JP7218961B2 - Method for producing enzymatic hydrolyzate of royal jelly and enzymatic hydrolyzate of royal jelly - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an enzymatic hydrolyzate of royal jelly and an enzymatic hydrolyzate of royal jelly. Specifically, the present invention relates to a method for producing a royal jelly enzymatic hydrolyzate with reduced allergenicity and suppressed browning. The present invention also relates to a method for reducing allergenicity and suppressing browning of a royal jelly enzymatic decomposition product. Furthermore, the present invention relates to a royal jelly enzymatic hydrolyzate that can have antioxidant activity.

Royal jelly is a useful natural material, but it is also known to cause allergic reactions. Therefore, various methods for reducing the amount of allergens by degrading or reducing the molecular weight of proteins that can be allergens have been investigated.

For example, a method for reducing the amount of allergens in royal jelly, in other words, a method for preparing low-allergen royal jelly, is a method of one-step enzymatic treatment of royal jelly with an alkaline peptidase having both endopeptidase and exopeptidase actions (Patent Document 1). ) have been proposed.

However, royal jelly that has been degraded by peptidase to make it hypoallergenic (royal jelly enzymatic hydrolyzate) is more susceptible to temperature and humidity than royal jelly that has not been treated with enzymes, and is susceptible to harsh conditions such as heating and humidification. Underneath, it is colored and browning progresses. Such coloring is considered to be due to the Maillard reaction that occurs between the amide groups of peptides or amino acids fragmented by enzymatic decomposition and the anomeric carbons of reducing sugars.

Various methods have been reported for suppressing such coloring of enzyme-treated royal jelly (Patent Documents 2 to 6). Patent Document 2 reports that browning caused by the enzyme treatment is significantly suppressed by performing the enzyme treatment under high pressure conditions of 40 to 600 MPa. Patent Document 3 reports that discoloration of the appearance over time can be prevented by dissolving royal jelly and at least one selected from starch and dextrin having a dextrose equivalent of 18 or less in a solvent, followed by freeze-drying. ing. Patent Documents 4 and 5 report that yeast fermentation treatment of royal jelly suppresses discoloration of the appearance of royal jelly over time. Patent Document 6 reports that enzymatic decomposition using an acidic protease having an optimum pH near the pH of an aqueous suspension of royal jelly results in less yellowness than conventional enzymatic methods. .

However, in the methods described in Patent Documents 2 to 5, in order to suppress browning, high pressure conditions (Patent Document 2), addition of starch and dextrin (Patent Document 3), fermentation treatment with yeast (Patent Documents 4 and 5) ) are required respectively.

Further, as described in Patent Document 6, Patent Document 1 describes that when royal jelly is enzymatically treated with an acid protease, allergenicity increases.

Furthermore, proteins contained in royal jelly are degraded into peptides or free amino acids by enzymatic degradation, and although it is expected that the composition of the peptides obtained will differ depending on the enzymes used (Patent Documents 7 and 8), no such proteins have been produced so far. The composition of the peptide and the details of its functionality have not been clarified.

JP 2007-295919 A JP 2009-254363 A JP 2011-152103 A JP 2018-108116 A JP 2018-102292 A JP-A-2003-334003 JP 2005-006533 A JP-A-2005-255670

In the methods described in Patent Documents 2 to 5, it is not described that browning can be suppressed during and after the production of enzymatic decomposition products of royal jelly. In addition, the method described in Patent Document 6 cannot reduce allergenicity because an acidic protease is used.

The present invention provides a method for producing a royal jelly enzymatic hydrolyzate in which allergenicity is reduced and browning during and after production is suppressed, as well as a royal jelly enzymatic hydrolyzate with reduced allergenicity and suppression of browning during and after production. The purpose is to provide a method.

Royal jelly has long been known to be effective for indefinite complaints such as hypertension, diabetes, menopausal symptoms, and pain, but its physiologically active substances are largely unknown. In the present invention, by enzymatically decomposing royal jelly proteins, it is possible to provide a royal jelly enzymatic hydrolyzate that is less allergenic, suppresses browning during and after production, and contains at least a peptide having antioxidant activity. With the goal.

As a result of intensive research to achieve the above object, the present inventors have found that treating with a peptidase having endopeptidase and exopeptidase actions can reduce allergenicity and reduce brown spots during and after production. We obtained knowledge that changes can be suppressed.

The present invention has been completed through further studies based on these findings. It provides a method.

(I) Method for producing enzymatic hydrolyzate of royal jelly
(I-1) A royal jelly enzymatic hydrolyzate having reduced allergenicity and suppressed browning during and after production, characterized by treating royal jelly with a peptidase having endopeptidase activity and exopeptidase activity. manufacturing method.
(I-2) The method according to (I-1), wherein the peptidase is an Aspergillus oryzae-produced peptidase.
(I-3) The method according to (I-1) or (I-2), wherein the optimum temperature of the peptidase is 50°C or higher.

(II) Method for reducing allergenicity of royal jelly enzymatic decomposition product and suppressing browning during and after production
(II-1) A method for reducing allergenicity of a royal jelly enzymatic hydrolyzate and suppressing browning during and after production, which comprises treating royal jelly with a peptidase having endopeptidase and exopeptidase actions.
(II-2) The method according to (II-1), wherein the peptidase is an Aspergillus oryzae-produced peptidase.
(II-3) The method according to (II-1) or (II-2), wherein the optimum temperature of the peptidase is 50°C or higher.

(III) Royal jelly enzymatic degradation product
(III-1) A royal jelly enzymatic hydrolyzate produced by any one of the methods (I-1) to (I-3), wherein 100 mg of Tyr-Asp is added to 100 g of the dried enzymatic hydrolyzate of royal jelly. A royal jelly enzymatic decomposition product containing the above.
(III-2) An enzymatic hydrolyzate of royal jelly containing 100 mg or more of Tyr-Asp per 100 g of dried enzymatic hydrolyzate of royal jelly.
(III-3) The royal jelly of (III-1) or (III-2) further containing 40 mg or more of Val-Pro and 0.5 mg or more of Met-Pro per 100 g of dried enzymatic decomposition product of royal jelly. enzymatic decomposition product.
(III-4) Furthermore, one or more selected from the group consisting of Pro-Ile and/or Pro-Ile-Leu, Pro-Val, Ile-Pro and/or Ile-Leu-Pro, and Pro-Met The enzymatic hydrolyzate of any one of (III-1) to (III-3) containing a peptide.

(IV) Food compositions, quasi-drugs or pharmaceutical compositions
A food composition, quasi-drug or pharmaceutical composition comprising the royal jelly enzymatic hydrolyzate of any one of (III-1) to (III-4).

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for producing a royal jelly enzymatic hydrolyzate in which browning caused by peptidase decomposition during and after production is suppressed while realizing improved safety by reducing allergenicity. . Moreover, according to the present invention, it is possible to provide a method for reducing the allergenicity of royal jelly enzymatically decomposed products and suppressing browning during and after production. Furthermore, according to the present invention, a royal jelly enzymatic hydrolyzate having at least antioxidant activity can be provided. The royal jelly enzymatic hydrolyzate of the present invention can also be effective against diabetes and hypertension.

1 is a graph showing the results of histamine release tests in Examples and Comparative Examples. The vertical axis indicates the histamine release amount (ng/mL) of the subject. 2 is a graph showing the results of browning evaluation of Examples and Comparative Examples. The vertical axis indicates luminance (%) in the HSL color space.

The present invention will be described in detail below.

As used herein, the term "comprise" includes both the meaning of "essentially consist of" and the meaning of "consisting of".

(1) Method for Producing Enzymatically Degraded Product of Royal Jelly The method for producing enzymatically degraded product of royal jelly of the present invention is a method for producing an enzymatically degraded product of royal jelly in which allergenicity is reduced and browning during and after production is suppressed. and treating royal jelly with a peptidase having an endopeptidase action and an exopeptidase action.

Royal jelly is a milky white jelly-like substance produced by mixing the secretions secreted from the hypopharyngeal and mammary glands of 3- to 12-day-old worker bees among honeybees. Main physiologically active ingredients in royal jelly include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as "decenoic acid") unique to royal jelly, proteins, lipids, sugars, B vitamins, Vitamins such as folic acid, nicotinic acid and pantothenic acid, and various minerals are included. As physiological activities and pharmacological activities of this royal jelly, antibacterial activity, immunoenhancing activity, antidepressant activity, antitumor activity, anti-inflammatory activity, blood flow increasing activity and the like are known. It has also been reported to reduce the side effects of anticancer drugs and to prolong life after radiation injury.

The royal jelly used in the present invention is not particularly limited, and may be, for example, raw royal jelly or a processed royal jelly product obtained by processing raw royal jelly.

Processed royal jelly products include royal jelly concentrates or dilutions obtained by concentrating or diluting raw royal jelly, royal jelly powder obtained by drying and pulverizing raw royal jelly, aqueous extracts obtained by extracting royal jelly with water or water-containing ethanol, or organic solvent extracts. etc. can be mentioned. The treated royal jelly may be one subjected to multiple treatments.

A royal jelly concentrate can be obtained, for example, by removing water from raw royal jelly. Royal jelly dilutions can be obtained, for example, by adding water to raw royal jelly.

Royal jelly powder can be obtained, for example, by pulverizing fresh royal jelly by methods known in the art such as freeze drying and spray drying. As a drying method, any known method employed in general food processing can be used, such as natural drying such as ventilation drying or sun drying, forced drying in which drying is performed by heating with electricity or the like, and freeze drying. Lyophilization is preferred. There are no particular restrictions on the drying time. In the case of natural drying such as ventilation or sun drying, it takes about 3 days, and in the case of forced drying by heating with electricity, it takes about 1 to 3 days at about 50°C. can be mentioned. Usually, it is preferable to dry so that the water content is 10% by mass or less, preferably 5% by mass or less. If it is difficult to reduce the water content to 10% by mass or less as in the case of natural drying such as ventilation or sun drying, the water content may be further reduced by using a freeze dryer after that.

The pulverization treatment (powdering treatment) is performed by any known method such as pulverization using a pulverizer (for example, a pin mill, hammer mill, ball mill, jet mill, etc.), or grinding using a stone mill. you can go

For the drying treatment and pulverization treatment of the raw royal jelly, known methods used in the art as described above can be widely adopted. can.

A royal jelly organic solvent extract can be obtained, for example, by extracting raw royal jelly or royal jelly powder using an organic solvent such as ethanol, methanol, propanol, or acetone as a solvent. The extraction time can be appropriately set according to the form of raw royal jelly used as a raw material, the type and amount of solvent, the temperature and stirring conditions during extraction, and the like. After extraction, solids may be removed by filtration, centrifugation, or the like. Moreover, the extracted solution may be used as it is, or the solvent may be removed from the solution and used as a concentrated liquid or powder. The royal jelly organic solvent extract is preferably a royal jelly ethanol extract.

Raw royal jelly can be obtained, for example, as a beekeeping product according to a conventional method. The place of production of royal jelly is not limited, and may be any of Japan, China, Brazil, European countries, Oceania countries, America and the like.

The peptidase used in the present invention is a peptidase having both endopeptidase and exopeptidase actions. According to the enzymatic treatment using such a peptidase, proteins can be made into low-molecular-weight proteins by a one-step enzymatic treatment, so that the operation is simple and the physiological activities of useful components contained in royal jelly can be eliminated or greatly reduced. It has the advantage of being able to prevent

The peptidase used in the present invention has an EC number (enzyme number) of Group 3.4, and its origin is not particularly limited as long as the effect of the present invention is exhibited. Peptidases derived from viruses, fungi (molds, yeast, mushrooms, etc.), algae, etc.) can be widely used. Among these, peptidases derived from bacteria or fungi are preferred, and more preferred are Aspergillus oryzae-produced peptidases, Streptomyces griseus-produced peptidases, or Aspergillus melleus-produced peptidases. Aspergillus oryzae-produced peptidases are particularly preferred.

"Exopeptidases" are classified into "aminopeptidases" and "carboxypeptidases." In addition, peptidases are sometimes given the terms acidic, neutral, and alkaline, respectively, depending on the optimum pH, such as "acidic exopeptidase," "neutral aminopeptidase," and "alkaline endopeptidase." may also be described in

As a peptidase having both an endopeptidase action and an exopeptidase action, a commercially available one can be used, and as such a commercial product, Aspergillus oryzae-produced peptidase (trade name: Sumiteam LP-G, Proteax, Flavorzyme, Protease A), Streptomyces griseus peptidase (trade name: actinase AS), Aspergillus melleus peptidase (trade name: protease P) can be exemplified.

As the peptidase used in the present invention, one derived from Aspergillus oryzae and having an optimum temperature of 50° C. or higher and an optimum pH of 7 to 8 is particularly preferable. As the peptidase used in the present invention, the protein obtained by extraction with Tris-HCl buffer, precipitation with trichloroacetic acid, and washing with acetone is digested with trypsin, desalted with a solid-phase column, and subjected to liquid chromatography. By mass spectrometry, when analyzed under the following analysis conditions, characteristic molecular weight compound ions (m / z: 970.5705 [MH], 980.5435 [MH], 541.3723 [M -H], 860.3850 [M - H], 855.4021 [M - H], 815.4538 [M - H], 1154.6102 [M + H], 595.5052 [M + H], 1208.9261 [M -H], 1209.2603 [M - H], each within ±5 ppm), and such peptidases include Proteax.

(Analysis conditions)
Column: Acquity BEH C18 (Waters: 2.1 x 100 mm, id 1.7 µm)
Column temperature: 40°C
Solvent A: 0.1% formic acid Solvent B: Acetonitrile Flow rate: 0.3 mL/min
Gradient: 5% B (0-2 min), 5-50% B (2-12 min), 50-100% B (12-22 min), 100% B (22-25 min), 5% B (25-30 min )

The amount of peptidase to be used relative to royal jelly varies depending on the concentration of royal jelly used, enzyme titer, reaction temperature and reaction time, and cannot be generalized. In general, it is preferable to use peptidase at a ratio of 50 to 10,000 action units per 1 g of protein contained in royal jelly. At this time, the peptidase may be added to the royal jelly at once, or may be added in small portions.

The pH of royal jelly for peptidase treatment is selected from the range of pH 2 to 12, preferably pH 7 to 10, more preferably pH 7 to 8.5, corresponding to the optimum pH of the enzyme used. Specifically, before adding the peptidase to the royal jelly, depending on the type of enzyme used, an acid or alkaline agent is added so that the pH is within the range of 2 to 12, preferably pH 7 to 10, more preferably pH 7 to 8.5. Alternatively, the desired pH is adjusted by the addition of a buffer. In this case, acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, etc.; alkaline agents include sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; Acid buffers and the like can be respectively exemplified.

The temperature for the peptidase treatment is not particularly limited, and is preferably within a practically applicable range including the optimal temperature range in which both endopeptidase action and exopeptidase action are expressed, that is, usually from the range of 20 to 70°C. selected. It is preferably in the range of 40 to 70°C. The peptidase treatment time is not particularly limited and depends on the type of enzyme used, reaction temperature, pH and other reaction conditions.

Royal jelly can be subjected to peptidase treatment as it is or in a state of being dissolved or dispersed in water. When the royal jelly is in dry form, it is preferably dissolved in water before being subjected to peptidase treatment.

Termination of peptidase treatment is accomplished by inactivating or removing the peptidase. The deactivation operation can be performed by heat treatment (for example, at 80° C. for 15 minutes).

In the present invention, it suffices to treat royal jelly with at least a peptidase as described above. A degrading enzyme treatment or the like can also be performed.

The enzymatic hydrolyzate of royal jelly produced by the production method of the present invention may then be prepared in the form of liquid preparations such as drinks and syrups, semi-liquid forms, or solid forms. Semi-liquid forms include pasty and jelly-like forms, solid forms include lyophilisates (e.g., lyophilized powders), tablets (including troches, chewable tablets, dragees, etc.), capsules, granules, and the like. be able to. The lyophilized product can be prepared by subjecting a peptidase-treated royal jelly enzyme hydrolyzate to a lyophilization treatment. A freeze-drying process can be performed according to a conventional method.

(2) Method for reducing allergenicity of enzymolyzed product of royal jelly and suppressing browning during and after production The method for reducing allergenicity of enzymatically resolved product of royal jelly and inhibiting browning during and after production of the present invention comprises using royal jelly as an endopeptidase. It can be carried out by treating with a peptidase having an action and an exopeptidase action.

The target royal jelly, the type of peptidase to be used, and the conditions for peptidase treatment are as described in (1) above.

Although the enzymatic hydrolyzate of royal jelly obtained by the method has a low-molecular-weight protein similar to the enzymatic hydrolyzate of royal jelly obtained by the conventional method, browning occurs both during and after production. significantly suppressed.

(3) Enzymatic degradation product of royal jelly The enzymatic degradation product of royal jelly of the present invention contains 100 mg or more of Tyr-Asp (tyrosine-aspartic acid, YD) per 100 g of dried enzymatic degradation product of royal jelly, and has antioxidant activity. Here, the enzymatic hydrolyzate of royal jelly is obtained by treating royal jelly or a protein of royal jelly with a predetermined proteolytic enzyme, and is also referred to as an enzymatically treated product of royal jelly, and contains multiple types of peptides having a predetermined composition. The enzymatic hydrolyzate of royal jelly is a proteolytic hydrolyzate of royal jelly in which the peptide composition after enzymatic treatment has not been adjusted.

The enzymatic hydrolyzate of royal jelly of the present invention is preferably obtained by treating the above-described royal jelly with a peptidase having endopeptidase and exopeptidase actions, particularly proteax. In addition, the enzymatic hydrolyzate of royal jelly of the present invention is preferably an enzymatic hydrolyzate of royal jelly produced by the method for producing an enzymatic hydrolyzate of royal jelly of the present invention.

The enzymatic hydrolyzate of royal jelly of the present invention may be in the form of a solution, or may be in the form of a powder after drying such as freeze-drying or air-drying.

The amount of each peptide contained in the enzymatic hydrolyzate of royal jelly of the present invention may vary depending on the raw material royal jelly. For example, YD may be 100 mg or more, preferably 120 mg or more, 150 mg or more, or 170 mg or more, and may be 500 mg or less, 300 mg or less, or 200 mg or less per 100 g of the dried product of the enzymatic decomposition product of royal jelly. may

The enzymatic hydrolyzate of royal jelly of the present invention further contains 40 mg or more of Val-Pro (valine-proline, VP) and 0 mg of Met-Pro (methionine-proline, MP) per 100 g of dried enzymatic hydrolyzate of royal jelly. .5 mg or more may be included. For example, VP may be 40 mg or more, preferably 50 mg or more, 55 mg or more, or 60 mg or more, and 200 mg or less, 150 mg or less, or 100 mg or less, per 100 g of the dried product of the enzymatic decomposition product of royal jelly. may MP may be 0.5 mg or more, for example, preferably 0.6 mg or more or 0.7 mg or more, and MP is 10 mg or less, 5 mg or less, or 2 mg or less with respect to 100 g of the dried product of the royal jelly enzymatic decomposition product. There may be.

The royal jelly enzymatic hydrolyzate of the present invention further includes Pro-Ile (PI) and/or Pro-Leu (PL), Pro-Val (PV), Ile-Pro (IP) and/or Leu-Pro (LP), and It may contain one or more peptides selected from the group consisting of Pro-Met (PM). In the enzymatic hydrolyzate of royal jelly of the present invention, for example, PI and/or PL may be 2 mg or more, 5 mg or more, or 8 mg or more, and may be 20 mg or less, 15 mg or less, or It may be 10 mg or less. The PV may be 10 mg or more, 15 mg or more, or 20 mg or more, and may be 40 mg or less, 30 mg or less, or 25 mg or less. IP and/or LP may be 20 mg or more, 25 mg or more, or 30 mg or more, and may be 50 mg or less, 45 mg or less, or 40 mg or less. PM may be 0.2 mg or more, 0.4 mg or more, or 0.5 mg or more, and may be 2 mg or less, 1.5 mg or less, or 1 mg or less.

Royal jelly enzymatic hydrolyzate of the present invention is further selected from the group consisting of Asp-Tyr (DY), Arg-Leu (RL), Arg-Ile (RI), Leu-Arg (LR), and Ile-Arg (IR) may comprise one or more peptides that are In the enzymatic hydrolyzate of royal jelly of the present invention, for example, DY may be 5 mg or more or 8 mg or more, and may be 20 mg or less or 15 mg or less, and RL may be 0 per 100 g of dried royal jelly enzymatic hydrolyzate. may be 5 mg or more or 1 mg or more; may be 5 mg or less or 2 mg or less; RI may be 1 mg or more or 2 mg or more; and may be 4 mg or less or 3 mg or less; It may be 5 mg or more or 2 mg or more, it may be 4 mg or less or 3 mg or less, and the IR may be 10 mg or more or 12 mg or more, and it may be 20 mg or less or 16 mg or less.

The amount of each peptide in the enzymatic hydrolyzate of royal jelly can be measured by a commonly used method. When the royal jelly enzymatic decomposition product is in a liquid or powder state, for example, after first dispersing it in a 0.1% formic acid aqueous solution, it is centrifuged at 15,000 rpm for 10 minutes, the supernatant is recovered, and further, 0.1% formic acid methanol The solution is added, centrifuged at 15,000 rpm for 10 minutes, and the supernatant is recovered to obtain a solution of peptides contained in the enzymatic hydrolyzate of royal jelly. If necessary, this peptide solution is treated with a solid phase column and the unadsorbed fraction is used as a sample. The content of each peptide in the sample was analyzed by water-soluble liquid chromatography-mass spectrometry (LC-MS), and using the peptide acid concentration in the sample and the calibration curve obtained with the standard solution, royal jelly enzyme The content of each peptide in the digest can be determined.

Since the enzymatic hydrolyzate of royal jelly of the present invention contains YD having antioxidant activity, it can have antioxidant activity. The enzymatic hydrolyzate of royal jelly of the present invention may further have antioxidant activity and antidiabetic activity by containing MP and VP having antioxidant activity and antidiabetic activity.

(4) Food composition, quasi-drug, or pharmaceutical composition The food composition, quasi-drug, or pharmaceutical composition of the present invention may contain the royal jelly enzyme hydrolyzate and have antioxidant activity.

The above-mentioned royal jelly enzymatic decomposition product may be used as it is as a product such as a pharmaceutical composition, a quasi-drug, or a food composition, or as a component of a product such as a pharmaceutical composition, a quasi-drug, or a food composition. may be used. A product containing a royal jelly enzymatic hydrolyzate can be obtained, for example, by adding the above-described royal jelly enzymatic hydrolyzate to an intermediate product in the production process of the product.

When a royal jelly enzymatic hydrolyzate is used as one component of a pharmaceutical composition, quasi-drug, or food composition, the pharmaceutical composition, quasi-drug, or food composition contains other components other than the royal jelly composition, such as pharmaceutical ingredients (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents), food-acceptable ingredients (e.g., Minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, coloring agents, fragrances, stabilizers, preservatives, slow release modifiers, surfactants, solubilizers, wetting agents).

The product containing the royal jelly enzymatic hydrolyzate may be in liquid, semi-liquid or solid form. Liquid forms include drinks, syrups, liquids, suspensions, emulsions, and injections (including cases where they are mixed with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use to prepare them as liquids). The semi-liquid form may be a paste or jelly form, and the solid form may be a freeze-dried product (e.g., freeze-dried powder), tablet (uncoated tablet, sugar-coated tablet, effervescent tablet, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders (powders), fine granules, granules, and the like. These various formulations can be prepared, for example, by mixing the enzymatic hydrolyzate of royal jelly obtained by the method described above and, if necessary, other ingredients, and molding them into the dosage forms described above.

When the above-described royal jelly enzymatic hydrolyzate is used as a food composition or as one component of the food composition, the food composition preferably emphasizes the tertiary function of food, that is, the function of regulating physical condition. Examples of products emphasizing the tertiary functions of foods include health foods, foods with functional claims, foods with nutrient function claims, dietary supplements, supplements, and foods for specified health uses.

Examples of food compositions include soft drinks such as coffee, juice and tea drinks, milk drinks, lactic acid drinks, yogurt drinks, carbonated drinks, and beverages such as sake, western liquor, fruit wine and honey wine; custard cream Spreads such as fruit paste; Western sweets such as chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes and puddings; , ice candy and sherbet; Curry, beef bowl, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam and other cooked foods; is mentioned.

The content of the enzymatic hydrolyzate of royal jelly in the food composition, quasi-drug, or pharmaceutical composition of the present invention is 0.1 part by weight or more of the enzymatic hydrolyzate of royal jelly when the dry weight of the composition is 100 parts by weight. 10 parts by weight or more, 25 parts by weight or more, 40 parts by weight or more, or 50 parts by weight or more; parts or less, and may be 55 parts by weight or less.

EXAMPLES The present invention will be described in detail below with reference to examples. However, the present invention is by no means limited to these examples.

[Example 1]
474 g of fresh royal jelly was weighed into a 3 L flask, 400 ml of ion-exchanged water was added, and the mixture was stirred until uniform to prepare a royal jelly diluted solution. A 2N NaOH aqueous solution was added to adjust the pH of the royal jelly dilution to 7.8. Next, a solution obtained by dissolving 4.75 g of Proteax (Amano Enzyme Co., Ltd.) having both endopeptidase action and exopeptidase action in 20 mL of ion-exchanged water was added to the royal jelly diluted solution, and further ion-exchanged water was added to make the total amount 980 g. added so that The reaction mixture was stirred with a propeller and reacted at 50° C. (constant temperature water bath) for 2 hours for hydrolysis. After raising the temperature of the constant temperature water bath to 80° C. and stirring for 15 minutes to deactivate the enzyme, 11.73 g of crystalline cellulose and 5.85 g of locust bean gum were mixed and cooled with water.

[Comparative Example 1]
A royal jelly enzymatic hydrolyzate was obtained in the same manner as in Example 1, except that Actinase AS (Kaken Pharmaceutical Co., Ltd.) was used as the enzyme instead of Proteax and the pH was adjusted to 8.8.

<Measurement of enzymatic degradation>
The degree of enzymatic degradation was confirmed by measuring the molecular weight of the compound after decomposition using gel filtration analysis. After the enzymatic reaction, 20 μL of the solution was weighed and added with 9.5 mL of 50 mM NaPi/0.3 M NaCl buffer (pH 7.0) to prepare an analysis sample. Using cytochrome C (nacalai tesque, M.W.=12,384) as an index, the ratio of components eluted earlier than cytochrome C was calculated. Analysis conditions are shown below.
Column: Shodex PROTEIN KW-802.5 (5 μm, 8.0 mm id×300 mm, Showa Denko Co., Ltd.),
Guard column: Shodex PROTEIN KW-G (5 μm, 8.0 mm id×10 mm, Showa Denko Co., Ltd.), column oven: 30° C., flow rate: 0.5 mL/min, mobile phase: A; 50 mM Na phosphate / 0.3 M NaCl buffer (pH 7.0), B; none, elution: B 0% (60 min hold), injection volume: 10 μL, detection: UV (220 nm).

Table 1 shows the results. In Example 1, no clear peak was observed before cytochrome C, and it was confirmed that the resolution was as high as in Comparative Examples.

<Histamine release test (confirmation of allergenicity)>
For the enzymatic hydrolyzate of royal jelly obtained in Example 1 and Comparative Example 1, the amount of released histamine was measured by the CAST method (cellular antigen stimulation test).

Specifically, an enzymatic hydrolyzate of raw royal jelly was applied to a blood sample of a subject who had experienced an allergic reaction to raw royal jelly, and the amount of released histamine was measured. The test subjects will be given a written explanation of the content of the test, fully explained to them about the purpose and content of the test, and only those who have obtained written consent to participate in the test of their own free will will be allowed to participate in the test. did Free histamine was measured using a commercially available ELISA kit (Histamine ELISA (manufactured by IMMUNO-BIOLOGICAL LABORATORIES)).

The results are shown in FIG. In Example 1, the histamine release value (ng/mL) is 1/2 or less compared to Comparative Example 1, and the royal jelly enzymatic hydrolyzate obtained by this method is superior to the royal jelly enzymatic hydrolyzate obtained by the conventional method. It is considered that the allergenicity has been reduced in the past.

<Evaluation of browning>
The royal jelly solutions after the enzymatic reaction in Examples and Comparative Examples were freeze-dried and pulverized, and browning evaluation (brightness analysis) was performed using images taken with a digital camera. Photographs were taken after pulverization and after storage at 50°C for 7 days. The luminance was calculated using "FlatColorPicker", which is color picker software, and the luminance (%) in the HSL color space was used as the value.

Table 2 shows the results after powdering, and FIG. 2 shows the results after storage at 50° C. for 7 days. The decrease in brightness and browning caused by enzymatic decomposition in the conventional method are not observed in the royal jelly enzymatically degraded product obtained by the method of the present invention, and the same browning occurs even after storage at 50°C for 7 days. had been suppressed.

[Example 3 Composition analysis of royal jelly enzymatic decomposition product]
(Sample preparation)
According to the method of Example 1, a Proteax-treated royal jelly solution was obtained and further freeze-dried to obtain two lots (181030, 190510) of royal jelly enzymatic hydrolyzate powder. For comparison, instead of Proteax, Actinase AS (Kaken Pharmaceutical Co., Ltd.), protease A-treated royal jelly, and protease P-treated royal jelly were used, and the pH was set to 8.8, 7.8, and 7.8, respectively. Except for this, in the same manner as in Example 1, a freeze-dried powder of each royal jelly enzymatic hydrolyzate was obtained. In addition, as commercially available enzyme-treated royal jelly, MF4 (Api Co., Ltd.), RJ peptide powder (Api Co., Ltd.), water-soluble royal jelly powder (Morikawa Kenkodo Co., Ltd.), black royal jelly powder (Maruo Shokuken Co., Ltd.), water-soluble Royal jelly powder (Nakahara Co., Ltd.) and Nisshin Royal Jelly P (Nissin Honey Co., Ltd.) were used. In addition, dry powder of non-enzymatically treated royal jelly was also used.

To 100 mg of each freeze-dried powder, 800 μL of 0.1% formic acid aqueous solution was added, 5 mm diameter zirconia beads were added, and the mixture was homogenized with a bead homogenizer at 25 Hz for 2 minutes. Centrifuge at 15,000 rpm for 10 minutes, collect 100 μL of the supernatant, add 300 μL of 0.1% methanol formic acid solution, centrifuge at 15,000 rpm for 10 minutes, collect the supernatant, and obtain the resulting peptide. The solution was used as a sample for analysis.

(Solid-phase column treatment)
Add 100 μL of 0.1% formic acid methanol solution to the MonoSpin C18 column, centrifuge at 5,000 g for 2 minutes, add 100 μL of 75% methanol solution with 0.1% formic acid added to the column, and further add 5,000 g and centrifuged for 2 minutes. 100 μL of the sample (peptide solution) obtained above was added to the treated column and centrifuged at 5,000 g for 2 minutes to collect the column effluent (unadsorbed fraction). 100 μL of 75% methanol solution containing 0.1% formic acid was added to the total volume of column effluent (approximately 100 μL) and filtered through a 0.2 μm syringe filter.

ＭＳ測定条件
使用機器：Ｑ Ｅｘａｃｔｉｖｅ ｆｏｃｕｓ
Ｉｏｎ ｓｏｕｒｃｅ：ＨＥＳＩ
Ｓｃａｎ ｒａｎｇｅ：７０－１０００ｍ／ｚ
Ｆｕｌｌ ｓｃａｎ ｉｓｏｌａｔｉｏｎ：７０，０００(Liquid chromatography-mass spectrometry (LC-MS) analysis)
The entire amount of the above filtrate was sealed in an undiluted HPLC vial, 2 μL of which was injected into LC-MS, and LC-MS was performed under the following conditions.
Equipment used: Ultimate3000 (Thermofisher)
Column: XBridge BEH Amide (2.1 x 150 mm, i.d. 2.5 µm Waters)
Column oven: 60°C
Solvent A: 10 mM ammonium formate (pH 2.5)
Solvent B: 10 mM ammonium formate 95% acetonitrile Flow rate: 0.2 mL/min Injection volume: 2 μL
Gradients are shown in Table 3.

MS measurement conditions Equipment used: Q Exactive focus
Ion source: HESI
Scan range: 70-1000m/z
Full scan isolation: 70,000

A calibration curve is obtained from each concentration of the standard solution and the area of each peak obtained from the chromatogram. From the calibration curve, the concentration of each peptide acid in the test solution was calculated, and the content of each peptide in the sample was obtained from the following formula.
Content (%) = Y = X x V x E/W/1000000 x 100
Content (mg/100g) = Y x 100/100 x 1000
Y: peptide content (%)
X: Concentration (ng/mL) of each peptide in the sample solution obtained from the calibration curve
V: volume of the final diluted solution of the sample (mL)
E: Dilution ratio of the final diluted solution of the sample W: Sample amount (mg)

(result)
Results for specific peptides contained in the royal jelly of each sample are shown in Table 4. Table 4 shows that the Proteax-treated royal jelly enzymatic hydrolyzate of the present invention contains more peptides than other royal jelly, MP (0.76-0.94 mg/100 g enzymatic hydrolyzate of royal jelly), VP ( 61.48-74.76mg/100g royal jelly enzymatic hydrolyzate), YD (173.02-180.99mg/100g royal jelly enzymatic hydrolyzate), PI (L) (8.71-8.88mg/100g royal jelly enzymatic hydrolyzate ), PV (20.53-22.36mg/100g royal jelly enzymatic decomposition product), I (L) P (32.67-38.21mg/100g royal jelly enzymatic decomposition product), PM (0.56-0.96mg/ 100 g royal jelly enzymatic decomposition product) and the like were confirmed. MP and VP are known to have antidiabetic activity and antioxidant activity, and YD is known to have antioxidant activity. It was suggested that it has oxidative activity.

Claims (8)

A method for producing a royal jelly enzymatic hydrolyzate with reduced allergenicity and suppressed browning during and after production, comprising treating royal jelly with a peptidase having an endopeptidase action and an exopeptidase action, The production method, wherein the peptidase is an Aspergillus oryzae-produced peptidase, and the optimum temperature of the peptidase is 50°C or higher. A method for reducing allergenicity of a royal jelly enzymatic hydrolyzate and suppressing browning during and after production, comprising treating royal jelly with a peptidase having an endopeptidase action and an exopeptidase action, wherein the peptidase is produced by Aspergillus oryzae. A method, wherein the peptidase has an optimum temperature of 50°C or higher. An enzymatic hydrolyzate of royal jelly containing 100 mg or more of Tyr-Asp per 100 g of dry matter of the enzymatic hydrolyzate of royal jelly. 4. The enzymatic hydrolyzate of royal jelly according to claim 3 , further comprising 40 mg or more of Val-Pro and 0.5 mg or more of Met-Pro per 100 g of the dried enzymatic hydrolyzate of royal jelly. Furthermore, Pro-Ile and / or Pro-Leu, Pro-Val, Ile-Pro and / or Leu-Pro, and Pro-Met comprising one or more peptides selected from the group consisting of, claim 3 or 4 Royal jelly enzymatic hydrolyzate according to . A food composition comprising the royal jelly enzymatic hydrolyzate according to any one of claims 3 to 5 . A quasi-drug containing the royal jelly enzyme hydrolyzate according to any one of claims 3 to 5. A pharmaceutical composition comprising the enzymatic hydrolyzate of royal jelly according to any one of claims 3 to 5.

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