KR101418067B1 - Preparation method of hypoallergenic royal jelly by enzyme treatment - Google Patents

Abstract

The present invention relates to a manufacturing method of hypoallergenic royal jelly by enzyme treatment wherein the royal jelly contains reduced allergenic material. By the preparation method, without separate processes such as pH control, 10-HDA (10-hydroxy-2-decenoic acid), which is a bioactive component included in royal jelly, and allergenic materials are effectively decomposed so that hypoallergenic royal jelly with allergenic material reduced to an amount which substantially does not express allergenic components is easily manufactured.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for preparing hypoallergenic royal jelly,

The present invention relates to a method for producing a low allergic royal jelly which reduces allergenic substances contained in royal jelly.

Royal Jelly is a milky, opaque creamy substance that is secreted from the pharynx after the worker is ingesting honey and pollen and between 3 and 12 days of age. The average content of water is 50 ~ 60%, protein is 18%, carbohydrate is 15%, fat is 3 ~ 6%, and carbohydrate is 3 ~ 6% 10-HDA (trans-10-hydroxy-2-decenoid acid), which is a specific fatty acid of royal jelly, is used as a representative index component of royal jelly. In addition, polyphenols polyphenol compounds such as apigenin, acacetin, galangin, kaempferol, quercetin and the like are contained in royal jelly.

The efficacy of royal jelly has been reported in the prevention of hyperlipidemia and arteriosclerosis, fatigue recovery, liver and kidney toxicity mitigation, aging prevention, immunity enhancement, antidiabetic, anti-cancer, inflammation relief, nerve cell protection effect, The results of research on skin antioxidation, moisturizing, and wrinkle relieving effects by ingestion have been reported. Studies have shown that 10-HDA content of royal jelly affects the physiological activities related to skin cosmetics (Journal of Medicinal Food. 14 (9): 899-906 (2011)), In functional foods, 10-HDA content is used as a reference standard for manufacturing royal jelly raw materials and products.

On the other hand, Royal Jelly contains various allergens (Allergen) that cause an allergic reaction to the body. The allergenic substance contained in the royal jelly can be classified into various protein bands in the vicinity of 47 to 55 kDa as analyzed by SDS-PAGE. A protein having a molecular weight of 55 kDa is known as a major allergic substance, and 4.5 kDa It has been studied that over peptide components also contain ingredients that act as allergic substances. (Tropical Biomedicine 25 (3): 243-251 (2008)) Due to these ingredients, royal jelly may cause allergies, and thus, despite the various usefulness of the royal jelly described above, the royal jelly It is causing the food industry to shrink.

Therefore, it is important to reduce the allergic properties of royal jelly. Among them, royal jelly is treated with proteolytic enzyme and saccharolytic enzyme to destroy or modify the structure of the allergic substance acting as an antigen, There is a way to reduce.

However, the above method is characterized in that royal jelly is divided into first and second reactions in different reaction pH and temperature conditions. In this case, a preparation process such as adding salts unnecessary for physiological activity to control the reaction pH, There is a fear that the physiologically active substance useful for the human body is lost during the primary / secondary reaction.

Therefore, the present invention can easily and easily reduce the allergenicity of royal jelly by performing the enzyme treatment in a single reaction bath without adjusting the pH separately, and also can be used as a useful physiologically active ingredient, 10-hydroxy-2-decenoic acid acid, which is capable of effectively inhibiting the degradation of the allergic royal jelly.

In order to achieve the above object, the inventors of the present invention have found that when the proteolytic enzyme and the non-oligosaccharide degrading enzyme are simultaneously treated in a single reaction vessel during royal jelly processing, each of the enzymes efficiently decomposes the substrate in a site- (10-hydroxy-2-decenoic acid), a useful physiologically active ingredient contained in royal jelly, in a single process under a single temperature condition without any additional steps such as pH adjustment, It is possible to provide a hypoallergenic royal jelly having allergenicity reduced to such an extent that the allergen component is not substantially expressed, thereby completing the present invention.

Accordingly, a preferred embodiment of the present invention provides a method for producing a hypoallergenic royal jelly, which comprises simultaneously treating a royal jelly with a protease and a non-oligosaccharide degrading enzyme.

The royal jelly which can be used is not particularly limited, and it is possible to use the raw royal jelly in the form of jelly, or the royal jelly powder which is obtained by lyophilizing the jelly in a state of being collected from the undiluted solution.

The major allergens included in royal jelly are the 55 kDa molecular weight proteins and the proteins with a molecular weight of 4 kDa or more and less than 55 kDa which are suspected of being allergic.

Therefore, the hypoallergenic royal jelly prepared according to a preferred embodiment of the present invention is preferably composed of a protein having a molecular weight of less than 4 kDa or a protein component of not less than 4 kDa and less than 55 kDa substantially not being expressed .

The treatment of the proteolytic enzyme and the non-electrolytic polysaccharide degrading enzyme is preferably performed by a single treatment process, that is, a one-pot process, in the pH range when the royal jelly is dissolved in water without pH adjustment step.

As described above, according to a preferred embodiment of the present invention, there is no need to prepare royal jelly as a pretreatment or an intermediate step in a primary / secondary reaction by varying the reaction pH and temperature conditions. Therefore, It is possible to prevent the introduction of unnecessary salts and to simplify the manufacturing process and to prevent the loss of physiologically active substances useful in the human body.

Preferably, the enzyme treatment may be carried out at 40 to 60 < 0 > C. If the temperature is lower than the above range, the efficiency of the enzyme is lowered and the overall reaction efficiency is lowered. If the temperature is higher than the above range, the enzyme may be denatured.

Also, preferably, the enzyme treatment can be carried out for 2 to 15 hours, more preferably 7 to 12 hours for sufficient decomposition of the allergen.

Preferably, the enzyme treatment can be carried out while stirring to further facilitate the contact between the allergen and the enzyme. The stirring may preferably be performed at 250 to 350 rpm.

The protease may preferably be at least two selected from the group consisting of a serine endo-type protease, a proline endo-type protease, and an exo-type protease.

These three types of proteolytic enzymes decompose the inside and the outside of the protein to effectively decompose the proteins that can become allergens. Therefore, preferably, the enzyme treatment step may include using two or more types of endo-type proteases or exo-proteases as the protease, and more preferably the above three types.

As the serine-based endo-type protease, pepsin derived from pigs can be preferably used. As the proline endo-type protease, preferably, it is derived from Aspergillus niger The exo-type protease can be preferably selected from the group consisting of Aspergillus oryzae ) can be used.

As the non-electrolytic polysaccharide degrading enzyme, those derived from Aspergillus niger can be preferably used, and more preferably, pectinase derived from Aspergillus niger ( pectinase, cellulase, hemicellulase, or beta-glucosidase may be used.

Preferably, the protease is treated at a concentration of 0.2 to 4.5 parts by weight, more preferably 0.2 to 3 parts by weight, based on 100 parts by weight of royal jelly. When two or more kinds of protease are used together, Preferably, each enzyme may be treated at a concentration of 0.1 to 1.5 parts by weight, preferably 0.1 to 1 part by weight, based on 100 parts by weight of royal jelly.

Preferably, the non-electrolytic polysaccharide degrading enzyme is treated at a concentration of 0.1 to 1.5 parts by weight, preferably 0.1 to 1 part by weight based on 100 parts by weight of royal jelly.

If the concentration of the enzyme is lower than the above range, the reaction rate becomes too slow and the reaction is not completely completed. If the concentration of the enzyme is higher than the above range, economical efficiency is lowered compared to the effect obtained.

After the enzyme treatment process is completed, the process may further include a step of inactivating (inactivating) or removing the enzyme for commercialization.

The enzyme inactivation step may be carried out using conventional physicochemical and enzymatic deactivation methods known in the art such as temperature elevation and pH adjustment. For example, in the temperature range of 65 to 100 ° C, preferably 90 to 100 ° C, Deg.] C for 60 minutes, preferably 10 to 30 minutes, to deactivate the protease and non-oligosaccharide degrading enzyme used. However, when the enzymes participating in the reaction at each step are completely reacted, the enzyme inactivation step may be omitted as necessary.

According to the preparation method of the present invention, 10-HDA (10-hydroxy-2-decenoic acid), which is a useful physiologically active ingredient contained in royal jelly, can be effectively decomposed while retaining the content of 10-hydroxy- It is possible to more easily and easily produce the hypoallergenic royal jelly in which the allergenicity is reduced to such an extent that the allergen component is not substantially expressed.

FIG. 1 shows SDS-PAGE analysis of the degree of reduction of allergenic substances according to Example 1 of the present invention (control group: 2-fold dilution of raw royal jelly (enzyme-free group)).
FIG. 2 is a graph showing SDS-PAGE analysis of the degree of reduction of allergenic substances according to the simultaneous treatment of proteolytic enzymes and carbohydrase according to Example 2 of the present invention (Control group: 2-fold dilution of raw royal jelly Treated group), enzyme mix: enzyme mixture itself before treatment with royal jelly).

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to facilitate understanding of the present invention. However, the following examples are merely illustrative of the present invention in order to more clearly understand the present invention, and the scope of the present invention is not limited by the following examples.

< Example  1> Hypoallergenic Royal jelly  Selection of enzymes for manufacture

The following process was performed to select enzymes that efficiently decompose a protein of 55 kDa molecular weight, which is a major allergen contained in royal jelly, and a protein whose molecular weight is more than 4 kDa and less than 55 kDa, which are suspected to be allergens.

First, a solution prepared by diluting raw royal jelly (purchased from Ansung Bee Farm) used as a raw material with water corresponding to 2 times the weight of the raw royal jelly was prepared and then heated to 50 캜 without any pH adjustment Then, 0.4 part by weight of the protease and glucosease of Table 1, purchased from Vision Biochem Co., Ltd., were added to 100 parts by weight of raw royal jelly, respectively, and reacted at 50 ° C. After completion of the enzyme reaction, And the enzyme was inactivated and used as a sample for SDS-PAGE.

Selection of enzymes for the production of hypoallergenic royal jelly Classification Enzyme Product Name Enzyme derived Action site Experiment 1 protein
Lytic enzyme BC Pepsi 1: 3000 Porcine gastric mucosa Serine protease Experiment 2 Pan creep sin Porcine pancreas protease,
α-amylase, lipase Experiment group 3 Delvolase Bacillus licheniformis Protease Experiment group 4 Protex 7L Bacillus amyloliquefaciens endo-peptidase Experiment group 5 Prozyme 2000P Aspergillus oryzae exo-peptidase Experiment group 6 Brewers Clarex Aspergillus niger Proline-specific
endo-protease Experiment group 7 Collupulin MG Carica papaya endo-protease Experiment group 8 Bromelain 1200GDU pineapple endo peptidase Experiment group 9 Non-functional
Polysaccharide
Lytic enzyme Filtrase Premium L Trichoderma reesei β-glucanase.
Cellulase Experiment group 10 Rohament CL Trichoderma reesei cellulase,
β-glucanase, hemicellulase Experiment group 11 Cytolase PCL5 Aspergillus niger pectinases cellulase, hemicellulase,
β-glucosidase Control group Fresh royal jelly 2 times dilution (Enzyme free treatment group)

The sample obtained by treating with the enzyme shown in Table 1 was prepared by adding 5 × SDS sample buffer containing bromophenol blue and heating at 100 ° C. for 5 minutes to prepare a 10 to 14% polyacrylamide gel SDS-PAGE analysis was performed using an electrophoresis system (BIO-RAD protean® system). After electrophoresis, the gel was stained with Brilliant blue and decolorized with a solution containing 30% (v / v) methanol and 10% (v / v) acetic acid. The photograph of the stained and discolored gel is shown in FIG. 1, and the degree of color development of a protein band having a molecular weight of 55 kDa and a protein band having a molecular weight of 4 kDa or more and less than 55 kDa was visually confirmed and shown in Table 2 below. In Table 2, the bands developed through the staining and discoloration process were shown in five stages (+) to (+++++) according to the degree of banding compared with the control group, and for the examples showing no significant difference, .

Assess the degree of reduction of allergenic substances 55 kDa 4 kDa or more and less than 55 kDa Experiment 1 + +++ Experiment 2 no difference no difference Experiment group 3 no difference no difference Experiment group 4 no difference no difference Experiment group 5 ++++ +++++ Experiment group 6 ++ ++ Experiment group 7 no difference no difference Experiment group 8 no difference no difference Experiment group 9 no difference no difference Experiment group 10 no difference no difference Experiment group 11 +++++ +

According to the results shown in Table 2, when the peptide was reacted at the same pH (3.3) and at the temperature (50 ° C), the peptides of 5 and 11 in the test groups were degraded and the degree of color development of the band was markedly decreased. , 6, and 11 showed that the peptides of 4 kDa or more and less than 55 kDa were completely degraded, and it was confirmed that they are effective enzymes to reduce allergic substances of royal jelly.

As can be seen from the results of Table 2, it can be confirmed that there is a difference in the degree of degradation of allergenic substances according to specific enzyme types among general protein and non-ionic polysaccharide degrading enzymes. The difference is that there is no study on allergenic substances for domestic royal jelly, and there is no suitable enzyme treatment condition, indicating a need to propose a new manufacturing method for allergenic substance decomposition of domestic royal jelly.

< Example  2> Protease and Non-functional  Reduction of allergic substances by simultaneous use of polysaccharide degrading enzyme

In order to reduce the allergic substances of royal jelly, protease and non-oligosaccharide degrading enzyme are separately reacted through the first and second reaction as in the conventional method, and at the same pH and temperature , Three kinds of protease enzymes (serine protease, exo-type protease derived from Aspergillus oryzae , and Aspergillus niger- derived proline-specific endo-type protease) and one non-oligosaccharide degrading enzyme ( Aspergillus niger- derived non- ionic polysaccharide degrading enzyme) was added at a concentration of 0.4 parts by weight to the total amount of 1.6 parts by weight based on 100 parts by weight of raw royal jelly, and the mixture was reacted at the same time, followed by SDS-PAGE, Was evaluated. A sample was taken and analyzed according to the reaction time in order to understand the degree of decomposition according to the reaction time, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that as the reaction time was longer, the 55 kDa peptide was degraded and the degree of color development of the band was decreased. Also, the color development degree of the band of 4 kDa or more and less than 55 kDa was also decreased. In addition, when compared with the results obtained by using protease alone or nonpolysaccharide degrading enzyme alone, the degree of color development of the band was markedly decreased by the joint use, and it was confirmed that the mixed use was more effective than using the enzyme alone. Above all, it was confirmed that the enzymes were treated separately at the same time by treating the enzymes simultaneously, and the reduction effect of the allergens was excellent without complicated processes in which the enzymes were separately reacted through the first and second reactions and subjected to the pH adjustment process for each reaction.

< Example  3> Enzyme treatment Royal jelly  The physiologically active substance (10- HDA ) analysis

In order to reduce the allergenic substance of royal jelly, a sample obtained through the simultaneous treatment of protease and non-oligosaccharide degrading enzyme according to Example 2 was used to change the component of 10-HDA, a physiologically active ingredient of royal jelly Respectively. This is to confirm that the decrease in allergic properties of royal jelly, which can be caused by the enzyme treatment, is suppressed.

The content of 10-HDA was analyzed by 10-HDA content in the sample of Example 2 using standard 10-HDA (Nacalai tesque, Japane) reagent using Liquid Chromatography.

A sample corresponding to 200 mg was taken in the above-mentioned pretreatment process, put into a 100 ml volumetric flask, and 10 ml of water was added, and the mixture was heated to 50 캜 and shaken. About 70 ml of methanol was added to the mixture, and the mixture was subjected to ultrasonic shaking (BRANSONIC® using a sonicator) for about 30 minutes, and methanol was added thereto to adjust the volume to 100 ml. This solution was filtered and used as the HPLC analytical solution. As a control, raw royal jelly was used as a comparative example through the same pretreatment process.

The column used for the analysis was μ-Bondapak C-18, and the mobile phase was detected at 214 nm absorbance at a flow rate of 1.4 ml / min using 0.02 M (NH ₄ ) ₂ HPO ₄ and methanol 7: 3 mixture. The 10-HDA content was expressed as a percentage by weight in terms of the amount of 10-HDA detected in each of the weighed samples (Table 3).

10-HDA analysis of enzyme-treated royal jelly Enzyme treatment
Reaction time In the enzyme-treated reaction solution of Example 2
10-HDA content (wt%) Comparative Example
10-HDA content (wt%) Comparative Example
(Raw royal jelly) 0.84 100.0 0 hr 0.88 105.9 1 hr 0.87 103.9 2 hr 0.81 96.8 3 hr 0.84 100.5 4 hr 0.82 98.5 5 hr 0.86 102.7 6 hr 0.86 102.9 7 hr 0.85 102.2 8 hr 0.86 103.2 12 hr 0.86 102.9

(* Above 100% by weight in Table 3 represents the calculated value by error)

As shown in Table 3 above, the 10-HDA content of the enzyme-treated royal jelly according to Example 2 was analyzed, and it was confirmed that the content of 10-HDA was kept at the same level within the error range as compared with the raw royal jelly .

Accordingly, it is possible to provide royal jelly which is excellent in health and beauty functional efficacy without fear of causing allergies by keeping the physiologically active ingredient of royal jelly as it is, as well as reducing allergic substances according to a preferred embodiment of the present invention .

Claims (12)

A method for producing a hypoallergenic royal jelly characterized by treating royal jelly together with a proteolytic enzyme and a non-polar polysaccharide degrading enzyme derived from Aspergillus niger ,
Wherein the protease comprises a serine endo-type protease, a proline endo-type protease, and an exo-type protease, wherein the molecular weight of the royal jelly is from 4 kD to 55 kD &Lt; / RTI &gt;
The non-polar polysaccharide degrading enzyme is selected from the group consisting of pectinase, cellulase, hemicellulase, and beta-glucosidase derived from Aspergillus niger . &Lt; / RTI &gt; delete The method of claim 1, wherein the enzyme treatment is performed at 40 to 60 占 폚. delete The method according to claim 1, wherein the serine-based endoprotease is pepsin derived from pigs. 2. The method according to claim 1, wherein the proline-based endo-type protease is derived from Aspergillus niger . The method according to claim 1, wherein the exo-type protease is selected from the group consisting of Aspergillus oryzae. &lt; / RTI &gt; delete delete The method according to claim 1, wherein the protease is treated at a total concentration of 0.2 to 4.5 parts by weight based on 100 parts by weight of the royal jelly. The method according to claim 1, wherein the non-electrolytic polysaccharide degrading enzyme is treated at a concentration of 0.1 to 1.5 parts by weight based on 100 parts by weight of the royal jelly. The method according to claim 1, wherein the royal jelly is fresh royal jelly or freeze-dried royal jelly

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