JP2005287411A - Low-allergenized royal jelly and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low-allergenized royal jelly capable of easily reducing allergic property while suppressing reduction of useful physiological activities of the royal jelly and to provide a method for producing the same. <P>SOLUTION: This low-allergenized royal jelly comprises active components such as 10-hydroxy decenoic acid, together with a peptide component originating from the royal jelly having ≤4.5 kDa of molecular weight. The low-allergenized royal jelly is effectively produced by subjecting royal jelly to a protease treatment and a glycolytic enzyme treatment using β-mannosidases. The protease treatment preferably comprises a first decomposing process subjecting a diluted royal jelly solution with adjusted pH at 6.5-8.0 to an endo type neutral protease treatment, a pH adjusting process adjusting the pH of the royal jelly diluted solution after the first decomposing process at 6.5-8.0 and a second decomposing process subjecting the diluted royal jelly solution after adjusting pH to an endo type neutral protease treatment. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a low allergenized royal jelly and a method for producing the same.

Conventionally, as this kind of low allergenized royal jelly, low allergenized royal jelly is known in which allergenicity is reduced to such an extent that it is not substantially expressed by subjecting royal jelly to glycolytic enzyme treatment and proteolytic enzyme treatment. (See Patent Document 1). In this allergen-reduced royal jelly, the pH of the solution is adjusted to 6.3 to 7.3, and a fraction containing a 55 kDa protein having a molecular weight of 55 kDa, which is the main allergen, is precipitated and removed using isoelectric point precipitation. As a result, allergic reactions can be suppressed. On the other hand, Patent Document 2 discloses a lipid metabolism improving agent containing a degradation product of royal jelly by protease as an active ingredient. In the degradation product of this lipid metabolism improving agent, those having a molecular weight of 10,000 or more are almost lost, and a peptide having a molecular weight of 500 to 3000 is the main component.
JP 2002-127715 A JP 2001-2577 A

  The present invention has been made on the basis of the knowledge that the allergenicity contained in the royal jelly could be eliminated very effectively as a result of intensive studies by the present inventors. An object of the present invention is to provide a low allergenized royal jelly that can easily reduce allergenicity while suppressing a decrease in useful physiological activity of the royal jelly, and a method for producing the same.

  In order to achieve the above object, the invention of a low allergenized royal jelly according to claim 1 is a low allergenized royal jelly with reduced allergenicity, comprising 10-hydroxydecenoic acid and a royal jelly-derived peptide component. The gist is that the peptide component is contained from a component having a molecular weight of 4.5 kDa or less. According to this structure, since the component exceeding the molecular weight 4.5 kDa which is easy to cause allergy among the peptide components derived from a royal jelly is not contained, it becomes easy to reduce allergenicity. Furthermore, since 10-hydroxydecenoic acid is contained, it exhibits a high effect on health and beauty.

  The invention of the method for producing a low allergenized royal jelly according to claim 2 is the method for producing a low allergenized royal jelly according to claim 1, wherein the royal jelly is subjected to proteolytic enzyme treatment and / or glycolytic enzyme treatment. Is the gist. According to this structure, since it is produced using an enzyme having a high substrate specificity, a component exceeding a molecular weight of 4.5 kDa is effectively decomposed among the peptide components derived from royal jelly, and the influence on other components is also achieved. Less is. For this reason, the useful physiological activity of royal jelly can be suppressed while reducing allergenicity easily.

  The method for producing a reduced allergenized royal jelly according to claim 3 is the invention according to claim 2, wherein the proteolytic enzyme treatment is performed at pH 6.5 to 8.0 using endo-type neutral protease. This is the gist. According to this configuration, the endo-type neutral protease can effectively decompose components exceeding a molecular weight of 4.5 kDa among the peptide components derived from royal jelly at the optimum pH.

  The method for producing a reduced allergenized royal jelly according to claim 4 is the invention according to claim 3, wherein the proteolytic enzyme treatment is performed on a royal jelly diluted solution whose pH is adjusted to 6.5 to 8.0. A first decomposition step for performing an endo-type neutral protease treatment, a pH adjustment step for adjusting the pH of the royal jelly diluent after the first decomposition step to 6.5 to 8.0, and a royal jelly dilution after the pH adjustment step The gist is to include a second decomposition step in which the liquid is subjected to endo-type neutral protease treatment. According to this configuration, the proteolytic activity of the endo-type neutral protease can be easily increased.

  The invention of the method for producing a low allergenized royal jelly according to claim 5 is a method for producing a low allergenized royal jelly, wherein a royal jelly diluted solution obtained by diluting a royal jelly with water or a buffer is subjected to a proteolytic enzyme treatment and a β-mannosidase treatment. The proteolytic enzyme treatment is performed at pH 6.5 to 8.0 using endo-type neutral protease. According to this configuration, allergenicity can be easily reduced because components that are likely to cause allergy among the peptide components derived from royal jelly are effectively decomposed. Furthermore, since 10-hydroxydecenoic acid is contained, it exhibits a high effect on health and beauty.

  According to the present invention, it is possible to provide a low allergenized royal jelly that can easily reduce allergenicity while suppressing a decrease in useful physiological activity of the royal jelly, and a method for producing the same.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments embodying the present invention will be described in detail. In the present embodiment, “royal jelly” is abbreviated as “RJ”.
RJ is a milky white jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and the greater vagina by bees aged 3 to 12 days. This RJ is known to have a favorable physiological activity on the human body. The main physiologically active components in the RJ include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as decenoic acid) unique to RJ, proteins, lipids, saccharides, vitamin Bs and folic acid. Vitamins such as nicotinic acid and pantothenic acid, and various minerals. As the physiological activity and pharmacological action of RJ, antibacterial action, immunity enhancing action, effective tumor action, anti-inflammatory action, blood flow increasing action and the like are known. In addition, the side effects of anticancer drugs are reduced and the life-prolonging effect at the time of radiation injury is also reported.

  As a raw material used for manufacturing the allergen-reduced RJ of the present embodiment, raw RJ or RJ powder obtained by drying and pulverizing the raw RJ is used. The production area of RJ may be any of China, Brazil, European countries, Oceania countries, the United States, and the like. This RJ contains various allergens that cause allergic reactions in the living body. Examples of allergens contained in the RJ include multiple types of protein bands that are found in the vicinity of a molecular weight of 47 to 55 kDa by SDS-PAGE under reducing conditions. Of these, a protein with a molecular weight of 55 kDa is a major allergen in RJ. Moreover, the component which acts as an allergen is also contained in the peptidic component having a molecular weight exceeding 4.5 kDa.

  The hypoallergenic RJ of the embodiment contains an active ingredient derived from raw RJ and a peptide component derived from raw RJ, and is configured to be absorbed into the body by oral ingestion. This allergen-reduced RJ may contain other components derived from raw RJ, such as carbohydrates and lipids, in addition to the active ingredient and peptide component, and other ingestible additives. It does not matter. The hypoallergenic RJ may contain a protein that is not derived from raw RJ. Examples of the usage form of this allergen-reduced RJ include, for example, liquid (drink), lyophilized, powdered by drying under reduced pressure or other powdering means, tablets, capsules added with excipients, and cosmetics. Examples include creams and lotions mixed with a base.

  The active ingredient has the effect of activating vital activities mainly by oral ingestion to promote health and beauty, contains at least decenoic acid, and contains ingredients specific to RJ such as parothin and popterin It is preferable. Furthermore, as this active ingredient, physiologically active substances such as γ-aminobutyric acid, carbachol, acetylcholine, phosphate compounds such as ATP, vitamins B1, B2, B6, B12, niacin, pantothenic acid, nicotinic acid, folic acid, biotin, Vitamins such as Pichiosi, Inositol, Minerals such as Phosphorus, Calcium, Copper, Manganese, Iron, Magnesium, Zinc, Taurine, Choline, Essential amino acids such as Lysine, Phenylalanine, Leucine, Methionine, Valine, Threonine, Tryptophan It is preferable that various L-α-amino acids are contained. In addition, it is preferable that this low allergen RJ is commercialized as adjustment RJ containing 0.18 mass% or more of decenoic acid.

  The peptidic component is derived from raw RJ, and examples thereof include simple proteins, complex proteins, polypeptides, and oligopeptides. This peptidic component may contain a component having a molecular weight exceeding 4.5 kDa, but preferably comprises only a component having a molecular weight of 4.5 kDa or less in order to reduce allergenicity. The allergen-reduced RJ composed only of components having a molecular weight of 4.5 kDa or less is a molecular weight of 4.5 kDa in Coomassie staining, preferably silver staining, in an electrophoresis gel after performing SDS-PAGE under reducing conditions. A protein band exceeding 1 is not detected. For example, when the raw RJ is subjected to SDS-PAGE, the amount of protein is equivalent to the amount of protein with a molecular weight of 55 kDa clearly visible by Coomassie staining (in terms of dry weight or in terms of absorbance at 280 nm). It means that when a low allergenized RJ is electrophoresed under the same conditions, a protein band having a molecular weight exceeding 4.5 kDa is not visually recognized. In addition, although the protein band exceeding the said molecular weight 4.5 kDa means that it is not visually recognized by Coomassie dyeing | staining, it is especially preferable not to be visually recognized also by silver dyeing | staining. The term “not detected” means that a peak exceeding a molecular weight of 4.5 kDa is not detected in a chromatogram at a detection wavelength of 220 nm when analyzed by high performance liquid chromatography (HPLC) using a gel filtration column. Means that no peptidic component exceeding a molecular weight of 4.5 kDa is detected.

  This allergen-reduced RJ may be produced by removing a peptidic component having a molecular weight of more than 4.5 kDa using a separation and purification technique such as gel filtration or ultrafiltration, particularly a technique for purification according to the molecular weight. Although it is good, since the recovery rate of the active ingredient is high, it is most preferable that it is produced by performing a treatment for enzymatically decomposing the peptide component. As the enzyme decomposing treatment, a proteolytic enzyme treatment and / or a saccharolytic enzyme treatment is preferably used. This enzymatic decomposition treatment is carried out by adding a proteolytic enzyme and / or a glycolytic enzyme to the raw material (liquid), preferably an RJ diluted solution obtained by diluting the raw material with water or a buffer solution. In addition, when performing a proteolytic enzyme treatment and a glycolytic enzyme treatment, each treatment may be performed separately or simultaneously.

  Proteolytic enzymes are enzymes that hydrolyze peptide bonds of peptidic components (proteins) and rapidly reduce the molecular weight (peptone) of high-molecular peptidic components. Examples of this proteolytic enzyme include microorganism-derived or plant-derived thiol proteinases (papain, phytin, promeline, etc.), aspartic proteinases, serine proteinases (pepsin, trypsin, chymotrypsin, pancreatin, etc.), and metal proteinases. Proteolytic enzymes are classified into acidic protease, neutral protease or alkaline protease according to their optimum pH, and are classified into endoprotease type (endo type) or exoprotease type (exo type) based on substrate specificity.

  Among these proteolytic enzymes, neutral protease or alkaline protease is preferably used because of its high effect of reducing the protein in RJ. On the other hand, since raw RJ has a pH of 3.5 to 4, if protease treatment is performed at a high pH, a large amount of salt is generated, which is not preferable, and neutral protease is particularly preferably used. These proteases are preferably of the endoprotease type because they can efficiently decompose a polymer such as a protein having a molecular weight of 55 kDa.

  As a proteolytic enzyme satisfying such various conditions, an endo-type neutral protease derived from Bacillus subtilis or an endo-type neutral protease derived from Aspergillus oryzae is particularly preferably used. Both endo-type neutral proteases belong to metalloproteinases, and the optimum pH is generally in the range of 6.5 to 7.5. These proteolytic enzymes may be used alone or in combination of two or more. In addition, the purity of the enzyme used is not particularly limited, and for example, a microbial product producing the enzyme, a homogenate from a plant or an animal may be used.

  This proteolytic enzyme treatment is preferably carried out at the optimum pH of the proteolytic enzyme in order to effectively decompose the allergen contained in the raw RJ. When the pH fluctuates during the proteolytic enzyme treatment It is good to comprise so that pH adjustment may be performed on the way. At this time, the proteolytic enzyme treatment includes a first decomposing step of performing proteolytic enzyme treatment on a raw material or RJ diluted solution adjusted to an optimum pH of the proteolytic enzyme, and a raw material or RJ diluted solution after the first decomposing step. A pH adjusting step for re-adjusting the pH to the optimum pH and a second decomposing step for subjecting the raw material or RJ diluent after the pH adjusting step to a proteolytic enzyme treatment are performed.

  A saccharolytic enzyme is an enzyme that acts on polysaccharides, glycoproteins and the like to cleave sugar chains. Examples of the glycolytic enzyme include cellulase, avicellase, hemicellulase, glucosidase, mannanase, galactosidase, xylanase and the like. Among these glycolytic enzymes, β-mannosidase is most preferably used because of its high effect of reducing RJ allergenicity. These glycolytic enzymes may be used alone or in combination of two or more. In addition, the purity of the enzyme used is not particularly limited, and for example, a microbial product producing the enzyme, a homogenate from a plant or an animal may be used.

  In order to reduce the viscosity and smoothly carry out various enzyme reactions, it is preferable to use an RJ diluted solution obtained by diluting the raw material with a predetermined amount of water or a buffer solution and sufficiently mixing the enzyme decomposing treatment. At this time, when raw RJ is used as a raw material, it is preferably diluted with 0.2 to 1 times, particularly preferably 0.33 times the amount of water or buffer. When the amount of water or buffer added for dilution is less than 0.2 times the amount, the enzyme reaction does not proceed smoothly because the viscosity of the RJ dilution is high, and conversely when the amount exceeds 1 time However, it is uneconomical because it is necessary to enlarge the equipment for manufacturing. On the other hand, when RJ powder is used as a raw material, it is preferably diluted with 2 to 5 times the amount of water or buffer for the same reason as in the case of raw RJ. Furthermore, it is preferable to adjust the pH of the RJ dilution to around the optimum pH of the enzyme. For example, when using an endo-type neutral protease, the pH of the RJ dilution is preferably 7.0 to 8.0, particularly It is preferable to adjust to 7.5.

  In addition, the amount of enzyme added in this enzymatic decomposition treatment can be arbitrarily selected. For example, when it is desired to complete the reaction in a short time of about 5 hours, an enzyme of 1 to 20 mg per 1 g of RJ solid content may be used, but it varies depending on the purity of the enzyme used. On the other hand, for example, when the enzyme reaction is performed for one day, the amount of the enzyme may be about 0.1 to 2 mg. In addition, since many components are contained in RJ and may act inhibitory to the enzyme, it is preferable to use 10 times or more when performing enzyme treatment on a normal substrate. . However, this can also be arbitrarily selected in view of the reaction time. This enzymatic decomposition treatment is preferably performed at an optimum temperature (usually 40 to 60 ° C.). Further, the reaction between RJ and the enzyme is preferably carried out with stirring in order to make the allergen and the enzyme easy to contact. In the above treatment, when an insoluble matter is generated in the raw material or the RJ diluent, it is preferable to remove the insoluble matter by centrifugation or filtration as appropriate. If it is contained, it is preferably added again before commercialization. Furthermore, it is preferable to inactivate or remove the enzyme after completion of the enzymatic decomposition treatment.

The effects exhibited by the above embodiment will be described below.
-Since allergen-ized RJ of embodiment contains decenoic acid, it exhibits a high effect on health and beauty. Furthermore, since this allergen-reduced RJ contains only components having a molecular weight of 4.5 kDa or less among the peptide components derived from RJ, it is difficult to cause allergies. That is, it has been elucidated by the present inventors that the RJ-derived peptide component having a molecular weight exceeding 4.5 kDa has an effect of causing allergy.

  -The allergen-reduced RJ of the embodiment is obtained by subjecting RJ to glycolytic enzyme treatment and proteolytic enzyme treatment, which are two types of enzymes having different action sites, so that almost all macromolecular peptide components in RJ Is reduced in molecular weight, and allergenicity is reduced to such an extent that it is not substantially expressed. Furthermore, in this allergen-reduced RJ, the amino acid absorbability is remarkably improved because the peptide component is reduced in molecular weight.

  Further, as a result of intensive studies by the present inventors, even when two or more types of proteolytic enzymes having different action sites on the substrate are allowed to act on RJ, as in the treatment applied to RJ in Patent Document 1. It has been confirmed that allergenicity could not be reliably reduced by enzyme selection. In addition, it was confirmed that allergenicity in RJ could not be completely removed even when a protein with a molecular weight of 55 kDa, which is the main allergen in RJ, was fractionated and subjected to glycolytic enzyme treatment and proteolytic enzyme treatment. Yes.

  On the other hand, for example, a propolis product that has been reduced to such an extent that allergenicity is not substantially expressed by the action of a hydrolase or an oxidoreductase is disclosed. On the other hand, the main allergens contained in RJ are different from the allergens in the conventional propolis products and cannot be sufficiently decomposed by the hydrolase treatment and / or the oxidoreductase treatment. It has been confirmed by the results of earnest research.

  -Health promotion activity contained in RJ is related to various components such as decenoic acid, peptidic components, vitamins, minerals and amino acids. For this reason, removal of the peptide component may cause an unexpected decrease in activity, and the amount of amino acids is drastically reduced. Therefore, there is a high possibility that the health promoting activity of raw RJ cannot be sufficiently caused. Therefore, the enzymatic decomposition is most effective as a method for reducing the allergen with high substrate specificity that specifically decomposes only the component causing the allergen and does not affect the other components.

  -By using β-mannosidase as a glycolytic enzyme, the allergy of RJ can be easily and reliably reduced. In particular, a protein having a molecular weight of 55 kDa, which is a major allergen in RJ, is known to have a high mannose-type sugar chain, and it is very likely that it constitutes an antigenic determinant. Therefore, there is a high possibility that the antigenic determinant can be more effectively eliminated by hydrolyzing the peptide bond of the protein having a molecular weight of 55 kDa and decomposing the high-mannose sugar chain.

<< Examination of Allergen Degradation Conditions >>
The conditions for efficiently degrading a molecular weight 55 kDa protein, which is a major allergen contained in RJ, and a protein having a molecular weight of 4 kDa or more and less than 55 kDa suspected of being an allergen were examined.

<Enzyme selection>
Preliminary using only proteolytic enzymes.
(Test Example 1): RJ diluted solution was prepared by adding 330 ml of pure water to 1000 g of RJ produced in China and stirring until uniform. The RJ dilution was adjusted to pH 7.5 with sodium bicarbonate or calcium carbonate, and then dispensed into 9 beakers of 100 ml each. 1.67 g of proteolytic enzyme was added to each beaker and reacted at 50 ° C. for 16 hours. Any one type of proteolytic enzymes shown in Table 1 is added to the beaker. The proteolytic enzymes shown in Table 1 are Bacillus subtilis-derived endo-type neutral protease (Protein N manufactured by Amano Enzyme), Aspergillus oryzae-derived endo-type neutral protease (Protin P manufactured by Daiwa Kasei Co., Ltd.), Aspergillo Peptidase A (Morshin F manufactured by Shengshin Co., Ltd.), Pepsin (manufactured by Wako Pure Chemical Industries, Ltd.), Pancreatin (Pancreatin F manufactured by Amano Enzyme), Papain (Papain W-40 manufactured by Amano Enzyme), Promeline (Amano Enzyme) Promeline F), Actinase (Actinase E, Kaken Pharmaceutical Co., Ltd.), Alkalophilic Protease (Toyobo). After completion of the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 30 minutes and sterilized.

(Degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1)
SDS-PAGE was performed on each sample obtained in Test Example 1. Each sample was added with 5 × SDS sample buffer containing 1/4 volume of 200 mM Tris-HCl (pH 6.8), 250 mM dithiothreitol, 5% SDS, 22.5% glycerol and an appropriate amount of bromophenol blue, and 98 ° C. For 2 minutes. Thereafter, about 0.3-0.5 μl, SDS-PAGE was performed using a fully automated electrophoresis system PhastSystem ™ (Amersham Pharmacia Biotech) using an 8-25% gradient polyacrylamide gel having a thickness of 0.45 mm. went. As a molecular weight marker. LMW kit E (Amersham Pharmacia Biotech) was used. The gel after electrophoresis was stained with Coomassie Brilliant Blue R-350 and decolorized with a solution containing 30% methanol and 10% acetic acid. Regarding the stained and decolored gel, the decomposition of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa (described as> 4 kDa in the table) was visually confirmed. The results are shown in Table 1.

表１より、プロテアーゼＮ、プロチンＰ、モルシンＦ及びペプシンでは分子量５５ｋＤａ蛋白質の分解能が高く、その他のプロテアーゼでは低かった。また、プロテアーゼＮ及びプロチンＰは、モルシンＦ及びペプシンよりも分子量４ｋＤａ以上５５ｋＤａ未満の蛋白質の分解能が高かった。従って、プロテアーゼＮ、プロチンＰ、モルシンＦ及びペプシンがＲＪ由来のペプチド性成分の分解に有効であることが分かった。

Table 1 shows that protease N, protin P, morsin F and pepsin have a high molecular weight 55 kDa protein, and other proteases have low resolution. Protease N and protin P had higher resolution of proteins having a molecular weight of 4 kDa or more and less than 55 kDa than morsin F and pepsin. Therefore, it was found that protease N, protin P, morsin F, and pepsin are effective in degrading RJ-derived peptidic components.

<Examination of processing conditions>
The pH, reaction temperature, etc. suitable for the decomposition of the RJ-derived peptide component were examined. In this study, a proteolytic enzyme and a glycolytic enzyme were used.

  (Test Example 2): 100 ml of pure water was added to 100 g of Chinese-produced RJ to prepare an RJ dilution. This RJ diluted solution was adjusted to pH 6.5 with sodium hydrogen carbonate or calcium carbonate, and then the precipitated fraction was fractionated by isoelectric point precipitation, and 1.67 g of molsin F and 30 mg of β- Mannosidase (Sumiteam ACH manufactured by Shin Nippon Chemical Industry Co., Ltd.) was added simultaneously and reacted at 50 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 3): 100 ml of pure water was added to 100 g of RJ produced in China to prepare an RJ dilution. After adjusting this RJ dilution to pH 6.5, 1.67 g of molsin F and 30 mg of β-mannosidase were added simultaneously and reacted at 50 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 4): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ dilution, 1.67 g of molsin F and 30 mg of β-mannosidase were simultaneously added and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 5): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. 1.67 g of molsin F and 30 mg of β-mannosidase were simultaneously added to this RJ diluted solution, reacted at 45 ° C. for 16 hours, then heated to 55 ° C. and reacted for 2 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  Test Example 6: An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. 1.83 g of molsin F and 30 mg of β-mannosidase were simultaneously added to this RJ dilution, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 7) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ diluted solution, 1.67 g of acidic protease (Protein M manufactured by Amano Enzyme) and 30 mg of β-mannosidase were simultaneously added and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 8): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ dilution, 1.67 g of protease M and 30 mg of β-mannosidase were simultaneously added, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 9) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. This RJ diluted solution was adjusted to pH 6.0 with sodium hydrogen carbonate or calcium carbonate, 1.67 g of protease N and 30 mg of β-mannosidase were added simultaneously, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

(Degradation confirmation 2 of RJ-derived peptide component by SDS-PAGE 2)
Each sample obtained in Test Examples 2 to 9 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 2.

表２より、プロテアーゼＭは有効でないことが分かった。また、モルシンＦの添加量を増大させてもそれに見合った分解能は発揮されなかった。また、モルシンＦの添加量を増やしても溶液のｐＨが低ければ分解能は低下することが分かり、ｐＨが分解能に最も効いていることが容易に推察される。反応温度はさほど重要でないことも推察される。プロテアーゼＮについても全く同様に、添加量はさほど重要でなかった。従って、プロテアーゼ処理の進行とともに溶液のｐＨが低下し、それに起因して蛋白質の分解能が低下するものと考えられる。

Table 2 shows that protease M is not effective. Further, even when the amount of Morsin F added was increased, the resolution corresponding to that was not exhibited. Further, it can be seen that even if the amount of Morsin F added is increased, the resolution decreases if the pH of the solution is low, and it is easily assumed that the pH is most effective for the resolution. It is also speculated that the reaction temperature is not so important. Just as for protease N, the amount added was not very important. Therefore, it is considered that the pH of the solution decreases with the progress of protease treatment, resulting in a decrease in protein resolution.

<Examination of resolution by pH adjustment 1>
The effect on the resolution was studied by adjusting the pH of the solution during the enzymatic reaction. Note that this study was performed preliminarily using only proteolytic enzymes.

  (Test Example 10): RJ dilution was prepared by adding 330 ml of pure water to 1000 g of RJ produced in China. The RJ dilution was adjusted to pH 7.5 and then dispensed into 9 beakers of 100 ml each. In each beaker, 1.67 g of any one type of proteolytic enzyme shown in Table 3 below was added and reacted at 50 ° C. for 2 hours to carry out the first decomposition step. Subsequently, after the pH adjustment step was performed by readjusting the RJ diluted solution after the first decomposition step to pH 7.5 with sodium bicarbonate or calcium carbonate, the reaction was further performed at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

(Degradation confirmation 3 of RJ-derived peptidic component by SDS-PAGE 3)
Each sample obtained in Test Example 10 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 3.

表３より、ｐＨ調整工程を挟んで第１分解工程と第２分解工程とを行うことにより、少ない酵素添加量であっても全般に良好な分解能が発揮された。但し、蛋白質分解酵素のみでは、分子量４ｋＤａ以上５５ｋＤａ未満の蛋白質の分解は不十分であった。

From Table 3, by performing the 1st decomposition process and the 2nd decomposition process on both sides of a pH adjustment process, even if it was a small enzyme addition amount, the good resolution was generally exhibited. However, only proteolytic enzymes were insufficient in degrading proteins having a molecular weight of 4 kDa or more and less than 55 kDa.

<Examination of resolution by pH adjustment 2>
The effect on the resolution was studied by adjusting the pH of the solution during the enzymatic reaction. In this study, a proteolytic enzyme and a glycolytic enzyme were used.

  (Test Example 11) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting the RJ dilution to pH 7.5, 1.67 g of protease N and 30 mg of β-mannosidase were added and reacted at 50 ° C. for 2 hours to perform the first decomposition step. Subsequently, the RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the second decomposition step was performed by further reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  Test Example 12: An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protin P and 30 mg of β-mannosidase were added, and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, the RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the second decomposition step was performed by further reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 13) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protease N was added and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, after performing the pH adjustment step by readjusting the RJ diluted solution after the first decomposition step to pH 7.5, 30 mg of β-mannosidase was added, and further reacted at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 14): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protin P was added, and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, after performing the pH adjustment step by readjusting the RJ diluted solution after the first decomposition step to pH 7.5, 30 mg of β-mannosidase was added, and further reacted at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

  (Test Example 15): 40 ml of pure water was added to 10 g of RJ freeze-dried powder made in China and stirred until uniform to prepare an RJ dilution. After adjusting the RJ dilution to pH 7.5, 1.67 g of protease N and 30 mg of β-mannosidase were added and reacted at 50 ° C. for 2 hours to perform the first decomposition step. The RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the pH adjustment step was performed. Then, the second decomposition step was performed by reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.

(Degradation confirmation 4 of RJ-derived peptidic component by SDS-PAGE 4)
Each sample obtained in Test Examples 11 to 15 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 4.

表４より、ｐＨ調整工程を挟んで第１分解工程と第２分解工程とを行うことにより、少ない酵素添加量であっても非常に良好な分解能が発揮された。さらに、蛋白質分解酵素及び糖分解酵素の両方を用いることにより、分子量４ｋＤａ以上５５ｋＤａ未満の蛋白質の分解が極めて良好に進行することが確認された。なお、糖分解酵素処理は第１又は第２分解工程のいずれか一方のみで十分であることも分かった。

From Table 4, by performing the 1st decomposition process and the 2nd decomposition process on both sides of a pH adjustment process, very good resolution was exhibited even with a small enzyme addition amount. Furthermore, it was confirmed that the degradation of a protein having a molecular weight of 4 kDa or more and less than 55 kDa proceeds extremely well by using both a proteolytic enzyme and a glycolytic enzyme. It was also found that only one of the first and second decomposition steps is sufficient for the glycolytic enzyme treatment.

<Examination of IgE antibody response in RJ immunized rats>
Measurement of RJ-specific IgE by 48-hour passive skin anaphylaxis (PCA) reaction Test Examples 11 and 12 in which significant effects were observed in the degradation of proteins having a molecular weight of 4 kDa or more in Example 1 and tests in which no effect was observed Examples 2 and 3 were evaluated for rat IgE antibody response. Prior to the test, antiserum against RJ was obtained as follows. First, a 6-week-old female Wistar rat was purchased from Japan SLC, and quarantined for 7 to 10 days. Next, based on the method of Bradford (M. Bradford, Anal. Biochm., 72, 248-254, 1976), the protein concentrations of Test Examples 2, 3, 11, and 12 were quantified using bovine γ globulin as a standard. It was. Test Examples 2, 3, 11, 12 or 3 mg of freeze-dried RJ powder were mixed with 10 mg of aluminum hydroxide gel (Alum Adjuband), respectively, to prepare an emulsion so that the total amount was 1 ml. As a control, ovalbumin (OVA) 0.1 mg was mixed with aluminum hydroxide gel 10 mg to prepare an emulsion so that the total amount was 1 ml. Each emulsion was intraperitoneally administered 1 ml per rat, and immunized 3 times at 2-week intervals. Blood was collected from the carotid artery by 8 animals in each group 35 days after the start of immunization. The blood was centrifuged at 3000 rpm for 10 minutes to remove the clot, and antiserum was obtained. Serum was stored at −40 ° C. until measurement. In addition, during the entire breeding period, the rats in a semi-barrier system breeding room set to conditions of temperature 23 ± 2 ° C., humidity 55 ± 15%, all fresh ventilation 12 times / hour, lighting from 8 am to 12 hours, Five to six animals were housed with a PC gauge, and were fed by free intake of solid feed (CE-2, Nippon Claire) and filtered tap water.

  Next, the hair of the back of 6-week-old female Wistar rats (2 per group) was shaved, and 0.1 ml of RJ-sensitized antiserum (rat No. 1-8) obtained from each individual was placed on the back of the rat ( 8 sites / one animal) were injected intradermally. OVA-sensitized antiserum is mixed with 0.1 ml of each individual antiserum, diluted 1/2 series with physiological saline, and intradermal injection of 0.1 ml of antiserum and antiserum diluted solution into the back of the rat did. 48 hours after the injection of antiserum, lyophilized RJ powder, an equal mixture of Test Examples 2 and 3, an equal mixture of Test Examples 11 and 12, or 10 mg of OVA and 5 mg of Evans Blue dye were used as an antigen in 1 ml of physiological saline. Each liquid mixture was prepared so as to be contained, and 1 ml per mouse was intravenously administered. Thirty minutes later, the skin on the back was peeled off, and the presence or absence of blue stain at the site where the antiserum was injected intradermally was observed from the inside of the skin. The results are shown in Table 5.

表５より、ＯＶＡ感作群のＩｇＥ抗体価は予備試験で１６倍と弱かったことから、ＲＪ感作群では抗血清原液を用いてＰＣＡ反応を観察した。生じた青染斑の強さを−（反応なし）、＋（反応あり）、＋＋（直径２０ｍｍ以上の強い反応）の３段階に分けて評価した。その結果、試験例１１，１２では８例中１例のみが陽性を示し、３群中で最も反応が弱かった。また、反応強度においても凍結乾燥ＲＪ＞試験例２，３＞試験例１１，１２の順であり、試験例１１，１２のＩｇＥ抗体産生能は最も弱かった。

From Table 5, since the IgE antibody titer of the OVA sensitized group was 16 times weak in the preliminary test, the PCA reaction was observed using the antiserum stock solution in the RJ sensitized group. The intensity of the resulting blue stain was evaluated in three stages:-(no reaction), + (with reaction), and ++ (strong reaction with a diameter of 20 mm or more). As a result, in Test Examples 11 and 12, only 1 out of 8 cases was positive, and the reaction was the weakest among the 3 groups. Further, the reaction intensity was freeze-dried RJ> Test Examples 2 and 3> Test Examples 11 and 12 in this order, and the IgE antibody producing ability of Test Examples 11 and 12 was the weakest.

<Examination of IgG antibody response in RJ immunized rats>
Prior to the test, antiserum against RJ was obtained as follows. First, a 6-week-old female Wistar rat was purchased from Japan SLC, and quarantined for 7 to 10 days. Next, in the same manner as in Example 2 above, an equal mixture of Test Examples 11 and 12 or 3 mg of freeze-dried RJ powder was mixed with 10 mg of aluminum hydroxide gel, respectively, to prepare an emulsion so that the total amount was 1 ml. As a control, OVA was mixed with 10 mg of aluminum hydroxide gel in the same manner as in Example 2 to prepare an emulsion so that the total amount was 1 ml. Each emulsion was intraperitoneally administered 1 ml per rat, and immunized 3 times at 2-week intervals. Blood was collected from the carotid artery 8 animals in each group 14 days (before the second sensitization), 21 days, 28 days (before the third sensitization), 35 days and 42 days after the start of immunization. The blood was centrifuged at 3000 rpm for 10 minutes to remove the clot, and antiserum was obtained. Serum was stored at −40 ° C. until measurement.

Next, freeze-dried RJ powder, a mixture of equal amounts of Test Examples 11 and 12, or OVA in phosphate buffer (PBS; 137 mM NaCl, 8.1 mM NaHPO ₄ .12H ₂ O, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ The antigen solution adjusted to 2 mg / ml in 50 μl was added to a 96-well plate (Nunc). After incubating at room temperature for 2 hours, the antigen solution in the 96-well plate was discarded and the wells were washed 3 times with PBS. Subsequently, a PBS-skimmed milk solution was prepared so that the skimmed milk concentration was 5%, 200 μl was added per well of a 96-well plate, incubated for 1 hour at room temperature, the PBS-skimmed milk solution was discarded, and the wells were washed with PBS. The inside was washed 3 times.

  Next, starting with the antiserum diluted to 1/5 in PBS, 50 μl each of the samples diluted in series of 1/2 were added to the wells, incubated for 2 hours at room temperature, The antiserum was discarded and the wells were washed 3 times with 0.05% Tween20-PBS solution. 50 μl of an HRP-labeled anti-rat IgG, IgM, IgA antibody solution (CAPPEL # 5574.lot # 01809, 21 mg / ml) diluted 2000 times with PBS was added. The antibody solution in the wells was discarded, and the wells were washed 3 times with 0.05% Tween20-PBS solution. Finally, 100 μl of TMB solution (Wako) was added, color development reaction was performed for 30 minutes, and the absorbance at 450 nm was measured with a microplate reader. The antibody titer was defined as the maximum dilution at which the absorbance at the same wavelength of the antiserum is equivalent to that of the non-sensitized serum, with the absorbance at 450 nm of the non-sensitized serum as a control. The results are shown in Table 6.

表６より、各ＩｇＧ抗体の抗体価は経時的に増加したが、試験例１１，１２の抗体価は他の２つと比較して著しく低かった。

From Table 6, the antibody titer of each IgG antibody increased with time, but the antibody titers of Test Examples 11 and 12 were significantly lower than the other two.

<Histamine release test>
In the same manner as in Example 2, freeze-dried RJ powder, Test Examples 2, 3, 11, and 12 and ovalbumin-sensitized antiserum were obtained. Differences among individuals were eliminated by mixing equal amounts of each antiserum. A female Wistar rat weighing 200-250 g was exsanguinated and Tyrode's buffer (137 mM NaCl, 3.7 mM KCl, 1.8 mM CaCl ₂ , 1.1 mM MgCl ₂ , 11.9 mM NaHCO ₃ , 0.4 mM NaH ₂ PO ₄ , 5.6) was injected intraperitoneally. 10 ml of mM D-Glucose) was injected, and after 2 minutes of massaging, ascites was collected from the rat abdominal cavity. The cell recovery solution was collected in a tube, centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. Further, 20 ml of Tyrode buffer was added to the tube, centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. This process was performed twice to wash the cells. Suspend the cells in 5 ml of Tyrode's buffer, measure the number of cells with a hemocytometer, adjust the cell suspension so that the final cell count is 1 × 10 ⁷ cells / ml, and add 0 to a 24-well plate. Dispensed 5 ml each.

Either non-sensitized serum, RJ-sensitized serum, or 0.5 ml of Tyrode's buffer was added to the cell suspension and incubated at 36 ° C. for 3 hours in a CO ₂ incubator. The cell suspension was collected from the 24-well plate, 2 ml of Tyrode buffer was added and centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. This process was performed twice to wash the cells. Cells were suspended in 0.7 ml of Tyrode's buffer and transferred to a 24-well plate. In the cell suspension, freeze-dried RJ powder, an equal amount mixture of Test Examples 2 and 3, or an equal amount mixture of Test Examples 11 and 12, respectively, 0.25 mg / ml, Tyrode buffer 0.2 ml, and 0.1 mg / ml 0.1 ml of phosphatidylserine (PS solution) was added and incubated at 36 ° C. for 20 minutes in a CO ₂ incubator. After stopping the reaction with ice cooling, the cell suspension was collected from the 24-well plate, centrifuged (1200 rpm, 5 min), and 0.8 ml of the supernatant was obtained with Pipetman, which was used as a sample for histamine determination. Histamine quantification was performed by ELISA using Histamine Enzyme Immunoassay Kit from SPI-bio. The results are shown in Table 7.

表７より、感作抗血清によるヒスタミン遊離量はＯＶＡ＞凍結乾燥ＲＪ＞試験例２，３の等量混合物＞試験例１１，１２の等量混合物の順となり、試験例１１，１２の等量混合物におけるヒスタミン放出量が最も少なかった。

From Table 7, the amount of histamine released by the sensitized antiserum was in the order of OVA> freeze-dried RJ> equal mixture of test examples 2 and 3> equal mixture of test examples 11 and 12, and equivalent amounts of test examples 11 and 12. The amount of histamine released in the mixture was the least.

<Allergen test using human antibody-producing cells>
An allergen test using human blood was performed. Prior to the test, blood was provided from allergic patients YY, allergic patients YM, and healthy persons AO who were confirmed to have allergies by RJ. These blood samples were centrifuged at 3000 rpm for 10 minutes to remove clots and used as serum. Serum was stored at −40 ° C. until measurement.

  Freeze-dried RJ powder, equal volume mixture of Test Examples 2 and 3, or equal volume mixture of Test Examples 11 and 12, each adjusted to a solid content of 15 mg / ml with PBS, 100 μl in a 96-well plate (Nunc) Added. After incubating at room temperature for 1 hour, the antigen solution in the 96-well plate was discarded, and the wells were washed 3 times with PBS. A PBS-BSA solution was prepared so that the fetal bovine serum (BSA) concentration was 1%, 200 μl per well of a 96-well plate was added and incubated at room temperature for 1 hour, and then the PBS-BSA solution was discarded.

  Human serum was serially diluted 1/2, 1/5, 1/10 with PBS containing 1% BSA, and 50 μl was added to the wells, followed by incubation at room temperature for 2 hours. The antiserum in the wells was discarded and the wells were washed 3 times with 0.05% Tween20-PBS solution. 50 μl of HRP-labeled anti-human IgE (0.5 mg / ml) diluted 500-fold with PBS containing 1% BSA was added. The antibody solution in the well was discarded, and the well was washed 3 times with 0.05% Tween20-PBS solution. Detection was performed using a Protein Detector ELISA Kit, and the absorbance at 405 nm was measured with a microplate reader. In addition, 1% BSA was used instead of human serum as a blank. The results are shown in Table 8.

表８より、凍結乾燥ＲＪ、試験例２，３の等量混合物に対する反応性は、いずれもＹＭ＞ＹＹ＞ＡＯとなり、健常者であるＡＯは各ＲＪにほとんど反応しなかった。また、試験例１１，１２の等量混合物に対する反応性は被験者全てにおいて全く見られなかった。この結果より、抗原性は未処理の凍結乾燥ＲＪ＞試験例２，３の等量混合物＞試験例１１，１２の等量混合物の順となり、試験例１１，１２の抗原性は限りなく低減されていた。

From Table 8, the reactivity with respect to the equal amount mixture of freeze-dried RJ and Test Examples 2 and 3 was YM>YY> AO, and AO as a healthy person hardly reacted to each RJ. Moreover, the reactivity with respect to the equal amount mixture of Test Examples 11 and 12 was not seen at all in all subjects. From this result, the antigenicity is in the order of untreated lyophilized RJ> equal mixture of test examples 2 and 3> equal mixture of test examples 11 and 12, and the antigenicity of test examples 11 and 12 is reduced as much as possible. It was.

<Amino acid absorption ability comparison test by inversion intestine test>
Guinea pigs fasted overnight were sacrificed by exsanguination and the intestine was removed. The intestinal tract was inverted and cut into three equal parts: the upper intestine, the middle intestine, and the lower intestine. One end of the inverted intestinal tract was tied with a thread, and 2 ml of 0.9% NaCl solution containing 0.3% glucose was injected into it, and the other end was tied to form an inverted sac. The inverted sac was placed in a flask containing 30 ml of a 5% freeze-dried RJ, an equal amount mixture of Test Examples 2 and 3, or an equal amount mixture solution of Test Examples 11 and 12, and aerated with oxygen for 10 minutes. Thereafter, the mixture was incubated at 37 ° C. for 1 hour, and the intestinal fluid was taken out and used as a sample for amino acid absorption ability test.

Using these samples, the amount of amino acids was measured by the Folin & Ciocalteu method. That is, each sample or 0.5 ml of L-tyrosine standard solution was placed in a centrifuge tube, and 0.5 ml of 20% trichloroacetic acid was added thereto. After thorough mixing and centrifugation, 0.1 ml of the supernatant was collected and placed in a test tube. To this, 5 ml of 2% Na ₂ CO ₃ /0.1N NaOH was added, 0.5 ml of phenol reagent was further added and mixed, and the mixture was allowed to stand for 30 minutes. Absorbance at 660 nm was measured, a calibration curve was prepared with L-tyrosine, and the amount of amino acid absorption (as L-tyrosine amount) was calculated from the absorbance of each sample. Since there was no difference in the absorption rate of amino acids in the upper, middle and lower parts of the intestinal tract, the average value thereof was taken (n = 3) and the L-tyrosine content was calculated. For L-tyrosine, a calibration curve was prepared in advance to confirm linearity. The results are shown in Table 9.

表９より、試験例２，３の等量混合物は凍結乾燥ＲＪの２．２倍、試験例１１，１２の等量混合物は試験例２，３の等量混合物の２．９倍のアミノ酸吸収量があった。これらの状況から、試験例２，３は凍結乾燥ＲＪの２〜３倍の吸収能、試験例１１，１２は４〜６倍の吸収能があることが認められた。

From Table 9, the equivalence mixture of Test Examples 2 and 3 was 2.2 times the freeze-dried RJ, and the equivalence mixture of Test Examples 11 and 12 was 2.9 times the amino acid absorption of the equivalence mixture of Test Examples 2 and 3. There was an amount. From these situations, it was confirmed that Test Examples 2 and 3 have an absorption capacity 2 to 3 times that of lyophilized RJ, and Test Examples 11 and 12 have an absorption capacity 4 to 6 times.

<Molecular weight analysis of enzyme-treated RJ>
About the equal amount mixture of Test Examples 2 and 3, or the equal amount mixture of Test Examples 11 and 12, the molecular weight was measured by HPLC using a gel filtration column. HPLC conditions were as follows: column; Superdex-peptide 10/300 GL (1.0 cm × 30 cm), mobile phase; 30% CH ₃ CN, 0.1% TFA / H ₂ O, pump; TOYOSODA CCPM, flow rate; 0.3 ml / Min, detection wavelength; 220 nm. The result of the equivalence mixture of Test Examples 2 and 3 is shown in FIG. 1B, and the result of the equivalence mixture of Test Examples 11 and 12 is shown in FIG. FIG. 1 (a) shows molecular weight markers, in which the molecular weight is 12.5 kDa (retention time Y = 26.958), molecular weight 6.5 kDa (retention time Y = 31.242), molecular weight 1274 Da (retention time Y = 38.993) in order from the left peak. ), Molecular weight 393 Da (retention time Y = 49.5), and molecular weight 132 Da (retention time Y = 56.435). From the calibration curve obtained by semi-log plotting the result of gel filtration shown in FIG. 1 (a), Y = 87.495-6.4744 Ln (X), R ² = 0.990, Y; retention time, X; It was molecular weight.

  In the chromatogram shown in FIG. 1 (b), a retention time Y = 33.008 peak (molecular weight 4523Da) and a retention time Y = 42.09 peak (molecular weight 1111Da) were confirmed, and the chromatogram shown in FIG. 1 (c). Then, the peak with the molecular weight of 4523 Da and the peptide component disappeared, and the peak with the molecular weight of 1111 Da remained. Furthermore, all of Test Examples 11 and 12, including a protein band having a molecular weight of 4 kDa or more by SDS-PAGE (Coomassie staining and silver staining) in a preliminary test as shown in Table 4 above, for example, It has been confirmed that no protein band was detected. Therefore, it was confirmed in Test Examples 11 and 12 that there was no peptide component having a molecular weight of 4.5 kDa and no peak having a molecular weight of 1.2 kDa or more.

<Examination of anti-fatigue effect>
In order to confirm the anti-fatigue effect of the lyophilized RJ powder and the equivalent mixture of Test Examples 11 and 12, the duration of the swimming exercise was measured in a state where a forced swimming exercise load was applied to the mouse. Five-week-old ddy male mice (body weight of about 28 g) were purchased from Japan SLC, and were allowed to freely ingest food and water except for the test time. Next, each sample was orally administered to mice once a day for 3 days at the doses shown in Table 10 below, and forced swimming for 5 minutes in a water bath (diameter 19 cm, depth 27 cm, water temperature 25 ° C.) on the 4th day ( The first time) was tried. Immediately thereafter, each sample was orally administered to mice at the doses shown in Table 10 below, and after 60 minutes, the second forced swimming was tried for 5 minutes. The time during which the mouse was immobile in the water during the second forced swimming was measured, and the cumulative immobility time was used as an index of the anti-fatigue effect. Each sample was suspended in distilled water, and only distilled water was orally administered to the control group. The results are shown in Table 10.

表１０より、凍結乾燥ＲＪ及び試験例１１，１２の等量混合物はいずれも、対照に対し抗疲労効果が有意に発揮されるとともに、その強さはほぼ同程度であった。

As shown in Table 10, both the freeze-dried RJ and the equimolar mixture of Test Examples 11 and 12 exhibited a significant anti-fatigue effect with respect to the control, and the strength thereof was almost the same.

(A) to (c) are all chromatograms showing the results of Example 7.

Claims (5)

A low allergen royal jelly with reduced allergenicity,
A low allergenized royal jelly comprising a peptide component derived from 10-hydroxydecenoic acid and royal jelly, wherein the peptide component is composed of a component having a molecular weight of 4.5 kDa or less. A method for producing the reduced allergenized royal jelly according to claim 1,
A method for producing a low allergenized royal jelly, comprising subjecting royal jelly to proteolytic enzyme treatment and / or glycolytic enzyme treatment. The method for producing a low allergenized royal jelly according to claim 2, wherein the proteolytic enzyme treatment is performed at pH 6.5 to 8.0 using endo-type neutral protease. The proteolytic enzyme treatment includes a first decomposition step of subjecting a royal jelly diluent adjusted to pH 6.5 to 8.0 to an endo-type neutral protease treatment, and a pH of the royal jelly diluent after the first decomposition step. A pH adjustment step of adjusting the pH to 6.5 to 8.0, and a second decomposition step of subjecting the royal jelly diluted solution after the pH adjustment step to an endo-type neutral protease treatment, A process for producing the allergen-reduced royal jelly as described. A method for producing a low allergenized royal jelly, comprising subjecting a royal jelly diluted with water or a buffer to a royal jelly diluted solution, which is subjected to proteolytic enzyme treatment and β-mannosidase treatment,
The method for producing a low allergenized royal jelly, wherein the proteolytic enzyme treatment is performed at pH 6.5 to 8.0 using endo-type neutral protease.

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