JP3994120B1 - Method for producing low allergenized royal jelly - Google Patents

Abstract

The present invention provides a method for producing a low allergenized royal jelly in which allergenicity is reduced while suppressing a decrease in useful physiological action of royal jelly.
A method for producing a royal jelly with reduced allergenicity, characterized in that raw royal jelly is treated with an alkaline peptidase having an endopeptidase action and an exopeptidase action.
[Selection] Figure 1

Description

  The present invention relates to a method for producing a reduced allergenic royal jelly.

  Raw royal jelly is a useful natural material, but it is known that it can cause allergic reactions.

  As a low allergenized royal jelly, there is known a low allergenized royal jelly in which allergenicity is reduced to such an extent that it is not substantially expressed by subjecting the royal jelly to a glycolytic enzyme treatment and a proteolytic enzyme treatment (Patent Document 1). reference). Patent Document 2 discloses that allergenicity of royal jelly is reduced using endo-type neutral peptidase. However, royal jelly treated with these methods still has allergenicity, and there is a need for royal jelly with further reduced allergenicity.

Patent Document 3 describes that a protein causing food allergy is treated with both endopeptidase and exopeptidase.
JP 2002-127715 A JP 2005-287411 A JP 2001-333794 A

  An object of this invention is to provide the manufacturing method of the low allergen-ized royal jelly which reduced allergenicity, suppressing the fall of the useful physiological activity which fresh royal jelly has.

  As a result of repeated investigations to achieve the above-mentioned objective, it is surprisingly useful to treat raw royal jelly with a single alkaline peptidase having both an endopeptidase action and an exopeptidase action. It has been found that a low allergenized royal jelly can be obtained with reduced allergenicity while maintaining excellent physiological activity.

That is, this invention provides the manufacturing method of the following low allergen royal jelly.
1. A method for producing a royal jelly with reduced allergenicity, characterized in that raw royal jelly is treated with an alkaline peptidase having an endopeptidase action and an exopeptidase action.
2. Item 2. The method according to Item 1, wherein the treatment is performed under alkaline conditions.
3. Item 3. The method according to Item 1 or 2, wherein the alkaline peptidase is a Streptomyces griseus-producing peptidase.

  According to the present invention, it has been possible to solve the difficult problems of maintaining the usefulness of raw royal jelly and reducing allergenicity.

  When a protein is treated with peptidase, if an alkaline protease having an endopeptidase action and an exopeptidase action is used, it will be cleaved thoroughly both inside and at the end of the peptide. If you want to eliminate the antigenicity of the protein that causes food allergies, as in Patent Document 3, there is no problem with processing the protein in this way, but endopeptidase and exopeptidase are used together for useful substances such as raw royal jelly. In this case, it is considered that the physiological activity is greatly reduced or disappears.

  However, according to the present invention, an unpredictable result that not only the antigenicity can be lowered by the alkaline protease having endopeptidase action and exopeptidase action but also useful physiological activity is maintained.

  That is, the royal jelly-treated product of the present invention does not show a loss of decenoic acid content, which is said to be the main physiologically active component, as shown in the experimental examples described below, and further exhibits an antihypertensive action and an antidepressant action. It has the characteristic that there exists the outstanding effect that property is suppressed notably.

  In the following, 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. The main physiologically active components in 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, various minerals and the like. As the physiological activity and pharmacological action of RJ, antibacterial action, immune enhancing action, antidepressant action, antitumor 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.

  Raw RJ can be used as a raw material used for production of low allergenized RJ. The production area of RJ may be any of Japan, China, Brazil, European countries, Oceania countries, the United States, etc.

  In the present invention, raw RJ is treated with an alkaline peptidase having an endopeptidase action and an exopeptidase action.

Preferable examples of an enzyme having both exopeptidase activity and endopeptidase activity include, for example, Streptomyces griseus production peptidase (trade name: actinase AS), Aspergillus oryzae production peptidase (trade name) : Protease A, flavorzyme), Aspergillus melleus-produced peptidase (trade name: Protease P).
Since the enzyme treatment of raw RJ according to the present invention can be carried out by a one-step enzyme reaction, it is preferable from the viewpoint of the effect of reducing the allergen and the ease of operation. The alkaline peptidase produced by both the endopeptidase action and the exopeptidase action preferably has a sufficient endopeptidase action.

  The amount of alkaline protease having endopeptidase action and exopeptidase action on raw RJ varies depending on the raw RJ concentration, enzyme titer, reaction temperature, and reaction time, but generally 50 to 10,000 per gram of raw RJ protein. Hydrolysis is carried out by adding the enzyme in the proportion of action units. The enzyme may be added all at once, or may be added in small portions.

  The pH of the raw RJ to be treated with alkaline peptidase is selected from the range of pH 7.5 to 10, preferably pH 7.8 to 9, corresponding to the optimum pH of the enzyme used. Specifically, before the enzyme is added to the peptidase solution, the pH is 7.5 to 10, preferably within the range of pH 7.8 to 9, depending on the type of enzyme used. It is carried out by adjusting to. In this case, examples of the alkaline agent include sodium hydroxide, potassium hydroxide, and potassium carbonate, and examples of the buffer include a phosphate buffer and a citrate buffer.

The temperature of the alkaline peptidase treatment is not particularly limited, and is selected from a range that can be put to practical use including an optimum temperature range where the enzyme action is expressed, that is, a range of usually 30 to 70 ° C. By maintaining the temperature at a temperature lower or higher than the optimum temperature of the peptidase, for example, in the range of 50 to 60 ° C., spoilage in the peptidase treatment step can be prevented.
The peptidase treatment time depends on the reaction conditions such as the type of enzyme used, reaction temperature, pH, etc., and is not particularly limited.

  The peptidase treatment is stopped by inactivating or removing the peptidase. The deactivation operation can be performed by heat treatment (for example, at 85 ° C. for 15 minutes).

EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1
200 g of raw RJ was weighed into a 500 ml beaker, 100 ml of ion exchanged water was added and stirred until uniform to prepare an RJ dilution. 2N NaOH aqueous solution was added to adjust the pH of the RJ dilution to 8.7-8.9. Next, a solution obtained by dissolving 2 g of actinase AS (Kaken Pharmaceutical Co., Ltd.) having both endopeptidase action and exopeptidase action in 20 ml of ion-exchanged water is added to the RJ diluent, and ion-exchanged water is further added to a total amount of 200 ml. It was added to become. The reaction mixture was reacted at 50 ° C. (a constant temperature water bath) for 4 hours while stirring with a propeller to effect hydrolysis. The enzyme was inactivated by raising the temperature of the constant temperature bath to 80 ° C., and then cooled with water.

Example 2
3 g of raw RJ was weighed into a 50 ml Erlenmeyer flask, 1 ml of ion exchanged water was added and stirred until uniform to prepare an RJ dilution. The pH of the RJ dilution was adjusted to 7.8-8.0 by adding 10N NaOH aqueous solution. Next, 30 mg of protease P (Amano Enzyme) having both endopeptidase action and exopeptidase action was added to the RJ dilution, and ion exchange water was added so that the total amount of ion exchange water was 3 ml. The reaction mixture was reacted at 40 ° C. for 16 hours while shaking in a constant temperature water bath to effect hydrolysis. The enzyme was inactivated by raising the temperature of the constant temperature bath to 80 ° C., and then cooled with water.

Example 3
An RJ enzyme degradation product was obtained in the same manner as in Example 2 except that protease A (Amano Enzyme) having both endopeptidase action and exopeptidase action was used as the enzyme instead of protease P.

Comparative Example 1
200 g of raw RJ was weighed into a 500 ml beaker, 100 ml of ion exchanged water was added and stirred until uniform to prepare an RJ dilution. 2N NaOH aqueous solution was added to adjust the pH of the RJ dilution to 8.7-8.9. Next, a solution obtained by dissolving 3.4 g of protease N (Amano Enzyme), which is an endopeptidase, and 60 mg of β-mannosidase (Sumiteam ACH, manufactured by Shin Nippon Chemical Industry Co., Ltd.) in 20 ml of ion-exchanged water is added to the RJ diluent, and ion exchange Water was added so that the total amount of ion-exchanged water was 200 ml. The reaction mixture was reacted at 50 ° C. (a constant temperature water bath) for 4 hours while stirring with a propeller to effect hydrolysis. The enzyme was deactivated by raising the temperature of the constant temperature water bath to 80 ° C., and then cooled with water to obtain an RJ enzyme degradation product.

Comparative Example 2
5 g of raw RJ was weighed into a 100 mL Erlenmeyer flask, 5 mL of ion exchange water and 50 mg of Morsin F (Kikkoman) were added, and the mixture was stirred until uniform to prepare an RJ dilution. The reaction mixture was reacted at 40 ° C. (a constant temperature water bath) for 16 hours and 24 hours while stirring with a stirrer to effect hydrolysis. The enzyme was inactivated by raising the temperature of the constant temperature bath to 80 ° C., and then cooled with water.

Comparative Example 3
The enzyme treatment was performed in the same manner as described above except that Morsin F was changed to FA-107-1 (Sankyo Lifetech).

Experimental example 1
<I> Confirmation of disappearance of antigenic protein of peptidase-treated product of raw royal jelly The degradation products obtained in Examples 1 to 3 were subjected to immunoblotting to confirm the disappearance of the antigenic protein. Each sample was diluted 3 times with 3x sample buffer (187.5 mM Tris-HCl pH 6.8, 6% SDS, 75% glycerol, 0.03% bromophenol blue, 1.5M 2-mercaptoethanol) and heated at 95 ° C for 5 minutes did. Thereafter, SDS-PAGE was performed using a 15% acrylamide gel. SDS-PAGE Standards, Low Range (BIO RAD) was used as a molecular weight marker. The protein developed on the gel is transferred to a PVDF membrane (BIO RAD) using a semi-dry blotting apparatus and immersed in a blocking buffer (2% skim milk, 10 mM Tris-HCl pH 7.5, 100 mM sodium chloride, 0.05% Tween 20). Shake for 1 hour at room temperature. Thereafter, the membrane was immersed in patient serum diluted with blocking buffer and allowed to stand at 4 ° C. overnight. Further, it was immersed in an anti-human IgE antibody (KPL) diluted with a blocking buffer and allowed to stand at 37 ° C. for 1 hour. Finally, using an ECL plus kit (Amersham LIFE SCIENCE), the membrane was immersed in a luminescent solution prepared according to the attached protocol and detected by ECL Mini-Camera (Amersham LIFE SCIENCE).

  It was visually confirmed that the antigen protein that reacts with the patient serum disappeared for all the samples tested.

In addition, a manual describing the contents of this study was distributed to the serum donors, and after obtaining a written consent to participate in the voluntary volition of the subject after fully explaining the purpose and content of the study. Only serum was used in this study.
<II> Immunoblot of raw royal jelly treated with acidic protease and alkaline protease>
The RJ enzyme degradation products obtained in Comparative Examples 2 and 3 and Example 1 were subjected to immunoblotting according to the above <I>.
The results are shown in Table 1.

* The result did not change whether the reaction time was 16 hours or 24 hours.
From the results shown in Table 1, it became clear that treatment of raw royal jelly with an acid protease cannot be anti-allergenic.

Experimental example 2
<Histamine release test>
The amount of histamine released by the CAST method (cellular antigen stimulation test) for the untreated RJ, the RJ enzyme degradation product obtained in Example 1, the comparative example, the allergen mix (positive control), and the anti-IgE receptor antibody (positive control). It was measured.

  Specifically, the amount of histamine released was measured by applying a raw RJ, an enzyme hydrolyzate thereof or a positive control to a blood sample of a subject who had experienced an allergic reaction to the raw RJ. The study will be distributed only to those who have received a written statement describing the content of the study, fully explained the purpose and content of the study, and obtained consent to participate by the subject's voluntary will. Went.

The measurement of free histamine was performed using a commercially available ELISA kit (Histamine ELISA (manufactured by IMMUNO-BIOLOGICAL LABORATORIES)) The allergen mix includes the following allergens.
Allergen mix: Anemonefish, Yellow-faced Plover, Barley, Prunus serrata, Nagahagusa, Shiragayaya, Rye, White birch, Hazel, Mugwort, Psyllium, Alternaria, Butterfly mite, Pepper mite, Pepper mushroom, Egg white, Milk white, Milk
The results are shown in Table 2 and FIG.

EC ₅₀ (50% effective concentration) in Table 2 and FIG. 1 means the concentration of the test substance that releases 50% of the maximum value of histamine.

  From the above results, it was revealed that the antigenicity of RJ can be effectively reduced by using both endopeptidase action and exopeptidase action together.

Experimental example 3
<Comparison of the amount of decenoic acid between raw RJ and RJ ethanol precipitate>
The ethanol precipitate of RJ was prepared by adding 300 mL of 90% ethanol to 100 g of raw RJ, stirring at room temperature for about 1 hour, centrifuging at 12,000 × g for 30 min, and then freeze-drying.

  About the raw RJ and RJ ethanol precipitate, the amount of decenoic acid was measured by HPLC using a reverse phase column. HPLC conditions are: Column; YMC-Pack ODS-AM (inner diameter 4.6 mm, length 150 mm), mobile phase; 10 mM phosphate buffer and methanol mixture (56:44, pH 2.6), flow rate: 1 ml / min Detection wavelength: 210 nm. The results are shown in Table 3.

By carrying out ethanol precipitation, the decenoic acid content was reduced to about 1/8.

Experimental Example 4
<Evaluation of antidepressant action of enzyme-treated RJ>
In order to confirm the antidepressant effect for the untreated RJ and the RJ enzymatic degradation product obtained in Example 1, the fear-condition-induced immobility time of mice was measured.
Method Mice were subjected to electrical stimulation loading with a fair condition measurement device (Acti Metrics), and then the test specimen was administered for 7 days. After the administration, it was again placed in the fair condition measuring apparatus, and the immobility time of 4 minutes was measured under the condition without electrical stimulation. The results are shown in Table 4 and FIG.

  The vehicle control group showed a significant increase in immobility time from 0 to 240 seconds compared to the vehicle control (no electrical stimulation) group. In addition, the untreated and Example 1 treated RJ showed a significant reduction in immobility time compared to the vehicle control group. Furthermore, no decrease in action due to enzyme treatment was observed.

Experimental Example 5
<ACE inhibition by enzyme-treated RJ>
The RJ enzyme degradation product obtained in Example 1 and Comparative Example was measured for ACE inhibitory action.
○ Reagent preparation Borate buffer (pH 8.3): 200 mM boric acid (H ₃ BO ₃ ), 50 mM sodium tetraborate (Na2B4O7)
Substrate solution: 200 mM boric acid (H ₃ BO ₃ ), 50 mM sodium tetraborate (Na ₂ B ₄ O ₇ ), 1 M sodium chloride (NaCl)
Substrate solution: Bz-Gly-His-Leu · H ₂ O (Peptide Institute) was dissolved in a substrate solution to 12.5 mM.
Enzyme solution: Angiotensin converting enzyme (Sigma) derived from rabbit lung was dissolved in borate buffer to a concentration of 25 mU / mL.
○ Experimental procedure 50 µL of the enzyme solution was added to 25 µL of the sample solution, stirred, and allowed to stand at 37 ° C for 5 minutes. Further, 50 μL of the substrate solution was added and stirred, and allowed to stand at 37 ° C. for 1 hour. A test solution was prepared by adding 125 μL of 0.5 N hydrochloric acid solution to this solution and stirring to stop the reaction. On the other hand, 25 μL of sample solution, 50 μL of enzyme solution, and 125 μL of 0.5 N hydrochloric acid solution were mixed and allowed to stand at 37 ° C. for 5 minutes. Then, 50 μL of the substrate solution was added and stirred, and left at 37 ° C. for 1 hour. This was used as a blank solution. Then, add 750 μL each of ethyl acetate to the test solution and blank solution, vigorously stir for 15 seconds using a vortex, then centrifuge (room temperature, 2,000 rpm for 10 minutes), and separate the upper layer (ethyl acetate layer). 250 μL each was collected. After completely removing ethyl acetate by heating under reduced pressure, the precipitate was dissolved in 500 μL of 1 M sodium chloride solution. Separately, a solution operated in the same manner using purified water instead of the sample solution was used as a control solution. About these liquids, the light absorbency in wavelength 228nm was measured with the ultraviolet visible absorptiometry.
・ Calculation of ACE inhibition rate

Ac: Absorbance of the liquid that was operated by adding water instead of the sample solution
As: Absorbance of sample solution
Abc: Absorbance of the blank solution that was prepared by adding water instead of the sample solution
Abs: Absorbance of blank solution of sample solution The results are shown in Table 5 and FIG.

  Example 1 treated RJ was found to have a higher ACE inhibitory effect than Comparative Example treated RJ.

Test results of histamine release by enzyme-treated RJ Evaluation of antidepressant action of enzyme-treated RJ Comparison of ACE inhibitory action by enzyme-treated RJ

Claims (2)

A method for producing a royal jelly with reduced allergenicity, comprising using raw royal jelly with an alkaline peptidase having an endopeptidase action and an exopeptidase action derived from Streptomyces griseus or Aspergillus oryzae , pH 7.8-9, 40- A process characterized in that the treatment is carried out under reaction conditions of 50 ° C. The method according to claim 1, wherein the alkaline peptidase is a Streptomyces griseus-producing peptidase.

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