water, the temperature of a solvent is not specifically limited. In addition, what is necessary is just to perform extraction operation for about several hours, stirring at predetermined temperature. And after extracting a solvent melt | dissolution component, the insoluble fraction with respect to the solvent whose main component is water can be obtained from a bee by applying well-known solid-liquid separation methods, such as filtration and centrifugation.

  Here, the extraction solvent whose main component is water means a solvent in which the component having the highest existence ratio (volume%) in the solvent is water. And it is preferable that content of the water in a solvent is 50 volume% or more. Further, as a component other than water, for example, a small amount of a hydrophilic organic solvent such as ethanol may be contained.

The manufacturing process of the processed bee candy according to the present embodiment is as follows. First, an insoluble component (hereinafter referred to as a honey powder), a raw bee cub, or a bee kid extracted by an extraction solvent whose main component is water. These are generally referred to as bee pups), and a step of subjecting to protease treatment to obtain a first intermediate treatment product is performed. Next, the first intermediate treatment product is preferably subjected to a cellulase treatment to obtain a second intermediate treatment product. Finally, a processed bee-like product is obtained by acidifying the first intermediate processed product or the second intermediate processed product. The protease treatment is a treatment that hydrolyzes the peptide bond of the protein contained in the raw material of the bee using a protease to reduce the molecular weight. Proteases include exo-type proteases that hydrolyze from the end of the peptide and endo-type proteases that degrade from the middle of the peptide. Any protease can be used. In addition, the protease includes an acidic protease having an optimum pH in the vicinity of acidity (for example, below pH 6.0), in the vicinity of neutrality (for example, pH 5.0 to 9.0), and in the vicinity of alkalinity (for example, pH 8.0 or more). Neutral protease and alkaline protease are present, and any protease can be used. Furthermore, there are proteases derived from various sources such as microorganisms, animals, plants, etc., but proteases derived from any of them can also be used. Among these proteases, microorganism-derived proteases are preferably used from the viewpoint of improving solubility in water.

As an acidic protease, specifically, Aspergillus niger (Aspergillus niger)
) Derived acidic protease. As a commercial product, Sumiteam AP (manufactured by Shin Nippon Chemical Industry Co., Ltd.) can be used. Protease treatment using Sumiteam AP is preferably performed under conditions of 35 to 50 ° C, more preferably 40 to 50 ° C, and further preferably 45 to 50 ° C.

As a neutral protease, specifically, Bacillus subtilis (Bacillus subtilis)
) Derived neutral protease, Aspergillus oryzae neutral protease, Bacillus stearothermophilus
And neutral protease derived from papaya and protease derived from papaya. As a neutral protease derived from Bacillus subtilis, protease N “Amano” G (manufactured by Amano Enzyme) can be used as a commercial product. Protease N “Amano” G acts effectively in the range of pH 5.0 to 7.0, and the optimum pH is 7.0. Protease treatment using protease N “Amano” G is preferably performed at 40 to 60 ° C., more preferably 45 to 55 ° C., and even more preferably 50 to 55 ° C.

  Moreover, as a neutral protease derived from Aspergillus oryzae, as a commercial product, Umamizyme G (manufactured by Amano Enzyme) and Sumiteam FP (manufactured by Shin Nippon Chemical Industry Co., Ltd.) can be used. Umamizyme G acts effectively in the vicinity of neutrality, and its optimum pH is pH 8.0. The protease treatment using equinezyme G is preferably performed under conditions of 35 to 55 ° C, more preferably 40 to 55 ° C, and further preferably 45 to 55 ° C. Protease treatment using Sumiteam FP is preferably performed under conditions of 30 to 50 ° C, more preferably 35 to 50 ° C, and even more preferably 40 to 50 ° C.

  As a protease derived from Bacillus stearothermophilus, protease S “Amano” G (manufactured by Amano Enzyme) can be used as a commercially available product. Protease S “Amano” G acts effectively in the neutral to weakly alkaline region, and its optimum pH is pH 8.0. Protease treatment using protease S “Amano” G is preferably performed at 50 to 75 ° C., more preferably 60 to 75 ° C., and even more preferably 65 to 70 ° C. In addition, as a protease derived from papaya, papain W-40 (manufactured by Amano Enzyme) can be used as a commercial product. The protease treatment using papain W-40 is preferably performed under the conditions of 40 to 70 ° C, more preferably 45 to 60 ° C, and further preferably 45 to 55 ° C.

As an alkaline protease, specifically, Bacillus licheniformis (Bacillus
and alkaline protease derived from licheniformis). Protin SD-AC-10F (manufactured by Daiwa Kasei Co., Ltd.) can be used as a commercially available product. Protin SD-AC-10F acts effectively near alkalinity, and its optimum pH is pH 10.0 to 11.0. Protease treatment using protin SD-AC-10F is preferably performed at 40 to 60 ° C, more preferably 50 to 60 ° C, and further preferably 55 to 60 ° C.

Among these proteases, protease N “Amano” G, equinezyme G, and protin SD-AC-10F are preferably used from the viewpoint of improving solubility in water.
Protease treatment using various proteases is carried out by incubating a reaction solution containing a bee raw material, protease, and water (or buffer solution) under reaction conditions corresponding to the various proteases. The treatment time for the protease treatment is appropriately set depending on the reaction temperature, enzyme titer, etc., but is preferably 2 to 5 hours, more preferably 3 to 4 hours. If the treatment time is less than 2 hours, proteolysis by protease may not proceed sufficiently. On the other hand, if the treatment time exceeds 5 hours, the time required for protease treatment (production of a processed bee chick) is remarkably wasted, which is uneconomical. The treatment pH is preferably performed around the optimum pH of each enzyme. In this protease treatment, it is desirable to inactivate the protease by immediately heating the incubated reaction solution at 85 to 100 ° C. for 5 to 60 minutes.

  The reaction solution contains water (or a buffer solution) as a solvent for suppressing the increase in viscosity due to the raw material of the bees and allowing the protease treatment to proceed rapidly. It is desirable that the reaction solution contains 2 to 15 times, preferably 2 to 14 times, more preferably 3 to 10 times the amount of water (or buffer solution) with respect to the weight of the bee raw material. . When less than twice the amount of solvent is added relative to the weight of the bee raw material, the increase in the viscosity of the reaction solution caused by the bee raw material cannot be sufficiently suppressed, so that the protease treatment can be advanced rapidly. It becomes difficult. On the contrary, when a solvent exceeding 10 times the amount of the raw material of the honeybee is added, there is a disadvantage that it takes a lot of time to powder the processed honeybee product obtained through each subsequent treatment. appear.

  Subsequently, a cellulase treatment is performed on the first intermediate treatment product obtained by the protease treatment. A cellulase process is a process which hydrolyzes the glycosidic bond of (beta) -1, 4-glycans, such as a cellulose contained in a bee raw material, and makes it low molecular weight using a cellulase.

Cellulases include exo-type cellulase that hydrolyzes from the terminal of β-1,4-glycan and β
There are endo-type cellulases that degrade from the middle of 1,4-glycans, and any cellulase can be used. In addition, the cellulase has an optimum pH in the vicinity of acidity (for example, less than pH 6.0), in the vicinity of neutrality (for example, pH 5.0 to 9.0), and in the vicinity of alkalinity (for example, pH 8.0 or more). Neutral cellulase and alkaline cellulase are present, and any cellulase can be used.

Specific examples of cellulases include acidic cellulases derived from Aspergillus niger. Aspergillus niger
Cellulase A “Amano” 3 (manufactured by Amano Enzyme) can be used as a commercially available acidic cellulase. Cellulase A “Amano” 3 works effectively in the vicinity of acidity, and its optimum pH is pH 4.5. Cellulase treatment using cellulase A “Amano” 3 is preferably performed at 40 to 60 ° C., more preferably 40 to 55 ° C., and even more preferably 50 to 55 ° C.

  Cellulase treatment using cellulase is performed by adding cellulase to the reaction solution after the protease treatment and incubating under reaction conditions according to cellulase. The treatment time of the cellulase treatment is appropriately set depending on the reaction temperature, the enzyme titer, etc., but is preferably 0.1 to 6 hours, more preferably 0.5 to 2 hours. If the treatment time is less than 0.1 hour, the decomposition of β-1,4-glycan by cellulase may not proceed sufficiently. Conversely, when the treatment time exceeds 6 hours, it is uneconomical because the time required for the cellulase treatment (manufacture of the processed bees) is significantly wasted. In this cellulase treatment, it is desirable to inactivate the cellulase by immediately heating the incubated reaction solution at 85 to 100 ° C. for 5 to 60 minutes.

  Subsequently, an acidification treatment is performed on the second intermediate treatment product obtained by the cellulase treatment. The acidification treatment is a treatment for degrading saccharides and insoluble lipids such as chitosan and cellulose contained in the raw material of bees and reducing the molecular weight by acidifying the reaction solution after the cellulase treatment. Specifically, an acidic substance is added to the reaction liquid after cellulase treatment and stirred until the pH is stabilized. Examples of the acidic substance include citric acid and hydrochloric acid. Moreover, pH at the time of an acidification process becomes like this. Preferably it is pH 2.0-4.0, More preferably, it is pH 3.0-3.5. The time for the acidification treatment is appropriately set depending on the pH, reaction temperature, etc., but is preferably 0.1 to 2 hours, more preferably 0.2 to 0.5 hours.

  Subsequently, the solution after the acidification treatment is subjected to filtration treatment using filter paper, diatomaceous earth, or the like, thereby separating the solution in which the soluble component is dissolved from the insoluble component. And the honey content of this embodiment is obtained by drying and pulverizing the solute contained in the solution obtained by the filtration process using means, such as freeze-drying.

  The processed beehive of this embodiment has a high antioxidant action, angiotensin converting enzyme (ACE) inhibitory activity action, skin fibroblast proliferation promoting action, fatigue recovery action, and blood flow improving action. Therefore, it can be applied as an antioxidant, an ACE inhibitor or an antihypertensive agent, a dermal fibroblast proliferation promoter, a fatigue recovery agent, and a blood flow improving agent for the purpose of obtaining these effects.

  As a specific blending form, the antioxidant can be preferably applied as a food / beverage product, a cosmetic product and a pharmaceutical product. The ACE inhibitor or the blood pressure lowering agent can be preferably applied as a food or drink or a medicine having a higher blood pressure. The dermal fibroblast proliferation promoter can be preferably applied as a cosmetic or the like. The fatigue recovery agent can be preferably applied as a food and drink such as a nutritional supplement and a pharmaceutical product. The blood flow-improving agent is preferably applied as a food and drink, a pharmaceutical product and the like having symptoms caused by poor blood circulation such as coldness and tinnitus.

  Moreover, the allergenicity of the processed bee candy according to the present embodiment is reduced as compared with the raw material of the honey bee. Therefore, the processed bee candy of this embodiment is preferably applicable as a food or drink (allergy-reducing food or drink) having an allergic symptom such as an allergic patient.

  When applying the processed bee candy of this embodiment to cosmetics, it can manufacture by mix | blending with a cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. Cosmetic bases are generally blended in common with cosmetics. For example, oil, purified water, and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-seborrheic agents. At least one selected from an agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately blended.

  When applying the processed beehive of this embodiment to food and drink, it can be used by adding to various food materials or beverage materials. The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, solid and the like, and the dosage form is any of tablets, capsules, granules, and drinks. There may be. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  When the processed bee candy of this embodiment is used as a pharmaceutical product, any administration method such as intravascular administration or transdermal administration can be employed in addition to administration by taking (oral intake). It is desirable to administer the processed beehive of this embodiment by oral ingestion. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive.

Next, operational effects in the present embodiment will be described below.
(1) In the present embodiment, the protease treatment and the acidification treatment are included in the process for obtaining the processed bee-child product. Thereby, the processed bee child thing which improved the solubility with respect to water from a bee child raw material can be obtained with a high yield. Furthermore, in this embodiment, cellulase treatment is included in the process for obtaining the processed bee-child product in addition to the above treatment. Thereby, the yield of a processed bee child can be raised more. Moreover, the defoaming property and filterability of a reaction liquid improve by performing a cellulase process.

  (2) In this embodiment, a processed bee product obtained by subjecting a bee raw material to a protease treatment and an acidification treatment has a high antioxidant effect. Therefore, it can be preferably applied as an antioxidant to cosmetics, foods and drinks, pharmaceuticals and the like for the purpose of antioxidant action.

  (3) In the present embodiment, a processed bee product obtained by subjecting a bee raw material to a protease treatment and an acidification treatment has a high ACE inhibitory activity. Therefore, as an ACE inhibitor or an antihypertensive agent, it can be preferably applied to foods and drinks, pharmaceuticals and the like intended for blood pressure effect.

  (4) In this embodiment, a processed bee product obtained by subjecting a bee raw material to a protease treatment and an acidification treatment has a high skin fibroblast proliferation promoting effect. Therefore, as a dermal fibroblast proliferation promoter, it can be preferably applied to foods and drinks, pharmaceuticals and the like for the purpose of demonstrating human skin fibroblast proliferation promoting action.

  (5) In this embodiment, the processed bee product obtained by subjecting the bee raw material to protease treatment and acidification treatment has a high anti-fatigue action. Therefore, it can be preferably applied as a fatigue recovery agent to foods and drinks, pharmaceuticals and the like for the purpose of fatigue recovery.

  (6) In this embodiment, the processed bee product obtained by subjecting the bee raw material to a protease treatment and an acidification treatment has a high blood flow improving effect. Therefore, it can be preferably applied as a blood flow improving agent to foods and drinks, pharmaceuticals and the like for the purpose of improving blood flow.

  (7) In the present embodiment, the protease used for protease treatment is preferably any one of a neutral protease derived from Bacillus subtilis, a neutral protease derived from Aspergillus oryzae, and an alkaline protease derived from Bacillus licheniformis. The In this case, the antioxidant effect and the blood flow improving effect can be further enhanced.

  (8) In the present embodiment, the allergenicity of the processed bee product obtained by subjecting the bee material to protease treatment and acidification is reduced compared to the bee material. . Therefore, it can be preferably applied to allergy-reducing food and drink.

In addition, this embodiment can also be changed and embodied as follows.
-In this embodiment, the processed bee product was obtained by subjecting the bee raw material to the three-step process of protease treatment, cellulase treatment and acidification treatment. However, the protease was obtained without performing the cellulase treatment. You may make it obtain a bee-child processed material only by two-step process of a process and an acidification process. Even in this case, a processed bee product having improved solubility in water from the bee material can be obtained in high yield.

  In this embodiment, the protease treatment, the cellulase treatment, and the acidification treatment were sequentially performed on the bee raw material, but the order of these treatments is not particularly limited, and is performed in any order. May be. Further, protease treatment and cellulase treatment may be performed simultaneously.

In the present embodiment, the solute contained in the solution obtained by the filtration treatment is dried and powdered, but the solution obtained by the filtration treatment may be directly applied to foods and drinks, pharmaceuticals, cosmetics, and the like. . In addition, the bee-processed product of the present invention is not limited to a powder form, and may be processed into any form such as liquid or paste.

  -The processed bee candy in this embodiment can be applied not only to foods and drinks and medicines consumed by humans, but may also be blended as livestock feeds, supplements, nutritional foods, medicines, etc. .

  -When applied as an allergy-reducing food or drink, it is preferable that the processed bee candy is subjected to a diatomaceous earth filtration treatment. In this case, the allergenicity of the processed bee can be more effectively reduced.

  -Since allergenicity of the processed bee pup can be more effectively reduced, a protease derived from a microorganism, preferably a neutral protease derived from Bacillus subtilis, is used.

Although the test example is given and the said embodiment is demonstrated further more concretely below, this invention is not limited to these.
(Preparation of processed bees)
As shown in Examples 1 to 6 in Table 1, protease treatment and acidification treatment were performed using bee powder obtained from a Chinese bee cocoon as a raw material. First, 2.5 g of this bee powder was suspended in water so as to be 20% by mass to prepare an aqueous solution of bee raw material (125 ml). Next, 25 mg of protease corresponding to each example (1% by mass with respect to the bee powder) was added to the aqueous raw material of the bee, and the protease treatment was performed at 50 ° C. for 4 hours. Here, as protease, Sumiteam AP (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Protease N “Amano” G (manufactured by Amano Enzyme), Umamizyme G (manufactured by Amano Enzyme), Sumiteam FP (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Six types of protease S “Amano” G (manufactured by Amano Enzyme) and protin SD-AC-10F (manufactured by Amano Enzyme) were used. The pH of the reaction solution during the protease treatment is 3.0 when the protease is Sumiteam AP, 7.0 when the protease N “Amano” G, Umamizyme G, Sumiteam FP, and the protease S “Amano” G. In the case of SD-AC-10F, it was set to 10.0.

  Subsequently, hydrochloric acid was added to each reaction solution after the protease treatment until the pH reached 3.5. Then, acidification was performed by incubating at room temperature for 0.2 hours while maintaining the pH of the reaction solution. Then, the reaction solution after the acidification treatment is filtered through diatomaceous earth, and the solute contained in the obtained solution is freeze-dried to prepare each powdered bee-treated product, and the yield and recovery rate are measured. did.

  In addition to this, as Examples 7 and 8, bee pups treated with bee pods were prepared in three steps: protease treatment, cellulase treatment and acidification. At that time, protease N “Amano” G or papain W-40 (manufactured by Amano Enzyme) was used as the protease, and cellulase A “Amano” 3 (manufactured by Amano Enzyme) was used as the cellulase. First, protease treatment was carried out by the same method as described above. When papain W-40 was used as the protease, the pH of the reaction solution during the protease treatment was 7.0. Thereafter, 25 mg of cellulase A “Amano” 3 (1 mass% with respect to the bee powder) was added to the reaction solution after the protease treatment, and the cellulase treatment was performed at 55 ° C. for 1 hour. The pH of the reaction solution during cellulase treatment was 4.5.

  Then, the acidification process was performed with respect to the reaction liquid after cellulase A "Amano" 3 process by the method similar to the said method. Then, the reaction solution after the acidification treatment was filtered with diatomaceous earth, and the solute contained in the obtained solution was freeze-dried to prepare a powdered bee-treated product, and its yield and recovery rate were measured. .

Moreover, the comparative example 1 used the aqueous solution (125 ml) of the honeybee powder prepared by suspending 2.5 g of honeybee powder in water so that it might become 20 mass%. The comparative example 2 used what performed the acidification process with the method similar to the said Example with respect to the said bee kid aqueous solution (125 ml). The comparative example 3 used what performed the cellulase process and the acidification process with the method similar to Example 7 and 8 with respect to the said beehive aqueous solution. In Comparative Example 3, cellulase A “Amano” G was first used for cellulase treatment, and then acidified. And also about Comparative Examples 1-3, it filtered and freeze-dried similarly to each Example, respectively, and prepared the powdery bee-treated material, and measured the yield and the recovery rate. The results are shown in Table 1.

表１に示されるように、プロテアーゼ処理及び酸性化処理の２工程処理した実施例１〜６は、未処理の比較例１と比較して高い回収率を示し、水に対する溶解性が向上していることが確認された。とくに、プロテアーゼとして、プロテアーゼＮ「アマノ」Ｇ、ウマミザイムＧ、及びプロチンＳＤ−ＡＣ−１０Ｆをそれぞれ使用した実施例２、３及び６は、３０％以上の高い回収率を示した。中でもプロテアーゼＮ「アマノ」Ｇを使用した実施例２は、約４０％という非常に高い回収率を示した。同様に、プロテアーゼ処理、セルラーゼ処理及び酸性化処理の３工程処理した実施例７及び８も、未処理の比較例１と比較して高い回収率を示し、水に対する溶解性が向上していることが確認された。とくに、微生物由来のプロテアーゼを使用することが有効であることが確認された。

As shown in Table 1, Examples 1 to 6, which were treated with two steps of protease treatment and acidification treatment, showed a high recovery rate compared to untreated Comparative Example 1, and improved solubility in water. It was confirmed that In particular, Examples 2, 3 and 6 using protease N “Amano” G, equinezyme G and protin SD-AC-10F as proteases showed a high recovery rate of 30% or more. Among them, Example 2 using protease N “Amano” G showed a very high recovery rate of about 40%. Similarly, Examples 7 and 8 that were treated in three steps of protease treatment, cellulase treatment, and acidification treatment also showed a higher recovery rate compared to untreated Comparative Example 1, and improved solubility in water. Was confirmed. In particular, it has been confirmed that it is effective to use a protease derived from a microorganism.

  In addition, it was confirmed that the recovery rate of Comparative Example 3 subjected to cellulase A “Amano” 3 treatment and acidification treatment in two steps was almost unchanged as compared with Comparative Example 2 in which only acid treatment was performed. On the other hand, Example 7 which processed 3 steps of protease N "Amano" G processing, cellulase A "Amano" 3 processing, and acidification processing processed 2 steps of protease N "Amano" G processing and acidification processing. Compared to Example 2, it was confirmed that the recovery rate was improved. From this result, it was shown that the combined use of protease treatment and cellulase treatment is effective in improving the recovery rate, that is, improving the solubility in water. In addition, when cellulase treatment was performed, it was also confirmed that the defoaming property and filterability of the reaction solution were improved separately from the recovery rate (data not attached).

  Next, the processed bee products (freeze-dried powder) of Example 7 were used as test samples, and various physiological activities of the processed bee products were evaluated. Hereinafter, Example 7 is simply referred to as Example. Moreover, the bee powder used as a raw material is used as a comparative example.

(Test Example 1, radical scavenging ability test as antioxidant activity)
The antioxidant activity of the examples was evaluated by a radical scavenging ability test. This test utilizes the fact that DPPH (1,1-Diphenyl-2-picrylhydrazyl) having a maximum absorption of 517 nm in a radical state is reduced by an antioxidant and fades. Each sample solution was prepared by dissolving each of Examples and Comparative Examples in distilled water so as to be 20 mg / ml. To 100 μl of each sample solution, 1.9 ml of a 170 μM DPPH ethanol solution was added and mixed to obtain a DPPH ethanol sample solution, which was allowed to react for 15 minutes. Then, spectrophotometer (Shimadzu Corporation UV-12
00), the absorbance of each DPPH ethanol sample solution at a wavelength of 517 nm was measured. In addition, the light absorbency in 517 nm was similarly measured by using what mixed distilled water with the DPPH ethanol solution instead of the sample solution as a control. The DPPH radical scavenging rate was determined from the following equation. The results are shown in Table 2.

DPPH radical scavenging rate (%) = {(Ac−As) / Ac} × 100
Ac: absorbance at 517 nm of control As: (absorbance at 517 nm of DPPH ethanol sample solution)-(absorbance at 517 nm of sample solution)

表２に示されるように、実施例及び比較例ともにＤＰＰＨラジカル補足作用を示すものの、とくに実施例は比較例と比べて高いＤＰＰＨラジカル補足作用を示した。この結果から、プロテアーゼ処理、セルラーゼ処理及び酸性化処理を行なうことにより、ＤＰＰＨラジカル補足作用、すなわち抗酸化作用が向上することが確認された。

As shown in Table 2, although both Examples and Comparative Examples showed DPPH radical scavenging action, the Examples particularly showed higher DPPH radical scavenging action than Comparative Examples. From this result, it was confirmed that DPPH radical scavenging action, that is, antioxidant action, is improved by performing protease treatment, cellulase treatment and acidification treatment.

  Free radicals are considered as causative factors for various diseases such as malignant tumors, heart diseases, and cerebrovascular diseases. Therefore, the example which has the effect | action which removes a free radical can become an effective component for the prevention and treatment of those diseases. Therefore, it can mix | blend suitably as active ingredients, such as health food, cosmetics, and a pharmaceutical aiming at the exhibit of free radical removal ability.

(Test Example 2, angiotensin converting enzyme (ACE) inhibitory activity test)
The angiotensin converting enzyme (ACE) inhibitory action of the examples was evaluated. ACE is an enzyme involved in the production of the blood pressure increasing factor angiotensin II, and it has been confirmed that suppressing the action of this enzyme also leads to a decrease in blood pressure. The sample solution was prepared by dissolving in Examples and Comparative Examples in distilled water to be 0.08 mg / ml, 0.4 mg / ml, and 2 mg / ml, respectively.

The measurement of ACE inhibitory activity was performed according to the method of Toda et al. First, 50 μl of each sample solution and 150 μl of 5.83 mM ACE substrate solution (dissolved in Bz-Gly-His-Leu · H 2 O, 1M NaCl / pH 8.3 50 mM borate buffer) are mixed, and premixed at 37 ° C. for 5 minutes. Incubated. Subsequently, 50 μl of 120 mU / ml rabbit lung-derived angiotensin converting enzyme was added to each sample and incubated at 37 ° C. for 30 minutes, and then 250 μl of 1N hydrochloric acid was added to stop the reaction. Then, hippuric acid produced in each sample was extracted with ethyl acetate, concentrated and dried, redissolved in 2.5 ml of distilled water, and the absorbance at 228 nm was measured.

  Prepare a mixture of distilled water instead of the sample solution and use it as a control. Prepare each sample and control with 250 μl of 1N hydrochloric acid added before incubation. Were each blank. And also about control and each blank, the same extraction process, concentration drying process, and re-dissolution process were performed after incubation, and the light absorbency in 228 nm was measured. And the ACE inhibition rate in the sample solution of each density | concentration was calculated | required from the following formula | equation.

ACE inhibition rate (%) = [{(Ec−Ecb) − (Es−Esb)} / (Ec−Ecb)] × 100
Ec: Absorbance at 228 nm of control Ecb: Absorbance at 228 nm of control blank Es: Absorbance at 228 nm of each sample Esb: Absorbance at 228 nm of each sample blank and, based on the determined ACE inhibition rate at each concentration, ACE The concentration at which the inhibitory activity was inhibited by 50% (mg / ml) was calculated as the ACE inhibitory activity IC50 value. The results are shown in Table 3.

表３に示されるように、比較例ではＡＣＥ阻害活性については殆ど認められないのに対し、実施例では高いＡＣＥ阻害活性が認められた。この結果から、ＡＣＥ阻害活性は、プロテアーゼ処理、セルラーゼ処理及び酸性化処理を行なうことにより初めて発現することが示された。よって、ＡＣＥ阻害活性を有する実施例は、血圧降下作用の発揮を目的とする飲食品及び医薬品等の有効成分として好適に配合することができる。

As shown in Table 3, in the comparative example, almost no ACE inhibitory activity was observed, whereas in the examples, high ACE inhibitory activity was observed. From this result, it was shown that the ACE inhibitory activity was first expressed by protease treatment, cellulase treatment and acidification treatment. Therefore, the example which has ACE inhibitory activity can be suitably mix | blended as active ingredients, such as food-drinks and a pharmaceutical aiming at the exhibit of the blood pressure lowering effect.

(Test Example 3, skin fibroblast proliferation test)
The human skin fibroblast proliferation action of the examples was evaluated. The sample medium was prepared by dissolving the examples in DMEM medium containing 10% FBS so as to be 6 μg / ml, 30 μg / ml, and 150 μg / ml. In addition, a 10% FBS-containing DMEM medium not containing Examples was prepared and used as a control.

  First, human skin fibroblasts were cultured in a DMEM medium containing 10% FBS, prepared to 0.8 × 10 4 cells / ml, and seeded at 1 ml in each well of a 24 well plate. On the next day, the medium was replaced with the sample medium, and after culturing for 48 hours, the number of cells was measured with a hemocytometer. The results are shown in Table 4.

表４に示すように、実施例を３０μｇ／ｍｌ及び１５０μｇ／ｍｌの濃度で含有する試料培地においては、コントロールに対して有意な細胞数の増加が認められた。この結果から、実施例は、ヒト皮膚繊維芽細胞を増殖させる作用を有することが示された。よって、実施例は、ヒト皮膚繊維芽細胞増殖作用の発揮を目的とする飲食品及び医薬品等の有効成分として好適に配合することができる。

As shown in Table 4, in the sample medium containing Examples at concentrations of 30 μg / ml and 150 μg / ml, a significant increase in the number of cells was observed with respect to the control. From this result, it was shown that an Example has the effect | action which proliferates a human skin fibroblast. Therefore, an Example can be suitably mix | blended as active ingredients, such as food-drinks and a pharmaceutical aiming at the exhibit of the human skin fibroblast proliferation effect.

(Test Example 4, fatigue recovery test)
The fatigue recovery action of the examples was evaluated by a mouse forced swimming test. When the mouse is placed in the water, it swims with its limbs and tail for the first minute or so and tries to escape. However, after that, fatigue causes time for the mouse to stop swimming and float on the surface of the water. In this test, this is measured as immobility time. In this test, in addition to Examples and Comparative Examples, L-carnosine was also evaluated as a positive control.

  A ddY male mouse (5 weeks old) was used as a test animal, and each sample dissolved in distilled water was orally administered for 4 days. Each sample was administered at 500 mg / kg (body weight) per day for Examples and Comparative Examples, and was administered at 50 mg / kg (body weight) per day for L-carnosine. Moreover, what administered only distilled water was prepared and this was made into control. And the forced swimming test was implemented on the 5th day. For forced swimming, first, preliminary forced swimming for 5 minutes was tried, and immediately after that, each sample was orally administered in the same amount as the previous day. After 60 minutes from the oral administration, re-forced swimming was tried for 5 minutes, and during the 5-minute re-forced swimming, the time during which the mouse did not move in water was accumulated, and this was defined as the immobility time. The results are shown in Table 5.

表５に示されるように、実施例及び比較例ともに、コントロールと比較して有意な浮動時間の短縮が認められた。よって、実施例は、肉体疲労の改善作用の発揮を目的とする飲食品及び医薬品等の有効成分として好適に配合することができる。

As shown in Table 5, both the examples and the comparative examples showed a significant reduction in floating time compared to the control. Therefore, an Example can be suitably mix | blended as active ingredients, such as food / beverage products and a pharmaceutical aiming at the improvement effect of physical fatigue.

(Test Example 5, blood flow test)
The blood flow improving effect of the examples was evaluated. In this test, L-arginine was also evaluated as a positive control in addition to Examples and Comparative Examples. Examples, comparative examples, and L-arginine were orally administered to rats at 2 g / kg (body weight), and anesthesia injection was performed after 60 minutes, and blood flow at the base of the tail of rats was measured. Started. The blood flow was measured for 60 minutes, and the average blood flow was calculated. The results are shown in Table 6. The same operation was performed for the Examples, Comparative Examples, and rats not administered with L-arginine, and this was used as a control.

表６に示されるように、実施例及び比較例ともに有意な血流量の増加作用を示した。とくに、実施例は比較例と比べても高い血流量の増加作用を示した。この結果から、プロテアーゼ処理、セルラーゼ処理及び酸性化処理を行なうことにより、血流量増加作用がさらに向上することが確認された。よって、実施例は、血流量改善作用の発揮を目的とする飲食品及び医薬品等の有効成分として好適に配合することができる。

As shown in Table 6, both the example and the comparative example showed a significant blood flow increasing action. In particular, the Example showed a high blood flow increasing action as compared with the Comparative Example. From this result, it was confirmed that the blood flow increasing action was further improved by performing protease treatment, cellulase treatment and acidification treatment. Therefore, an Example can be suitably mix | blended as active ingredients, such as food-drinks and a pharmaceutical aiming at the exhibit of the blood flow improvement effect.

(Test Example 6, allergy test)
The presence or absence of allergenicity in the examples was evaluated. In this test, crustacean allergic substances (crustacean allergenic proteins) contained in Examples and Comparative Examples were measured by ELISA. Crustacean allergens were measured using FA Test EIA-Crustacea “Nissui” (Nissui Pharmaceutical Co., Ltd.). Sample solutions were prepared by dissolving in distilled water so that each of Examples and Comparative Examples was 2.5 mg / ml, and the crustacean allergen content in each sample solution was measured. For each sample solution, the crustacean allergen content is measured three times. If the average value of the crustacean allergen content is 20 ppm or more, allergic (positive), 1.0 ppm or more and less than 20 ppm. When allergy was suspected (positive) and less than 1.0 ppm, it was determined that there was no allergy (negative). The results are shown in Table 7.

表７に示されるように、比較例は甲殻類アレルギー性物質含量が多く、甲殻類アレルギーについて陽性を示した。これに対して、実施例は甲殻類アレルギー性物質含量が低減されており、甲殻類アレルギーについて陰性を示した。この結果から、プロテアーゼ処理、セルラーゼ処理及び酸性化処理を行なうことにより、甲殻類アレルギー性物質含量が低下し、アレルギー性が低減されることが確認された。よって、実施例はアレルギー低減飲食品として好適に適用することができる。

As shown in Table 7, the comparative example had a high crustacean allergic substance content and was positive for crustacean allergy. On the other hand, the Examples showed a reduced content of crustacean allergenic substances and were negative for crustacean allergies. From this result, it was confirmed that by performing protease treatment, cellulase treatment and acidification treatment, the content of crustacean allergenic substances is reduced and allergenicity is reduced. Therefore, an Example can be applied suitably as an allergy reduction food / beverage product.

Next, a technical idea that can be grasped from the above embodiment will be described.
-The cooling property improving agent characterized by including the processed product of the solubilized bee.
A tinnitus ameliorating agent characterized by containing the solubilized bee treatment product.

O Antioxidant characterized by containing the solubilized bee treatment product.
○ An antihypertensive agent comprising the solubilized bee treatment product.
A skin fibroblast growth promoter characterized by comprising the solubilized bee treatment product.

-Anti-fatigue agent characterized by containing the processed product of the solubilized bee.
(Circle) the blood-flow improving agent characterized by containing the said solubilized bee treatment thing.

Claims (9)

Solubilization characterized in that it is obtained by treating a bee or an insoluble component obtained by extracting bees with an extraction solvent whose main component is water by combining protease treatment and acidification treatment. Processed bee pups. It is obtained by treating a bee or an insoluble component obtained by extracting bees with an extraction solvent whose main component is water by combining protease treatment, cellulase treatment and acidification treatment. Solubilized bee processed product. The protease treatment is performed using at least one selected from a neutral protease derived from Bacillus subtilis, a neutral protease derived from Aspergillus oryzae, and an alkaline protease derived from Bacillus licheniformis. Or the solubilized bee treatment product according to claim 2. The processed product of solubilized bees according to any one of claims 1 to 3, wherein the processed product of solubilized bees has reduced allergenicity. A solubilized bee treatment characterized by treating a bee or an insoluble component obtained by extracting the bee with an extraction solvent whose main component is water in combination with a protease treatment and an acidification treatment. Manufacturing method. A solubilized bee characterized by treating a bee or an insoluble component obtained by extracting the bee with an extraction solvent whose main component is water by combining protease treatment, cellulase treatment and acidification treatment. Of manufacturing a child processed product. 6. The protease treatment is performed using at least one selected from a neutral protease derived from Bacillus subtilis, a neutral protease derived from Aspergillus oryzae, and an alkaline protease derived from Bacillus licheniformis. Or the manufacturing method of the solubilized bee treatment thing of Claim 6. A medicinal product, cosmetic or food-drink comprising the solubilized bee-treated product according to any one of claims 1 to 4. The food / beverage product according to claim 8, wherein the food / beverage product is an allergy-reducing food / beverage product.