JP2012202966A - Royal jelly quality evaluation method and royal jelly quality evaluation kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a royal jelly quality evaluation method and royal jelly quality evaluation kit capable of easily performing royal jelly quality evaluation in a short time in a royal jelly production place by using a new index which is alternative to decenoic acid.SOLUTION: Royal jelly is diluted by 1,000-fold to 20,000-fold and ultrafiltered with the use of an ultrafilter where molecular weight cutoff is a hundred thousand to two hundred thousands. Then, the percentage content of apisin contained in the royal jelly is measured by the ELISA method by using a polyclonal antibody or a peptide antibody specifically responsive to the apisin.

Description

  The present invention relates to a royal jelly quality evaluation method and a royal jelly quality evaluation kit.

  Royal jelly is a translucent milky substance that is produced in the head gland tissue consisting of hypopharyngeal and maxillary glands of bees (such as Apis mellifera). This secretion is a nutritional food exclusively for the queen bee and larvae, and the period during which this secretion is produced is limited to 6-12 days of age when the worker bee is referred to as a parenting bee. The queen bee lays 2,000 to 3,000 eggs a day using this royal jelly as a bait. In addition, the life of a worker bee that does not use royal jelly as a staple food is 30 to 45 days, whereas the life of a queen bee is as long as 3 to 4 years.

  Royal jelly contains 66% of water and contains various vitamins and minerals in a well-balanced manner, including protein (14%), carbohydrates (12%), and fatty acids (4%). Royal jelly has been clarified in nutritional significance and pharmacological action through many years of research, and clinical trials have been conducted to confirm its usefulness and safety. Royal jelly is widely used as a raw material for cosmetics and quasi-drugs in the form of raw milk, and as a health food material in the form of freeze-dried FD powder, for example, in tablets and powders.

  Conventionally, the content rate of decenoic acid (10-hydroxy-2-decenoic acid) is set as a quality control standard of royal jelly (for example, refer patent document 1). The content of decenoic acid is measured by high performance liquid chromatography (HPLC).

JP 2005-6629 A

  However, decenoic acid is extremely stable even when the royal jelly deteriorates, and can be added to and mixed with the deteriorated royal jelly, so that it is not suitable as an index for evaluating the quality of royal jelly such as deterioration over time. There was a problem. In addition, since HPLC requires a heavy centrifuge, it cannot be transported to royal jelly production sites such as Yamaku. For this reason, after collecting the royal jelly, it has to be transported to a place where the HPLC is located, and there is a problem that it takes time for analysis.

  The present invention has been made paying attention to such problems, and using a new index instead of decenoic acid, the quality evaluation of royal jelly can be performed in a short time and easily at the royal jelly production area. The object is to provide a method and a royal jelly quality evaluation kit.

  In order to achieve the above-mentioned object, the inventors consider that the protein is the largest among the components of royal jelly, 11% to 16%, and this protein plays a major part in the pharmacological action / efficacy of royal jelly. Focusing on this, we conducted an extensive study. As a result, the present inventors have developed a method for easily measuring the content of apicin in royal jelly by ELISA (Eliza) method using apicin occupying most of the royal jelly protein as an index.

  Apicin is a glycoprotein consisting of 6 subunits with a molecular weight of 55,000 and having a molecular weight of about 350,000, accounting for 40% to 65% of the royal jelly protein. Apicin has been reported to have a lot of reports such as growth promotion action and prolongation of survival days in cultured cells, and it is considered that it has important functionality and is deeply related to the quality of royal jelly.

  Apisin is weak against heat, and as shown in Table 1, when physical heat is applied to the raw milk of royal jelly, it has been confirmed that the amount of apicin decreases as the quality of royal jelly deteriorates. It has also been confirmed that when a high purity apicin aqueous solution is lyophilized and then dissolved again and the purity is measured, the amount of apicin is significantly reduced. Thus, since a strong correlation is recognized between the content ratio and the quality of royal jelly, apicin is considered to be a new quality standard marker for royal jelly instead of decenoic acid.

  Therefore, the method for evaluating the quality of royal jelly according to the present invention is characterized in that the content of apicin contained in royal jelly is measured by ELISA using an antibody that specifically reacts with apicin.

  Since the royal jelly quality evaluation method according to the present invention uses, as a new indicator, apicin, which has a strong correlation with the quality of royal jelly, instead of decenoic acid, the quality of royal jelly can be accurately evaluated. In addition, since the ELISA method is used, a heavy apparatus such as a centrifuge is not required, and the quality of the royal jelly can be evaluated in a short time and easily even in a royal jelly production area such as a mountain in China.

  In the method for evaluating the quality of royal jelly according to the present invention, the antibody may be any antibody as long as it reacts specifically with apicin, but is preferably a polyclonal antibody or a peptide antibody. Since the polyclonal antibody or the peptide antibody specifically reacts with apicin, the content of apicin can be accurately measured, and the evaluation accuracy can be improved.

  In the method of evaluating the quality of royal jelly according to the present invention, it is preferable to measure the content of apicin by ELISA after diluting and ultrafiltration of the royal jelly. In particular, it is preferable to dilute the royal jelly 1000 times to 20,000 times and then ultrafilter with an ultrafiltration membrane having a fractional molecular weight of 100,000 to 200,000. In this case, since ultra-fine filtration (monomers) having a small molecular weight other than apicin contained in royal jelly can be removed, the content of apicin can be measured more accurately.

The royal jelly quality evaluation kit according to the present invention is a quality evaluation kit for carrying out the royal jelly quality evaluation method according to the present invention, characterized by having an antibody that specifically reacts with apicin.
Since the royal jelly quality evaluation kit according to the present invention is capable of performing the royal jelly quality evaluation method according to the present invention, it is possible to easily and easily perform the royal jelly quality evaluation in a royal jelly production area such as a mountain in China. it can.

A royal jelly quality evaluation kit according to the present invention includes a diluting means for diluting royal jelly, an ultrafiltration means for ultrafiltering the royal jelly diluted by the diluting means, and an apicin configured to be able to perform an ELISA method. Measuring means, wherein the apicin measuring means comprises: an antibody that specifically reacts with apicin; a microplate that adsorbs the antibody and the royal jelly ultrafiltered by the ultrafiltration means; and the microplate on the microplate. The sample has a microplate reader capable of measuring the absorbance of color development or luminescence, and after diluting the royal jelly with the diluting means and ultrafiltering with the ultrafiltration means, the content of apicin in the diluted royal jelly is determined. Measured by ELISA using the above apicin measuring means, to the royal jelly before dilution For determining the content of Murrell Apishin is preferably quality evaluation kit royal jelly. In this case, the content of apicin can be measured particularly accurately, and the evaluation accuracy can be increased. The ultrafiltration means preferably comprises a desktop device that is easy to carry.
In the present invention, the ELISA method used may be a direct method, a sandwich method, or a competitive method.

  According to the present invention, there is provided a royal jelly quality evaluation method and a royal jelly quality evaluation kit capable of performing a quality evaluation of royal jelly in a royal jelly production area in a short time and easily using a new index instead of decenoic acid. Can do.

It is a graph which shows the titer of the polyclonal antibody used with the quality evaluation method of the royal jelly of embodiment of this invention, and the quality evaluation kit of a royal jelly. FIG. 2 is a diagram showing the results of SDS-PAGE electrophoresis and Western blotting of a polyclonal antibody used in the royal jelly quality evaluation method and the royal jelly quality evaluation kit shown in FIG. 1. (A) The graph which shows the HPLC pattern of apicin standard goods, (b) The graph which shows the HPLC pattern of the water-soluble component of a general royal jelly. It is the amino acid sequence of apicin, and the sequences of candidate peptide candidates (candidates 1 to 3) corresponding to the partial structure of apicin used in the royal jelly quality evaluation method and the royal jelly quality evaluation kit of the embodiment of the present invention.

  Hereinafter, examples using the royal jelly quality evaluation method and the royal jelly quality evaluation kit of the embodiment of the present invention will be described with reference to the drawings. In the Examples, the case where a polyclonal antibody is used and the case where a peptide antibody is used are respectively compared with the analysis values by HPLC.

  First, a polyclonal antibody that specifically reacts with apicin monomer to multimer was prepared, and the obtained polyclonal antibody was used to examine the content of apicin in royal jelly.

[Preparation of polyclonal antibody]
A polyclonal antibody against apicin was prepared as follows.
Pre-collection → (1 week) → Immunization → (2 weeks) → Immunization → (2 weeks) → Initial blood collection (5 mL) → (1 week) → Immunization → (2 weeks) → Second blood collection → Immunization → 2 weeks → All Blood collection For the injection of antigen (purified apicin), 0.3 mg / 0.5 mL + TiterMax 0.5 mL was emulsified and injected into three backs of rabbits. In whole blood collection, about 50 mL of whole body blood was collected, and serum was obtained by centrifugation at 3,000 rpm for 30 minutes.

  The titer of the antibody thus prepared is shown in FIG. The results of SDS-PAGE electrophoresis and Western blotting are shown in FIG.

[Preparation of royal jelly sample]
A royal jelly sample for quality evaluation was prepared as follows. First, 0.1 g of royal jelly raw milk was accurately weighed by dilution means, and water was added to make a total amount of 1 g. After this royal jelly diluted solution was well stirred, it was further diluted 100 to 1,500 times. When performing ultrafiltration, this diluted solution is passed through an ultrafiltration membrane (product name “apollo”) having a molecular weight cut-off of 150,000 by ultrafiltration means, and centrifuged (3,000 G). A sample was prepared for ELISA. When the ultrafiltration was not performed, the diluted solution was used as it was as a sample for ELISA. When the polyclonal antibody was diluted 15,000 times in the royal jelly and the peptide antibody was diluted 5,000 times in the royal jelly, it was within the range of the calibration curve.

[ELISA method]
The ELISA method was performed by the direct method or the sandwich method.
In the direct method, first, 50 μL of the prepared royal jelly sample and apicin standard (50 μg / mL to 300 μg / mL) are added to the wells of the ELISA microplate and adsorbed at 37 ° C. for 1 hour. To that well, add 2% skim milk solution in an amount that fills all wells. After the adsorption is stopped, the skim milk solution is removed, and a sufficient amount of PBS (including 0.05% tween 20) is added and washed. Repeat three times. After removing the washing solution, 50 μL of polyclonal antibody (0.01 mg / mL) is added and adsorbed at 37 ° C. for 1 hour. After removing the antibody solution, a sufficient amount of PBS (containing 0.05% tween 20) is added to wash all wells. Repeat three times. After removing the washing solution, 100 μL labeled (peroxidase) antibody (goat-derived anti-rabbit IgG antibody; manufactured by Funakoshi, diluted 1000-fold) is added and adsorbed at 37 ° C. for 1 hour. After removing the labeled antibody solution, an amount of PBS (including 0.05% tween 20) that fills all wells is added and washed. Repeat three times. After removing the washing solution, 100 μL of a substrate (hydrogen peroxide) solution is added, and color is allowed to develop at 37 ° C. for 15 minutes. Add 100 μL of stop solution (0.01% sodium azide) to stop color development. Absorbance (415 nm) is measured with a microplate reader.

  The sandwich method follows the direct method. A part of the antibody is used for the solid phase of the plate, the remaining antibody is labeled with biotin to prepare a sandwich method kit, and the absorbance is measured by the same operation as in the direct method.

  The polyclonal antibody, ELISA microplate and microplate reader constitute the apicin measurement means of the royal jelly quality evaluation kit of the embodiment of the present invention. Also, the royal jelly quality evaluation kit of the embodiment of the present invention is constituted by the diluting means, the ultrafiltration means and the apicin measuring means. The ultrafiltration means consists of a desktop device that is easy to carry.

[HPLC (High Performance Liquid Chromatography)]
In order to evaluate the analysis result by the royal jelly quality evaluation method of the embodiment of the present invention, chemical analysis by HPLC was also performed. HPLC was performed by a conventional method.

  The royal jelly supernatant used in HPLC was prepared as follows. First, 3 g of royal jelly raw milk was accurately weighed and water was added to make a total amount of 100 g. The royal jelly diluted solution is stirred well for about 30 minutes, and then centrifuged at 10,000 G for 30 minutes while cooling at 4 ° C. The obtained supernatant was passed through a filtration filter (product name “Ultrafree-MC”; manufactured by MILLIPORE) to prepare an HPLC sample.

The analysis conditions of HPLC are as follows.
Column: TSK-gel G3000SW (7.5mm ID. X 60cm)
Mobile phase: 0.1 M sodium phosphate buffer (pH 7.0)
+0.1 MNa ₂ SO ₄
Flow rate: 0.6ml / min
Detector: UV detector (280 nm)
Column temperature: room temperature Calibration curve: A calibration curve was prepared from the solid content weight of the apicin standard and the HPLC peak area of the apicin solution diluted at each stage.
In addition, the HPLC pattern of an apicin standard product is shown in FIG. 3 (a), and the HPLC pattern of the water-soluble component of a general royal jelly is shown in FIG. 3 (b).

[Apicin analysis results]
The amount of apicin contained in the royal jelly raw milk was measured by the royal jelly quality evaluation method of the embodiment of the present invention using a polyclonal antibody, and the results are shown in Table 2. For comparison, the results of analysis by HPLC are also shown in Table 2. The ELISA method was a direct method.

  As shown in Table 2, the amount of apicin measured by the method for evaluating the quality of royal jelly according to the embodiment of the present invention using a polyclonal antibody is an analytical value almost equivalent to the amount of apicin by HPLC method of 7.17 g. It was confirmed. The analytical value before passing through the ultrafiltration membrane is 9.73 g, whereas the analytical value of the amount of apicin after passing through the ultrafiltration membrane is 7.54 g, and royal jelly contains an apicin-related substance. It was confirmed that a more accurate amount of apicin can be analyzed by ultrafiltration.

  Next, peptide antibodies that specifically react with apicin monomers to multimers were prepared, and the apicin content in royal jelly was examined using the obtained peptide antibodies.

[Production of peptide antibody]
A peptide antibody against apicin was prepared in accordance with the polyclonal antibody production method of Example 1. First, a peptide corresponding to the partial structure of apicin was prepared, and a peptide antibody was prepared using the peptide. The amino acid sequence of apicin and the sequences of peptide antibody candidates (candidates 1 to 3) corresponding to the partial structure of apicin are shown in FIG.

[Apicin analysis results]
The amount of apicin contained in the royal jelly raw milk was measured by the royal jelly quality evaluation method of the present embodiment using peptide antibodies, and the results are shown in Table 3. For comparison, the results of analysis by HPLC are also shown in Table 3. The preparation of the royal jelly sample, the ELISA method, and the HPLC method were performed in the same manner as in Example 1. The sample for ELISA was prepared by diluting royal jelly 5,000 times. The ELISA method was a direct method.

  As shown in Table 3, the peptide antibody is considered to be more specific to apicin than an antibody prepared using apicin protein as an antigen.

  As described above, the royal jelly quality evaluation method and the royal jelly quality evaluation kit (ELISA method) according to the embodiment of the present invention obtained results consistent with the analysis results of the HPLC method. Since the HPLC method uses the standard product of apicin, the most accurate quantification is possible, but it may not be possible because it is not always equipped in the mountainous area overseas. On the other hand, the royal jelly quality evaluation method and the royal jelly quality evaluation kit according to the embodiment of the present invention do not require a heavy device such as a centrifuge and dilute with water at the time of sample preparation. Therefore, the quality of royal jelly can be easily confirmed on the spot. Even when ultrafiltration is performed, it can be carried easily by moving it to the back of a mountain by using a desktop apparatus. As described above, according to the royal jelly quality evaluation method and the royal jelly quality evaluation kit of the embodiment of the present invention, the royal jelly milked in an apiary such as a mountain back in China, or the royal jelly stored for making a lot. Can be immediately evaluated on the spot.

  Since the royal jelly quality evaluation method and the royal jelly quality evaluation kit according to the embodiment of the present invention use apicin as a new indicator instead of decenoic acid, the quality of the royal jelly is improved. Can be evaluated accurately.

In addition, when ultrafiltration is performed, it is possible to remove a low-molecular-weight contaminant (monomer) other than apicin contained in royal jelly, so that the content of apicin can be measured more accurately. it can. That is, royal jelly contains related substances such as monomer (molecular weight 55,000) in addition to apicin (molecular weight 350,000), and when the combined analysis results are reflected, the accurate amount of apicin is The value may not be calculated. In order to prevent this, the sample before the ELISA measurement is passed through an ultrafiltration membrane having a molecular weight cut-off of 150,000 and centrifuged (3,000 G), for example. (Mer) can be removed, and a more accurate value can be obtained.

Claims (6)

  A method for evaluating the quality of royal jelly, characterized by measuring the content of apicin contained in royal jelly by ELISA using an antibody that specifically reacts with apicin.   The method of claim 1, wherein the antibody is a polyclonal antibody or a peptide antibody.   3. The method for evaluating the quality of a royal jelly according to claim 1, wherein the royal jelly is diluted and ultrafiltered, and then the content of apicin is measured by an ELISA method.   The method for evaluating the quality of a royal jelly according to claim 3, wherein the royal jelly is diluted 1000 to 20,000 times and then ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 100,000 to 200,000.   5. A royal jelly quality evaluation kit for carrying out the royal jelly quality evaluation method according to claim 1, comprising an antibody that specifically reacts with apicin. Dilution means for diluting royal jelly,
Ultrafiltration means for ultrafiltration of royal jelly diluted by the dilution means;
An apicin measuring means configured to be able to perform the ELISA method,
The apicin measurement means comprises: an antibody that reacts specifically with apicin; a microplate that adsorbs the antibody and the royal jelly ultrafiltered by the ultrafiltration means; and the absorbance of color development or luminescence of the sample on the microplate A microplate reader capable of measuring
After diluting the royal jelly with the diluting means and performing ultrafiltration with the ultrafiltration means, the content of apicin in the diluted royal jelly is measured by the ELISA method using the apicin measuring means, and is contained in the royal jelly before dilution. A royal jelly quality evaluation kit for determining the content of apicine.