WO2020196583A1 - Method for producing enzymolyzed honeybee larvae - Google Patents

Method for producing enzymolyzed honeybee larvae Download PDF

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Publication number
WO2020196583A1
WO2020196583A1 PCT/JP2020/013215 JP2020013215W WO2020196583A1 WO 2020196583 A1 WO2020196583 A1 WO 2020196583A1 JP 2020013215 W JP2020013215 W JP 2020013215W WO 2020196583 A1 WO2020196583 A1 WO 2020196583A1
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Prior art keywords
bee
peptidase
enzyme
treated product
action
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PCT/JP2020/013215
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French (fr)
Japanese (ja)
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耕太朗 二平
颯 板谷
礼訓 八巻
雅之 山家
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株式会社山田養蜂場本社
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Priority to JP2021509480A priority Critical patent/JP7260203B2/en
Publication of WO2020196583A1 publication Critical patent/WO2020196583A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to a method for producing a bee pup enzyme-treated product in which allergenicity is reduced and browning is suppressed.
  • the present invention also relates to a method for reducing allergenicity and suppressing browning of a Wasp enzyme-treated product.
  • Health foods made from powder of dried and crushed bee cubs are known. This health food is rich in proteins, amino acids, vitamins and minerals possessed by bee cubs, and has been shown to have an improving effect on autonomic imbalance, menopause and the like.
  • bee cubs are useful natural materials, but on the other hand, they may cause allergic reactions due to the proteins contained in them.
  • decomposition of high molecular weight proteins using a proteolytic enzyme is generally performed (Patent Documents 1 and 2).
  • Patent Document 1 states that the fat of a bee cub is decomposed by adding a lipid-degrading enzyme, and then the protein of the bee cub is decomposed by adding a proteolytic enzyme.
  • Patent Document 1 after the protein decomposition step, a sugar-degrading enzyme such as ⁇ -mannosidase is added to decompose the sugar of the bee cub, whereby the oligosaccharide component can be efficiently obtained. It has been reported that it can be used as a highly nutritious processed food and drink for bee cubs.
  • a sugar-degrading enzyme such as ⁇ -mannosidase
  • Patent Document 2 by treating a bee or a processed product thereof in combination with two types of proteolytic enzymes, an acidic protease and a neutral protease, a high molecular weight protein can be efficiently decomposed and the amount of allergen is reduced. It has been reported that it can be done.
  • Patent Document 3 reports that the browning caused by the enzymatic treatment is significantly suppressed by performing the enzymatic treatment of bees under high pressure conditions of 40 to 600 Mpa.
  • Patent Document 3 requires high pressure conditions in order to suppress browning.
  • Patent Documents 1 and 2 it is necessary to use two or more kinds of enzymes in enzymatic decomposition. Further, the method described in Patent Document 3 does not describe that browning can be suppressed during and after the production of the bee pup enzyme-treated product.
  • the present invention provides a method for producing a bee pup enzyme-treated product, which can reduce allergenicity and suppress browning during and after production even when a single enzyme is used under normal pressure conditions, and a bee. It is an object of the present invention to provide a method for reducing allergenicity of a child enzyme-treated product and suppressing browning during and after production.
  • the present inventors used a single enzyme under normal pressure conditions by treating bee pups with peptidase having endopeptidase action and exopeptidase action. It was found that allergenicity can be reduced and browning during and after production can be suppressed even if there is any.
  • the present invention has been completed by further studying based on these findings, and the following method for producing a bee child enzyme-treated product, reducing allergenicity of the bee child enzyme-treated product, and during and after production. It provides a method for suppressing browning of.
  • II Method for reducing allergenicity of bee child enzyme-treated product and suppressing browning during and after production
  • II-1 A method for reducing allergenicity of a bee pup enzyme-treated product and suppressing browning during and after production, which comprises treating bee pups with peptidase having endopeptidase action and exopeptidase action. ..
  • 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.
  • the present invention even when a single enzyme is used under normal pressure conditions, browning during and after production caused by peptidase decomposition is realized while improving safety by reducing allergenicity. It is possible to provide a method for producing a bee-child enzyme-treated product in which is suppressed. Further, according to the present invention, a method that can be carried out using a single enzyme under normal pressure conditions to reduce the allergenicity of a Wasp enzyme-treated product and to suppress browning during and after production. Can be provided.
  • the method for producing a bee-koenzyme-treated product of the present invention is a bee-koenzyme treatment in which allergenicity is reduced and browning during and after production is suppressed.
  • a method for producing a product which comprises treating a bee cub with a peptidase having an endopeptidase action and an exopeptidase action.
  • the bee cub means a bee larva and a pupa.
  • the type of bee is not particularly limited, and known bees such as Japanese honey bee, Western honey bee, African honey bee, Wasp (including hornet), paper wasp, and bumblebee can be widely used. It is preferably a honey bee, and more preferably a western honey bee because of its availability. In addition, regardless of the distinction between males and females, males are preferable.
  • the larvae and pupae are not particularly limited as long as they are hatched from eggs. Of these, bee larvae and pupae 16 to 23 days after hatching are preferably used, and bee larvae and pupae 18 to 21 days after hatching are more preferably used.
  • Bee cubs have accumulated nutrients in their bodies.
  • male honeybees have been used as a material for Chinese medicine for a long time, and in addition to containing various amino acids including essential amino acids in a well-balanced manner, vitamins such as proteins, lipids, sugars, B vitamins, folic acid, nicotinic acid, and pantothenic acid.
  • vitamins such as proteins, lipids, sugars, B vitamins, folic acid, nicotinic acid, and pantothenic acid.
  • And is rich in minerals such as zinc and selenium.
  • As the physiological activity and pharmacological action of bee cubs antibacterial action, anti-inflammatory action, antiviral action, antigen insect action, tinnitus eliminating action and the like are known.
  • the bee cub is used in a state where the raw bee cub and the raw bee cub are processed.
  • Processed bee cubs specifically, crushed bee cubs (raw or dried), dried bee cubs (raw or crushed), bee cubs (raw, dried) It includes those obtained by heat-treating a product or crushed product, and those obtained by extracting bee cubs with water, hydrous ethanol, or the like.
  • the dry powder of the bee cub prepared by drying the bee cub (raw) and then pulverizing it can be mentioned.
  • bee cub includes, in addition to raw bee pups, processed products obtained by subjecting the bee pups to drying, crushing or heating, and bee pups. Includes those extracted (including raw, dried and crushed products) with water, hydrous ethanol, etc.
  • drying method known methods used in general food processing can be used, such as ventilation drying, natural drying such as sun drying, forced drying that heats and dries with electricity, and freeze drying. It is preferably freeze-dried.
  • the drying time is not particularly limited.
  • natural drying such as ventilation and sun drying
  • forced drying by heating with electricity, etc. it takes about 1 to 3 days at about 50 ° C.
  • the degree can be mentioned.
  • the crushing treatment can be performed by using a known method such as a method of crushing using a crusher (mill) or a method of grinding using a stone mill.
  • the method of heating the bee cub is not particularly limited, and a method of heat treatment at 70 to 120 ° C. can be mentioned. It is also possible to simply put bee cubs (including raw, dried and crushed products) into boiling water and heat-treat them. In order to avoid elution of active ingredients such as various vitamins and amino acids as much as possible, As a heat treatment, a method of steaming bee cubs with steam is preferably used.
  • the extraction method is to add water, hydrous ethanol, etc. to bee cubs (including raw, dried and crushed products), stir, and then centrifuge to obtain a supernatant, or filter with filter paper to extract the filtrate.
  • the method of obtaining is used.
  • the protein contained in the bee cub is reduced in molecular weight, and an enzyme-treated product (hypoallergenicizing enzyme-treated product) in which the allergic reaction caused by the protein is suppressed can be obtained. ..
  • the peptidase used in the present invention is a peptidase having both an endopeptidase action and an exopeptidase action at the same time. According to the enzymatic treatment using such peptidase, the protein can be reduced in molecular weight by the one-step enzymatic treatment, so that the operation is simple.
  • the peptidase used in the present invention has an EC number (enzyme number) of 3.4 group, and the origin is not particularly limited as long as the effect of the present invention is exhibited, and animals, plants, and microorganisms (bacteria, viruses, etc.) Peptidases derived from fungi (molds, yeasts, mushrooms, etc.), algae, etc.) can be widely used. Among these, peptidases derived from bacteria or fungi are preferable, and peptidases derived from Aspergillus oryzae are particularly preferable.
  • Exopeptidase is classified into “aminopeptidase” and “carboxypeptidase”.
  • peptidase may give each enzyme the terms acidic, neutral, and alkaline, respectively, depending on the optimum pH, for example, “acidopeptidase,” “neutral aminopeptidase,” and “alkaline endopeptidase.” It may be described in.
  • peptidase a commercially available product can be used, and as such a commercially available product, the product names "Sumiteam LP-G” (Nippon Chemical Industrial Co., Ltd.) and “Protease M” Amano “SD” are preferable. , “Protease P” Amano “3SD”, “Proteax” (Amano Enzyme Co., Ltd.), “Flavourzyme” (Novozymes Japan Co., Ltd.) can be exemplified.
  • peptidase used in the present invention those derived from Aspergillus oryzae, those having an optimum temperature of 50 ° C. or higher, and those having an optimum pH of 7 to 8 are particularly preferable, and such peptidases are Proteax. Can be mentioned.
  • the peptidase used in the present invention the protein obtained by extracting with Tris-HCl buffer, precipitating with trichloroacetic acid, and washing with acetone is digested with trypsin, desalted on a solid phase column, and then liquid chromatography.
  • the amount of peptidase used for bee pups varies depending on the bee pup concentration, enzyme titer, reaction temperature and reaction time used, and cannot be unequivocally stated. In general, it is preferable to use peptidase at a rate of 50 to 10,000 action units per gram of protein contained in bee pups. At this time, the peptidase may be added to the bee cubs at once, or may be added in small portions.
  • the pH of the bee cub solution and dispersion during the peptidase treatment is selected from the range of pH 2 to 12, preferably pH 7 to 10, and more preferably pH 7 to 8.5, depending on the optimum pH of the enzyme used. Specifically, before adding peptidase to the bee cub solution and dispersion, the pH should be in the range of pH 2 to 12, preferably pH 7 to 10, and more preferably pH 7 to 8.5 depending on the type of enzyme used. The pH is adjusted to the desired pH by adding an acid, an alkaline agent, or a buffer.
  • hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid and the like are used as the acid; sodium hydroxide, potassium hydroxide, potassium carbonate and the like are used as the alkaline agent; and phosphoric acid buffer and citric acid are used as the buffering agent.
  • Acid buffering agents and the like can be exemplified respectively.
  • the temperature of the peptidase treatment is not particularly limited, and is preferably from a range that can be put into practical use, that is, usually in the range of 20 to 70 ° C., including an optimum temperature range in which both endopeptidase action and exopeptidase action are exhibited. Be selected.
  • the temperature is preferably in the range of 40 to 70 ° C, more preferably 55 to 70 ° C.
  • the time of peptidase treatment depends on the type of enzyme used, the reaction temperature, the reaction conditions such as pH, and is not particularly limited.
  • the bee cub can be subjected to peptidase treatment as it is or in a state of being dissolved or dispersed in water.
  • the bee pups are in a dry form, they are preferably subjected to peptidase treatment in a state of being dissolved in water.
  • the peptidase treatment is stopped by inactivating or removing the peptidase.
  • the deactivation operation can be performed by heat treatment (for example, at 80 ° C. for 15 minutes).
  • the bee pups may be treated with peptidase at least as described above, and not only the peptidase treatment but also the combination treatment with other enzymes, for example, peptidase treatment as long as the effect of the present invention is not impaired. It can also be treated with a glycolytic enzyme.
  • the bee-child enzyme-treated product produced by the production method of the present invention may then be prepared in the form of a liquid such as a drink or a syrup, or may be prepared in a semi-liquid form or a solid form.
  • Semi-liquid forms include paste-like and jelly-like forms
  • solid forms include freeze-dried products (for example, freeze-dried powder), tablets (including troches, chewable tablets, sugar-coated tablets, etc.), capsules, granules, and the like. be able to.
  • the freeze-dried product can be prepared by subjecting the peptidase-treated bee-child enzyme-treated product to the freeze-drying treatment.
  • the freeze-drying treatment can be carried out according to a conventional method.
  • the target bee cubs, the type of peptidase used, the conditions for peptidase treatment, etc. are as described above in (1).
  • the bee-child enzyme-treated product obtained by the method is similar to the bee-child enzyme-treated product obtained by the conventional method, although the protein is reduced in molecular weight, both at the time of production and after the production.
  • the browning phenomenon is predominantly suppressed in. Furthermore, such an effect can be obtained even under normal pressure conditions and when a single enzyme is used.
  • Example 1 39.5 g of bee cubs were weighed in a glass beaker, 150 ml of ion-exchanged water was added, and the mixture was stirred until uniform to prepare a beak cub dilution solution.
  • the pH of the Wasp dilute solution was adjusted to 7.0 by adding a 2N NaOH aqueous solution.
  • a solution of 1180 mg of Proteax (Amano Enzyme Co., Ltd.), which has both endopeptidase action and exopeptidase action, in 10 ml of ion-exchanged water to the bee dilute solution, and add the total amount of ion-exchanged water.
  • Proteax Amano Enzyme Co., Ltd.
  • the reaction mixture was reacted at 70 ° C. (constant temperature water tank) for 2 hours while stirring with a propeller to carry out hydrolysis.
  • the temperature of the constant temperature water tank was raised to 80 ° C. and the mixture was stirred for 15 minutes to inactivate the enzyme.
  • Example 2 As an enzyme, protease P (Amano Enzyme Co., Ltd.) having both endopeptidase action and exopeptidase action was used instead of Proteax, and the reaction temperature was set to 40 ° C. in the same manner as in Example 1. A bee pup enzymatic degradation product was obtained.
  • Example A bee-child enzymatic decomposition product in the same manner as in Example 1 except that Actinase AS (Kaken Pharmaceutical Co., Ltd.) was used instead of Proteax and the pH was 8.8 and the reaction temperature was 50 ° C. Got
  • ⁇ Measurement of enzymatic decomposition degree> 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 ⁇ L of the solution was weighed, and 9.5 mL of 50 mM NaPi / 0.3M NaCl buffer (pH 7.0) was added to prepare an analysis sample. Using cytochrome C (nacalai tesque, MW 12,384) as an index, the proportion of components eluted earlier than cytochrome C was calculated. The analysis conditions are shown below.
  • the amount of histamine released was measured by allowing the enzyme hydrolyzate of the bee cub to act on the blood sample of the subject who had experienced an allergic reaction to the bee cub.
  • This test will be given only to those who have distributed a manual describing the contents of this test to the subjects, fully explained the purpose and contents of the test, and obtained a written consent to participate voluntarily by the subjects themselves. Was done.
  • the measurement of free histamine was carried out using a commercially available ELISA kit (Histamine ELISA (manufactured by IMMUNO-BIOLOGICAL LABORATORIES)).
  • Example 1 the histamine free value (ng / mL) was 3/5 or less as compared with the comparative example, and the bee-child enzyme-treated product obtained by the method was treated with the bee-child enzyme obtained by the conventional method. It is considered that the allergenicity can be reduced predominantly over the product.
  • Table 2 shows the results after powdering
  • Fig. 2 shows the results after storage at 50 ° C for 7 days.
  • the phenomenon of decrease in brightness and browning, which was observed by the conventional method due to enzymatic decomposition, is not observed in the Wasp enzyme-treated product obtained by the method of the present invention, and the same applies even after storage at 50 ° C. for 7 days. Browning was suppressed.

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Abstract

Disclosed are: a method for producing an enzyme-treated product of honeybee larvae which has reduced allergenic potency and does not undergo brownish discoloration during and after the production thereof, the method being characterized by treating honeybee larvae with a peptidase having both of the activity of an endopeptidase and the activity of an exopeptidase; and a method for reducing the allergenic potency of an enzyme-treated product of honeybee larvae and preventing the occurrence of brownish discoloration of the enzyme-treated product of honeybee larvae during and after the production of the enzyme-treated product of honeybee larvae, the method being characterized by treating honeybee larvae with a peptidase having both of the activity of an endopeptidase and the activity of an exopeptidase.

Description

酵素分解蜂の子の製造方法How to make enzymatically decomposed bee cubs
　本発明は、アレルギー性が低減され且つ褐変化が抑制されてなる蜂の子酵素処理物の製造方法に関する。また、本発明は、蜂の子酵素処理物のアレルギー性低減且つ褐変化抑制方法に関する。 The present invention relates to a method for producing a bee pup enzyme-treated product in which allergenicity is reduced and browning is suppressed. The present invention also relates to a method for reducing allergenicity and suppressing browning of a Wasp enzyme-treated product.
　蜂の子を乾燥して粉砕させた粉末を原料とする健康食品が知られている。この健康食品は、蜂の子の有するタンパク質、アミノ酸、ビタミン及びミネラルを豊富に含有し、自律神経失調症、更年期障害等に対する改善作用が認められている。 Health foods made from powder of dried and crushed bee cubs are known. This health food is rich in proteins, amino acids, vitamins and minerals possessed by bee cubs, and has been shown to have an improving effect on autonomic imbalance, menopause and the like.
　このように蜂の子は、有用な天然素材であるが、一方でそれらに含まれるタンパク質に由来してアレルギー反応を引き起こす場合がある。このようなアレルギー性を低減するために、タンパク質分解酵素を使用した高分子タンパク質の分解が一般的に行われている(特許文献１及び２)。 As described above, bee cubs are useful natural materials, but on the other hand, they may cause allergic reactions due to the proteins contained in them. In order to reduce such allergenicity, decomposition of high molecular weight proteins using a proteolytic enzyme is generally performed (Patent Documents 1 and 2).
　特許文献１には、蜂の子の脂肪を脂質分解酵素を添加して分解した後、タンパク質分解酵素を添加して蜂の子のタンパク質を分解することで、酵素分解の過程においてタンパク質の周囲に形成された脂肪を分解し、タンパク質とタンパク質分解酵素とを効率的に接触させて、蜂の子のタンパク質の分解をより効率的に行うことができること、それにより蜂の子加工飲食品の栄養的価値を高め且つアレルゲン性を効果的に改善することが可能となることが報告されている。 Patent Document 1 states that the fat of a bee cub is decomposed by adding a lipid-degrading enzyme, and then the protein of the bee cub is decomposed by adding a proteolytic enzyme. The ability to break down the formed fat and efficiently bring the protein into contact with the proteolytic enzyme to more efficiently break down the protein in the bee cub, thereby nutrition of the bee cub processed food and drink. It has been reported that it is possible to increase the value and effectively improve the allergenicity.
　さらに、特許文献１では、タンパク質の分解工程の後に、β－マンノシダーゼ等の糖質分解酵素を添加して蜂の子の糖質を分解すること、それによりオリゴ糖成分を効率的に得ることができ、栄養価の高い蜂の子加工飲食品とすることができることが報告されている。 Further, in Patent Document 1, after the protein decomposition step, a sugar-degrading enzyme such as β-mannosidase is added to decompose the sugar of the bee cub, whereby the oligosaccharide component can be efficiently obtained. It has been reported that it can be used as a highly nutritious processed food and drink for bee cubs.
　特許文献２では、ハチノコ又はその加工物を酸性プロテアーゼ及び中性プロテアーゼの2種類のタンパク質分解酵素を併用して処理することにより、効率的に高分子タンパク質を分解することができ、アレルゲン量を低減できることが報告されている。 In Patent Document 2, by treating a bee or a processed product thereof in combination with two types of proteolytic enzymes, an acidic protease and a neutral protease, a high molecular weight protein can be efficiently decomposed and the amount of allergen is reduced. It has been reported that it can be done.
　しかし、ペプチダーゼによりタンパク質を分解し、低アレルゲン化された蜂の子(酵素分解蜂の子)は、着色して褐変化が進行するという問題がある。 However, there is a problem that the bee pups (enzymatically decomposed bee pups) that have been hypoallergenic by decomposing proteins with peptidase are colored and browning progresses.
　このような酵素処理蜂の子の着色を抑制する各種方法が報告されている(特許文献３)。特許文献３では、ハチノコの酵素処理を４０～６００Ｍｐａの高圧条件下で行うことにより酵素処理によって生じる褐変化が有意に抑制されることが報告されている。 Various methods for suppressing the coloring of such enzyme-treated bee pups have been reported (Patent Document 3). Patent Document 3 reports that the browning caused by the enzymatic treatment is significantly suppressed by performing the enzymatic treatment of bees under high pressure conditions of 40 to 600 Mpa.
　しかしながら、特許文献３に記載の方法では、褐変化を抑制するために、高圧条件が必要である。 However, the method described in Patent Document 3 requires high pressure conditions in order to suppress browning.
日本国特開2009-254348号公報Japanese Patent Application Laid-Open No. 2009-254348 日本国特開2016-77259号公報Japanese Patent Application Laid-Open No. 2016-77259 日本国特開2009-254363号公報Japanese Patent Application Laid-Open No. 2009-254363
　特許文献１及び２に記載の方法では、酵素分解において2種以上の酵素を使用する必要がある。また、特許文献３に記載の方法では、蜂の子酵素処理物の製造時及び製造後において褐変化を抑制できることは記載されていない。 In the methods described in Patent Documents 1 and 2, it is necessary to use two or more kinds of enzymes in enzymatic decomposition. Further, the method described in Patent Document 3 does not describe that browning can be suppressed during and after the production of the bee pup enzyme-treated product.
　本発明は、常圧条件で単一の酵素を用いた場合であってもアレルギー性が低減でき且つ製造時及び製造後の褐変化を抑制できる、蜂の子酵素処理物の製造方法、並びに蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法を提供することを目的とする。 The present invention provides a method for producing a bee pup enzyme-treated product, which can reduce allergenicity and suppress browning during and after production even when a single enzyme is used under normal pressure conditions, and a bee. It is an object of the present invention to provide a method for reducing allergenicity of a child enzyme-treated product and suppressing browning during and after production.
　本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することにより、常圧条件で単一の酵素を用いた場合であっても、アレルギー性を低減できる上、製造時及び製造後の褐変化を抑制できるという知見を得た。 As a result of diligent research to achieve the above object, the present inventors used a single enzyme under normal pressure conditions by treating bee pups with peptidase having endopeptidase action and exopeptidase action. It was found that allergenicity can be reduced and browning during and after production can be suppressed even if there is any.
　本発明は、これら知見に基づき、更に検討を重ねて完成されたものであり、以下の蜂の子酵素処理物の製造方法、並びに蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法を提供するものである。 The present invention has been completed by further studying based on these findings, and the following method for producing a bee child enzyme-treated product, reducing allergenicity of the bee child enzyme-treated product, and during and after production. It provides a method for suppressing browning of.
　(I) 蜂の子酵素処理物の製造方法
(I-1) 蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することを特徴とする、アレルギー性が低減され且つ製造時及び製造後の褐変化が抑制されてなる蜂の子酵素処理物の製造方法。
(I-2) 前記ペプチダーゼがアスペルギルス・オリゼー産生ペプチダーゼである、(I-1)に記載の方法。
(I-3) 前記ペプチダーゼの至適温度が50℃以上である、(I-1)又は(I-2)に記載の方法。 (I) Method for producing Wasp enzyme-treated product
(I-1) A bee pup that is characterized by treating bee pups with a peptidase having an endopeptidase action and an exopeptidase action, wherein allergenicity is reduced and browning during and after production is suppressed. A method for producing a child enzyme-treated product.
(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) 蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法
(II-1) 蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することを特徴とする、蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法。
(II-2) 前記ペプチダーゼがアスペルギルス・オリゼー産生ペプチダーゼである、(II-1)に記載の方法。
(II-3) 前記ペプチダーゼの至適温度が50℃以上である、(II-1)又は(II-2)に記載の方法。 (II) Method for reducing allergenicity of bee child enzyme-treated product and suppressing browning during and after production
(II-1) A method for reducing allergenicity of a bee pup enzyme-treated product and suppressing browning during and after production, which comprises treating bee pups with peptidase having endopeptidase action and exopeptidase action. ..
(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.
　本発明によれば、常圧条件で且つ単一の酵素を用いた場合であっても、アレルギー性の低減による安全性の向上を実現しつつ、ペプチダーゼ分解によって生じる製造時及び製造後の褐変化が抑制されてなる蜂の子酵素処理物の製造方法を提供することができる。また、本発明によれば、常圧条件で単一の酵素を用いて実施することができる、蜂の子酵素処理物のアレルギー性を低減し且つ製造時及び製造後の褐変化を抑制する方法を提供することができる。 According to the present invention, even when a single enzyme is used under normal pressure conditions, browning during and after production caused by peptidase decomposition is realized while improving safety by reducing allergenicity. It is possible to provide a method for producing a bee-child enzyme-treated product in which is suppressed. Further, according to the present invention, a method that can be carried out using a single enzyme under normal pressure conditions to reduce the allergenicity of a Wasp enzyme-treated product and to suppress browning during and after production. Can be provided.
実施例及び比較例のヒスタミン遊離試験の結果を示すグラフである。縦軸は被験者のヒスタミン遊離量(ng/mL)を示す。It is a graph which shows the result of the histamine release test of an Example and a comparative example. The vertical axis shows the amount of histamine released (ng / mL) of the subject. 実施例及び比較例の褐変評価の結果を示すグラフである。縦軸はHSL色空間における輝度(%)を示す。It is a graph which shows the result of the browning evaluation of an Example and a comparative example. The vertical axis shows the brightness (%) in the HSL color space.
　以下、本発明の実施の形態について詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
　なお、本明細書において「含む(comprise)」とは、「本質的にからなる(essentially consist of)」という意味と、「のみからなる(consist of)」という意味をも包含する。 In addition, in the present specification, "comprise" also includes the meaning of "essentially consisting of" and the meaning of "consisting of only".
　（１）蜂の子酵素処理物の製造方法
　本発明の蜂の子酵素処理物の製造方法は、アレルギー性が低減され且つ製造時及び製造後の褐変化が抑制されてなる蜂の子酵素処理物の製造方法であって、蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することを特徴とする。 (1) Method for Producing Waspeptidase-treated Product The method for producing a bee-koenzyme- treated product of the present invention is a bee-koenzyme treatment in which allergenicity is reduced and browning during and after production is suppressed. A method for producing a product, which comprises treating a bee cub with a peptidase having an endopeptidase action and an exopeptidase action.
　ここで蜂の子とは、蜂の幼虫及びさなぎを意味する。蜂の種類は特に制限されず、ニホンミツバチ、西洋ミツバチ等のミツバチ、アフリカ蜂化ミツバチ、スズメバチ(クロスズメバチを含む)、アシナガバチ、マルハナバチ等、公知の蜂を広く用いることができる。好ましくはミツバチであり、より好ましくは入手の容易性から西洋ミツバチである。なお、雄と雌との別は問わず、好ましくは雄である。 Here, the bee cub means a bee larva and a pupa. The type of bee is not particularly limited, and known bees such as Japanese honey bee, Western honey bee, African honey bee, Wasp (including hornet), paper wasp, and bumblebee can be widely used. It is preferably a honey bee, and more preferably a western honey bee because of its availability. In addition, regardless of the distinction between males and females, males are preferable.
　幼虫及びさなぎは、卵から孵化したものであれば特に制限されない。中でも好ましくは孵化後16～23日経過した蜂の幼虫及びさなぎ、より好ましくは孵化後18～21日経過した蜂の幼虫及びさなぎが用いられる。 The larvae and pupae are not particularly limited as long as they are hatched from eggs. Of these, bee larvae and pupae 16 to 23 days after hatching are preferably used, and bee larvae and pupae 18 to 21 days after hatching are more preferably used.
　蜂の子は、体内に栄養素を蓄積している。特に、ミツバチの雄は古くから漢方の素材として使用されており、必須アミノ酸を含む各種アミノ酸をバランスよく含むほか、タンパク質、脂質、糖類、ビタミンB類、葉酸、ニコチン酸、パントテン酸等のビタミン類、及び亜鉛、セレン(セレニウム)等のミネラルを豊富に含んでいる。蜂の子の生理活性及び薬理作用としては、抗菌作用、抗炎症作用、抗ウィルス作用、抗原虫作用、耳鳴り解消作用等が知られている。 Bee cubs have accumulated nutrients in their bodies. In particular, male honeybees have been used as a material for Chinese medicine for a long time, and in addition to containing various amino acids including essential amino acids in a well-balanced manner, vitamins such as proteins, lipids, sugars, B vitamins, folic acid, nicotinic acid, and pantothenic acid. , And is rich in minerals such as zinc and selenium. As the physiological activity and pharmacological action of bee cubs, antibacterial action, anti-inflammatory action, antiviral action, antigen insect action, tinnitus eliminating action and the like are known.
　本発明において蜂の子は、生の蜂の子及び生の蜂の子を加工処理した状態で使用される。蜂の子を加工処理したものとして、具体的には、蜂の子(生又は乾燥物)を粉砕したもの、蜂の子(生又は粉砕物)を乾燥したもの、蜂の子(生、乾燥物又は粉砕物)を加熱処理したもの、蜂の子を水、含水エタノール等により抽出したもの等が含まれる。好ましくは、蜂の子(生)を乾燥した後、粉砕することによって調製される蜂の子の乾燥粉末を挙げることができる。 In the present invention, the bee cub is used in a state where the raw bee cub and the raw bee cub are processed. Processed bee cubs, specifically, crushed bee cubs (raw or dried), dried bee cubs (raw or crushed), bee cubs (raw, dried) It includes those obtained by heat-treating a product or crushed product, and those obtained by extracting bee cubs with water, hydrous ethanol, or the like. Preferably, the dry powder of the bee cub prepared by drying the bee cub (raw) and then pulverizing it can be mentioned.
　本発明でいう「蜂の子」という用語には、特に言及しない限り、生の蜂の子に加えて、当該蜂の子に乾燥、粉砕又は加熱の処理を施した加工物、並びに蜂の子(生、乾燥物及び粉砕物を含む)を水、含水エタノール等により抽出したものが含まれる。 Unless otherwise specified, the term "bee cub" as used in the present invention includes, in addition to raw bee pups, processed products obtained by subjecting the bee pups to drying, crushing or heating, and bee pups. Includes those extracted (including raw, dried and crushed products) with water, hydrous ethanol, etc.
　乾燥方法としては、通風乾燥、天日乾燥などの自然乾燥、電気などで加熱して乾燥させる強制乾燥、凍結乾燥など、一般食品加工で採用される公知の方法を使用することができる。好ましくは、凍結乾燥である。なお、乾燥時間は特に制限されず、例えば、通風、天日乾燥などの自然乾燥の場合は、約3日程度、電気等で加熱して強制乾燥させる場合は、50℃程度で1～3日程度を挙げることができる。通常、水分含量が10質量%以下、好ましくは5質量%以下になるように乾燥させることが好ましい。なお、通風、天日乾燥などの自然乾燥の場合のように水分含量を10質量%以下にすることが難しい場合は、その後、凍結乾燥機にかけて更に水分を下げる処理を行い得る。 As the drying method, known methods used in general food processing can be used, such as ventilation drying, natural drying such as sun drying, forced drying that heats and dries with electricity, and freeze drying. It is preferably freeze-dried. The drying time is not particularly limited. For example, in the case of natural drying such as ventilation and sun drying, it takes about 3 days, and in the case of forced drying by heating with electricity, etc., it takes about 1 to 3 days at about 50 ° C. The degree 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 and sun drying, then a freeze-dryer can be used to further reduce the water content.
　粉砕処理(粉末化処理)は、粉砕器(ミル)を用いて粉砕する方法、石臼を用いてすりつぶす方法等、公知の方法を使用して行い得る。 The crushing treatment (powdering treatment) can be performed by using a known method such as a method of crushing using a crusher (mill) or a method of grinding using a stone mill.
　蜂の子を加熱する方法は、特に制限されず、好ましくは70～120℃で熱処理する方法を挙げることができる。簡便には沸騰した水中に蜂の子(生、乾燥物及び粉砕物を含む)を投入して加熱処理することもでき、各種のビタミン、アミノ酸等の有効成分の溶出をできるだけ避けるためには、加熱処理として蜂の子を蒸気で蒸す方法が好適に使用される。 The method of heating the bee cub is not particularly limited, and a method of heat treatment at 70 to 120 ° C. can be mentioned. It is also possible to simply put bee cubs (including raw, dried and crushed products) into boiling water and heat-treat them. In order to avoid elution of active ingredients such as various vitamins and amino acids as much as possible, As a heat treatment, a method of steaming bee cubs with steam is preferably used.
　抽出方法は、蜂の子(生、乾燥物及び粉砕物を含む)に水、含水エタノール等を添加し、攪拌した後、遠心分離により上清を得る方法、ろ紙によるろ過を行い、ろ液を得る方法等が用いられる。 The extraction method is to add water, hydrous ethanol, etc. to bee cubs (including raw, dried and crushed products), stir, and then centrifuge to obtain a supernatant, or filter with filter paper to extract the filtrate. The method of obtaining is used.
　蜂の子はペプチダーゼで処理されることにより蜂の子に含まれるタンパク質が低分子化され、当該タンパク質に起因するアレルギー反応が抑制されてなる酵素処理物(低アレルゲン化酵素処理物)が得られる。 By treating the bee cub with peptidase, the protein contained in the bee cub is reduced in molecular weight, and an enzyme-treated product (hypoallergenicizing enzyme-treated product) in which the allergic reaction caused by the protein is suppressed can be obtained. ..
　本発明で用いられるペプチダーゼは、エンドペプチダーゼ作用とエキソペプチダーゼ作用の両方を同時に有するペプチダーゼである。かかるペプチダーゼを使用した酵素処理によれば、一段階酵素処理でタンパク質を低分子化することができるので、操作が簡便である。 The peptidase used in the present invention is a peptidase having both an endopeptidase action and an exopeptidase action at the same time. According to the enzymatic treatment using such peptidase, the protein can be reduced in molecular weight by the one-step enzymatic treatment, so that the operation is simple.
　また、本発明で用いられるペプチダーゼは、EC番号(酵素番号)が3.4群のものであり、本発明の効果を奏する限り、由来は特に制限されず、動物、植物、及び微生物(細菌、ウィルス、真菌類(カビ、酵母、キノコ等)、藻類など)に由来するペプチダーゼを広く使用できる。これらの中でも細菌又は真菌類由来のペプチダーゼが好ましく、アスペルギルス・オリゼー(Aspergillus oryzae)由来のペプチダーゼが特に好ましい。 The peptidase used in the present invention has an EC number (enzyme number) of 3.4 group, and the origin is not particularly limited as long as the effect of the present invention is exhibited, and animals, plants, and microorganisms (bacteria, viruses, etc.) Peptidases derived from fungi (molds, yeasts, mushrooms, etc.), algae, etc.) can be widely used. Among these, peptidases derived from bacteria or fungi are preferable, and peptidases derived from Aspergillus oryzae are particularly preferable.
　「エキソペプチダーゼ」は、「アミノペプチダーゼ」と「カルボキシペプチダーゼ」とに分類される。また、ペプチダーゼは、至適pHによって、それぞれ酸性、中性、アルカリ性という用語を各酵素につけることがあり、例えば、「酸性エキソペプチダーゼ」、「中性アミノペプチダーゼ」、「アルカリ性エンドペプチダーゼ」のように記載することもある。 "Exopeptidase" is classified into "aminopeptidase" and "carboxypeptidase". In addition, peptidase may give each enzyme the terms acidic, neutral, and alkaline, respectively, depending on the optimum pH, for example, "acidopeptidase," "neutral aminopeptidase," and "alkaline endopeptidase." It may be described in.
　ペプチダーゼとしては、市販のものを使用することができ、そのような市販品としては、好ましくは、商品名「スミチームLP-G」(新日本化学工業株式会社)、「プロテアーゼM『アマノ』SD」、「プロテアーゼP『アマノ』3SD」、「プロテアックス」(天野エンザイム株式会社)、「フレーバーザイム(Flavourzyme)」(ノボザイムズジャパン株式会社)を例示することができる。 As the peptidase, a commercially available product can be used, and as such a commercially available product, the product names "Sumiteam LP-G" (Nippon Chemical Industrial Co., Ltd.) and "Protease M" Amano "SD" are preferable. , "Protease P" Amano "3SD", "Proteax" (Amano Enzyme Co., Ltd.), "Flavourzyme" (Novozymes Japan Co., Ltd.) can be exemplified.
　本発明で用いられるペプチダーゼとしては、アスペルギルス・オリゼー由来のもの、至適温度が50℃以上であるもの、至適pHが7～8であるのものが特に好ましく、そのようなペプチダーゼとしてはプロテアックスが挙げられる。また、本発明で用いられるペプチダーゼとしては、Tris-HClバッファーにて抽出、トリクロロ酢酸にて沈殿、アセトンにて洗浄することで得たタンパク質をトリプシン消化、固相カラムで脱塩後、液体クロマトグラフィー質量分析法により、下記分析条件にて分析した際に、特徴的な分子量の化合物イオン(m/z:970.5705[M-H], 980.5435[M-H], 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], 各±5 ppm以内)を有するものも好ましく、そのようなペプチダーゼとしてはプロテアックスが挙げられる。 As the peptidase used in the present invention, those derived from Aspergillus oryzae, those having an optimum temperature of 50 ° C. or higher, and those having an optimum pH of 7 to 8 are particularly preferable, and such peptidases are Proteax. Can be mentioned. As the peptidase used in the present invention, the protein obtained by extracting with Tris-HCl buffer, precipitating with trichloroacetic acid, and washing with acetone is digested with trypsin, desalted on a solid phase column, and then liquid chromatography. When analyzed under the following analytical conditions by mass spectrometry, compound ions with characteristic molecular weights (m / z: 970.5705 [MH], 980.5435 [MH], 541.3723 [MH], 860.3850 [MH], 855.4021 [MH] ], 815.4538 [MH], 1154.6102 [M + H], 595.5052 [M + H], 1208.9261 [MH], 1209.2603 [MH], each within ± 5 ppm) is also preferable, and such a peptidase is a prote. Ax can be mentioned.
(分析条件)
カラム：Acquity BEH C18(Waters:2.1x100 mm, id 1.7μm)カラム温度：40℃
溶媒A：0.1%ギ酸
溶媒B：アセトニトリル
流速：0.3 mL／min
グラジェント: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) (Analysis conditions)
Column: Acquity BEH C18 (Waters: 2.1x100 mm, id 1.7 μm) Column temperature: 40 ° C
Solvent A: 0.1% Formic acid Solvent B: Acetonitrile Flow rate: 0.3 mL / min
Granant: 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)
　蜂の子に対するペプチダーゼの使用量は、使用する蜂の子濃度、酵素力価、反応温度及び反応時間により異なり一概には言えない。一般的には、蜂の子に含まれるタンパク質1 g当り50～10000作用単位の割合でペプチダーゼを用いることが好ましい。なお、このとき、ペプチダーゼの蜂の子への添加は、一度に添加してもよく、少量ずつ分割して添加してもよい。 The amount of peptidase used for bee pups varies depending on the bee pup concentration, enzyme titer, reaction temperature and reaction time used, and cannot be unequivocally stated. In general, it is preferable to use peptidase at a rate of 50 to 10,000 action units per gram of protein contained in bee pups. At this time, the peptidase may be added to the bee cubs at once, or may be added in small portions.
　ペプチダーゼ処理に際して蜂の子の溶液及び分散液のpHは、使用酵素の至適pHに対応して、pH2～12、好ましくはpH7～10、より好ましくはpH7～8.5の範囲から選択される。具体的には、前記蜂の子の溶液及び分散液にペプチダーゼを添加する前に、使用酵素の種類によりpH2～12、好ましくはpH7～10、より好ましくはpH7～8.5の範囲内になるように、酸、アルカリ剤、あるいは緩衝剤の添加により所望のpHに調整される。この場合、酸としては、塩酸、硫酸、硝酸、リン酸、酢酸等を；アルカリ剤としては水酸化ナトリウム、水酸化カリウム、炭酸カリウム等を；また、緩衝剤としては、リン酸緩衝剤、クエン酸緩衝剤等をそれぞれ例示することができる。 The pH of the bee cub solution and dispersion during the peptidase treatment is selected from the range of pH 2 to 12, preferably pH 7 to 10, and more preferably pH 7 to 8.5, depending on the optimum pH of the enzyme used. Specifically, before adding peptidase to the bee cub solution and dispersion, the pH should be in the range of pH 2 to 12, preferably pH 7 to 10, and more preferably pH 7 to 8.5 depending on the type of enzyme used. The pH is adjusted to the desired pH by adding an acid, an alkaline agent, or a buffer. In this case, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid and the like are used as the acid; sodium hydroxide, potassium hydroxide, potassium carbonate and the like are used as the alkaline agent; and phosphoric acid buffer and citric acid are used as the buffering agent. Acid buffering agents and the like can be exemplified respectively.
　ペプチダーゼ処理の温度は、特に制限はなく、好ましくはエンドペプチダーゼ作用及びエキソペプチダーゼ作用の両作用が発現する最適温度範囲を含む、実用に供せられ得る範囲、すなわち、通常20～70℃の範囲から選択される。好ましくは40～70℃、より好ましくは55～70℃の範囲である。ペプチダーゼ処理の時間は、使用酵素の種類、反応温度、pH等の反応条件に依存し、特に限定されない。 The temperature of the peptidase treatment is not particularly limited, and is preferably from a range that can be put into practical use, that is, usually in the range of 20 to 70 ° C., including an optimum temperature range in which both endopeptidase action and exopeptidase action are exhibited. Be selected. The temperature is preferably in the range of 40 to 70 ° C, more preferably 55 to 70 ° C. The time of peptidase treatment depends on the type of enzyme used, the reaction temperature, the reaction conditions such as pH, and is not particularly limited.
　なお、蜂の子は、そのまま、又は水に溶解若しくは分散させた状態でペプチダーゼ処理に供することができる。蜂の子が乾燥形態である場合は、水に溶解させた状態でペプチダーゼ処理に供することが好ましい。 The bee cub can be subjected to peptidase treatment as it is or in a state of being dissolved or dispersed in water. When the bee pups are in a dry form, they are preferably subjected to peptidase treatment in a state of being dissolved in water.
　ペプチダーゼ処理の停止は、ペプチダーゼを失活又は除去することにより行う。失活操作は加熱処理(例えば、80℃で15分間等)により行うことができる。 The peptidase treatment is stopped by inactivating or removing the peptidase. The deactivation operation can be performed by heat treatment (for example, at 80 ° C. for 15 minutes).
　本発明では、蜂の子を少なくとも前述するようにペプチダーゼで処理すればよく、ペプチダーゼ処理だけでなく、本発明の効果を損なわない限りにおいて、その他の酵素との組み合わせ処理、例えば、ペプチダーゼ処理と合わせて糖分解酵素処理などを行うこともできる。 In the present invention, the bee pups may be treated with peptidase at least as described above, and not only the peptidase treatment but also the combination treatment with other enzymes, for example, peptidase treatment as long as the effect of the present invention is not impaired. It can also be treated with a glycolytic enzyme.
　本発明の製造方法により製造される蜂の子酵素処理物は、その後、ドリンク剤、シロップ剤などの液剤の形態に調製されてもよいし、半液体形態、又は固体形態に調製されてもよい。半液体形態としてはペースト状及びゼリー状の形態が、固体形態としては凍結乾燥物(例えば、凍結乾燥粉末)、錠剤(トローチ、チュアブル錠、糖衣錠などを含む)、カプセル、顆粒などの形態を挙げることができる。なお、凍結乾燥物は、ペプチダーゼ処理を行った蜂の子酵素処理物を、凍結乾燥処理に供することによって調製することができる。凍結乾燥処理は、定法に従って行うことができる。 The bee-child enzyme-treated product produced by the production method of the present invention may then be prepared in the form of a liquid such as a drink or a syrup, or may be prepared in a semi-liquid form or a solid form. .. Semi-liquid forms include paste-like and jelly-like forms, and solid forms include freeze-dried products (for example, freeze-dried powder), tablets (including troches, chewable tablets, sugar-coated tablets, etc.), capsules, granules, and the like. be able to. The freeze-dried product can be prepared by subjecting the peptidase-treated bee-child enzyme-treated product to the freeze-drying treatment. The freeze-drying treatment can be carried out according to a conventional method.
　（２）蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法
　本発明の蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法は、蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することにより実施することができる。 (2) Method for reducing allergenicity of Waspeptidase-treated product and suppressing browning during and after production The method for reducing allergenicity of Waspeptidase-treated product and suppressing browning during and after production according to the present invention. It can be performed by treating the bee pups with a peptidase that has endopeptidase and exopeptidase effects.
　ここで対象とする蜂の子、使用するペプチダーゼの種類、ペプチダーゼ処理の条件などについては、（１）で前述した通りである。 The target bee cubs, the type of peptidase used, the conditions for peptidase treatment, etc. are as described above in (1).
　当該方法によって得られる蜂の子酵素処理物は、従来の方法によって得られる蜂の子酵素処理物と同様にタンパク質が低分子化されているにもかかわらず、製造時及び製造後の両方の時点において褐変現象が優位に抑制されている。さらに、このような効果は、常圧条件で且つ単一の酵素を用いた場合であっても得ることができる。 The bee-child enzyme-treated product obtained by the method is similar to the bee-child enzyme-treated product obtained by the conventional method, although the protein is reduced in molecular weight, both at the time of production and after the production. The browning phenomenon is predominantly suppressed in. Furthermore, such an effect can be obtained even under normal pressure conditions and when a single enzyme is used.
　以下、本発明を実施例により詳細に説明する。しかし、本発明はこれら実施例等になんら限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples and the like.
　実施例１
　蜂の子39.5 gをガラスビーカーに量りとり、イオン交換水150 mlを加えて均一になるまで攪拌して蜂の子希釈液を調製した。2N NaOH水溶液を加えて蜂の子希釈液のpHを7.0に調整した。次に、エンドペプチダーゼ作用とエキソペプチダーゼ作用の両方を有するプロテアックス(天野エンザイム株式会社) 1180 mgをイオン交換水10 mlに溶かした溶液を蜂の子希釈液に加え、さらにイオン交換水を全量が285 gになるように加えた。反応混合物をプロペラで撹拌しながら70℃(恒温水槽)で2時間反応させて、加水分解を行った。恒温水槽の温度を80℃に上げて15分間攪拌し酵素を失活させた。 Example 1
39.5 g of bee cubs were weighed in a glass beaker, 150 ml of ion-exchanged water was added, and the mixture was stirred until uniform to prepare a beak cub dilution solution. The pH of the Wasp dilute solution was adjusted to 7.0 by adding a 2N NaOH aqueous solution. Next, add a solution of 1180 mg of Proteax (Amano Enzyme Co., Ltd.), which has both endopeptidase action and exopeptidase action, in 10 ml of ion-exchanged water to the bee dilute solution, and add the total amount of ion-exchanged water. Added to 285 g. The reaction mixture was reacted at 70 ° C. (constant temperature water tank) for 2 hours while stirring with a propeller to carry out hydrolysis. The temperature of the constant temperature water tank was raised to 80 ° C. and the mixture was stirred for 15 minutes to inactivate the enzyme.
　実施例２
　酵素として、プロテアックスに代えて、エンドペプチダーゼ作用とエキソペプチダーゼ作用の両方を有するプロテアーゼP (天野エンザイム株式会社)を使用し、反応温度を40℃とした以外は、実施例１と同様にして、蜂の子酵素分解物を得た。 Example 2
As an enzyme, protease P (Amano Enzyme Co., Ltd.) having both endopeptidase action and exopeptidase action was used instead of Proteax, and the reaction temperature was set to 40 ° C. in the same manner as in Example 1. A bee pup enzymatic degradation product was obtained.
　比較例
　酵素として、プロテアックスに代えて、アクチナーゼAS (科研製薬株式会社)を使用しpHを8.8、反応温度を50℃とした以外は、実施例１と同様にして、蜂の子酵素分解物を得た。 Comparative Example A bee-child enzymatic decomposition product in the same manner as in Example 1 except that Actinase AS (Kaken Pharmaceutical Co., Ltd.) was used instead of Proteax and the pH was 8.8 and the reaction temperature was 50 ° C. Got
　＜酵素分解度測定＞
　ゲルろ過分析を用いて分解後の化合物の分子量を測定することで酵素分解度の確認を行った。酵素反応後溶液を20μL量りとり、9.5 mLの50 mM NaPi/0.3M NaCl buffer (pH7.0)を加え分析サンプルとした。チトクロムC (nacalai tesque、M.W.=12,384)を指標とし、チトクロムCより早く溶出される成分の割合を算出した。分析条件を以下に示す。
カラム：Shodex PROTEIN KW-802.5 (5μm, 8.0 mm i.d.×300 mm, 昭和電工株式会社)、ガードカラム：Shodex PROTEIN KW-G (5μm, 8.0 mm i.d.×10 mm, 昭和電工株式会社)、カラムオーブン：30℃、流速：0.5 mL/min、移動相：A；50 mMリン酸Na/0.3 M NaCl緩衝液(pH7.0), B；なし、溶出：B 0% (60 min hold)、注入量：10μL、検出：UV (220 nm)。 <Measurement of enzymatic decomposition degree>
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 9.5 mL of 50 mM NaPi / 0.3M NaCl buffer (pH 7.0) was added to prepare an analysis sample. Using cytochrome C (nacalai tesque, MW = 12,384) as an index, the proportion of components eluted earlier than cytochrome C was calculated. The analysis conditions are shown below.
Column: Shodex PROTEIN KW-802.5 (5 μm, 8.0 mm id × 300 mm, Showa Denko KK), Guard column: Shodex PROTEIN KW-G (5 μm, 8.0 mm id × 10 mm, Showa Denko KK), Column oven: 30 ° C, flow velocity: 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).
　結果を表１に示す。いずれの実施例においてもチトクロムC以前に見られたピークの割合は、比較例以上の高分解度となることが確認された(表１)。 The results are shown in Table 1. In all the examples, it was confirmed that the ratio of the peaks observed before cytochrome C was higher than that of the comparative example (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　＜ヒスタミン遊離試験(アレルゲン性確認)＞
　実施例及び比較例で得た蜂の子酵素分解物について、CAST法(cellular antigen stimulation test)によって、遊離するヒスタミン量を測定した。 <Histamine release test (confirmation of allergenicity)>
The amount of histamine released was measured by the CAST method (cellular antigen stimulation test) for the bee pup enzymatic decomposition products obtained in Examples and Comparative Examples.
　具体的には、蜂の子に対してアレルギー反応を経験したことのある被験者の血液サンプルに対し、蜂の子の酵素加水分解物を作用させて、遊離するヒスタミン量を測定した。被験者には、本試験内容を記載した説明書を配布し、試験の趣旨及び内容を十分に説明し、被験者本人の自由意志により参加する旨の同意書を得た者に対してのみ、本試験を行った。遊離ヒスタミンの測定は、市販のELISAキット(Histamine ELISA (IMMUNO-BIOLOGICAL LABORATORIES社製))を用いて行った。 Specifically, the amount of histamine released was measured by allowing the enzyme hydrolyzate of the bee cub to act on the blood sample of the subject who had experienced an allergic reaction to the bee cub. This test will be given only to those who have distributed a manual describing the contents of this test to the subjects, fully explained the purpose and contents of the test, and obtained a written consent to participate voluntarily by the subjects themselves. Was done. The measurement of free histamine was carried out using a commercially available ELISA kit (Histamine ELISA (manufactured by IMMUNO-BIOLOGICAL LABORATORIES)).
　結果を図１に示す。実施例１ではヒスタミン遊離値(ng/mL)が比較例に比べて3/5以下となっており、当該方法によって得られる蜂の子酵素処理物は従来の方法によって得られる蜂の子酵素処理物より優位にアレルゲン性を低下できていると考えられる。 The results are shown in Fig. 1. In Example 1, the histamine free value (ng / mL) was 3/5 or less as compared with the comparative example, and the bee-child enzyme-treated product obtained by the method was treated with the bee-child enzyme obtained by the conventional method. It is considered that the allergenicity can be reduced predominantly over the product.
　＜褐変評価＞
　実施例及び比較例の酵素反応後の蜂の子溶液を凍結乾燥し粉末化させ、デジタルカメラで撮影した画像を用いて褐変評価(輝度分析)を行った。撮影は粉末化後及び50℃にて7日間保管後に行った。輝度の算出はカラーピッカーソフトである「FlatColorPicker」を用いて行い、HSL色空間における輝度(%)をその値とした。 <Browning evaluation>
The bee cub solutions after the enzymatic reaction of Examples and Comparative Examples were freeze-dried and pulverized, and browning evaluation (luminance analysis) was performed using images taken by a digital camera. Photography was performed after pulverization and after storage at 50 ° C. for 7 days. The brightness was calculated using the color picker software "Flat Color Picker", and the brightness (%) in the HSL color space was used as the value.
　粉末後の結果を表２に、50℃にて7日間保管後の結果を図２に示す。従来の方法では酵素分解により見られていた輝度の低下、褐変の現象が本発明の方法によって得られる蜂の子酵素処理物では見られず、50℃にて7日間の保管後においても同様に褐変が抑制されていた。 Table 2 shows the results after powdering, and Fig. 2 shows the results after storage at 50 ° C for 7 days. The phenomenon of decrease in brightness and browning, which was observed by the conventional method due to enzymatic decomposition, is not observed in the Wasp enzyme-treated product obtained by the method of the present invention, and the same applies even after storage at 50 ° C. for 7 days. Browning was suppressed.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

Claims (6)

  1. 　蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することを特徴とする、アレルギー性が低減され且つ製造時及び製造後の褐変化が抑制されてなる蜂の子酵素処理物の製造方法。 A bee cub enzyme-treated product, which comprises treating bee pups with a peptidase having an endopeptidase action and an exopeptidase action, which reduces allergenicity and suppresses browning during and after production. Production method.
  2. 　前記ペプチダーゼがアスペルギルス・オリゼー産生ペプチダーゼである、請求項１に記載の方法。 The method according to claim 1, wherein the peptidase is an Aspergillus oryzae-produced peptidase.
  3. 　前記ペプチダーゼの至適温度が50℃以上である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the optimum temperature of the peptidase is 50 ° C. or higher.
  4. 　蜂の子をエンドペプチダーゼ作用とエキソペプチダーゼ作用とを有するペプチダーゼで処理することを特徴とする、蜂の子酵素処理物のアレルギー性低減且つ製造時及び製造後の褐変化抑制方法。 A method for reducing allergenicity of bee pup enzyme-treated products and suppressing browning during and after production, which comprises treating bee pups with peptidase having endopeptidase action and exopeptidase action.
  5. 　前記ペプチダーゼがアスペルギルス・オリゼー産生ペプチダーゼである、請求項４に記載の方法。 The method according to claim 4, wherein the peptidase is an Aspergillus oryzae-produced peptidase.
  6. 　前記ペプチダーゼの至適温度が50℃以上である、請求項４又は５に記載の方法。 The method according to claim 4 or 5, wherein the optimum temperature of the peptidase is 50 ° C. or higher.
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JP2009254348A (en) * 2008-03-28 2009-11-05 Shingy:Kk Method for producing food and drink of processed larva of bee, and food and drink of processed larva of bee
JP2009254363A (en) * 2008-03-28 2009-11-05 Yamada Bee Farm Corp Browning-reduced and enzyme-treated product of larva of bee, loyal jelly and extract thereof, and method for preparing the same
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JP2009254348A (en) * 2008-03-28 2009-11-05 Shingy:Kk Method for producing food and drink of processed larva of bee, and food and drink of processed larva of bee
JP2009254363A (en) * 2008-03-28 2009-11-05 Yamada Bee Farm Corp Browning-reduced and enzyme-treated product of larva of bee, loyal jelly and extract thereof, and method for preparing the same
JP2010030975A (en) * 2008-06-23 2010-02-12 Api Co Ltd Bee larva solubilized product, preparation method thereof, and pharmaceutical product, cosmetic, or food and drink containing the bee larva solubilized product
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