JP2002335884A - Marker for freshness of royal jelly - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a marker for indicating the freshness of royal jelly. SOLUTION: A low-molecular weight protein having a molecular weight of <10,000 contained in the royal jelly is usable as the freshness marker of the royal jelly. The freshness of a royal jelly is judged when the ratio of the low-molecular weight protein a molecular weight of <10,000 or thereabout into the total weight of the proteins in the royal jelly is <=44 wt.%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an indicator of freshness of royal jelly.

[0002]

[Prior art]

[0003] Royal jelly is known as a material having a nourishing and tonic effect, and health foods containing it as a main component are widely sold. However, all of its physiological activities have not yet been determined. Therefore, its physiological activities include
At present, the lot difference is large and the activity value is not controlled. Because it is a natural substance, there is a case where the composition of the content component varies, and it is a problem whether or not the active ingredient is correctly mixed. In particular, like royal jelly, its effectiveness is clearly recognized, and it is composed of multiple components.
It is difficult to determine which substances to analyze and evaluate for effectiveness and quality as indicators of freshness. Therefore, in terms of stability of preparations and quality, especially for royal jelly blended in health foods and the like, it has been desired to formulate an index of freshness as a standard for the degree of effectiveness of each lot and the deterioration of itself.

Meanwhile, in royal jelly, 1) TSK gel G3000SW (7.5 × 30 cm) manufactured by Tosoh Corporation
Was used as a column, and 0.3M sodium chloride, 0.1M was used.
0.1 M phosphate buffer containing 0.05% sodium azide (pH
7.0) was used as a developing solution, the flow rate was set to 0.3 ml / min, the column temperature was set to 35 ° C., and the retention time was 35 in high-performance liquid chromatography in which the absorbance was detected at 280 nm.
This protein has a peak at ~ 45 minutes and has a molecular weight of about 10
Low molecular weight proteins of less than 000. 2) Tricine SD
The existence of low-molecular-weight proteins having a molecular weight of less than about 10,000 as measured by S-polyacrylamide gel electrophoresis is not known, and the origin of the low-molecular-weight proteins having a molecular weight of less than about 10,000 is high due to heat. It was not known that it was produced by the decomposition of a molecular weight protein, and the various physiological activities of royal jelly were reduced accordingly. Furthermore, it has not been known that the low molecular weight protein having a molecular weight of less than about 10,000 serves as an indicator of the freshness of royal jelly.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and has as its object to provide an indicator substance of royal jelly freshness.

[0006]

In view of such a situation, the present inventors have made intensive research efforts, and as a result, the indicator substance of freshness in royal jelly is a low molecular weight protein having a molecular weight of less than about 10,000, and the protein has a low molecular weight. Molecular weight 100 due to heat
It has been found that various biological activities of royal jelly are reduced due to the decomposition of high molecular weight proteins of at least 00, and the present invention has been completed. Hereinafter, the protein may be simply referred to as “low molecular weight protein having a molecular weight of less than about 10,000” or “low molecular weight protein having a molecular weight of less than 10,000”. That is, the present invention relates to the following technology. (1) An indicator of royal jelly freshness, characterized by using a low molecular weight protein having a molecular weight of less than about 10,000 in royal jelly as an indicator. (2) The indicator substance of royal jelly freshness according to (1), wherein the low molecular weight protein having a molecular weight of less than about 10,000 has the following properties.
1) TSK Gel G3000SW (7.
5 × 30 cm) as a column, a developing solution of 0.1 M phosphate buffer (pH 7.0) containing 0.3 M sodium chloride and 0.05% sodium azide, and a flow rate of 0.3 ml
/ Min, a protein having a peak at a retention time of 35 to 45 minutes in high performance liquid chromatography in which the column temperature is set at 35 ° C. and the absorbance is detected at 280 nm.
Low molecular weight proteins having a molecular weight of less than about 10,000. 2)
It is a low molecular weight protein having a molecular weight of less than about 10,000 as measured by Tricine-SDS polyacrylamide gel electrophoresis. (3) The indicator substance of royal jelly freshness according to (1) or (2), wherein the indicator substance shows freshness of royal jelly. (4) The low-molecular-weight protein having a molecular weight of less than about 10,000, which is the indicator of freshness according to any one of (1) to (3), accounts for 44% by weight or less of the total protein mass in royal jelly. An indicator of royal jelly freshness, characterized by the following: Hereinafter, the present invention will be described focusing on embodiments.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (1) Royal jelly which is an essential component of the composition of the present invention The composition of the present invention is characterized by using royal jelly as an essential component. The chemical composition of royal jelly varies slightly depending on the place of production, but the water content is 65-70%, protein 15-20%, carbohydrate 10-15%, fat 1.7-6%, ash content 0.7-2. %. The biological and pharmacological effects of royal jelly include anti-aging, enzymatic, antibacterial, antitumor,
The effects on the circulatory system are known. The royal jelly of the present invention is characterized in that the ratio of low molecular weight proteins having a molecular weight of less than about 10,000 to the total protein mass in the royal jelly is 44% by weight or less. This protein is the first component identified in the royal jelly by the present inventors. This protein is produced by the decomposition of a high molecular weight protein having a molecular weight of about 10,000 or more by heat, and has a low molecular weight of less than about 10,000. As the protein content increases, various biological activities of royal jelly decrease. As a physiological activity of royal jelly, for example, it has been found that royal jelly exerts activities such as a limiting momentum enhancing effect, a hepatocyte proliferation promoting effect, a blood ammonia concentration suppressing effect, a blood lactate accumulation suppressing effect. Here, as for the content of the protein, in order to sufficiently exert the above-mentioned effect, the ratio of the low molecular weight protein having a molecular weight of less than about 10,000 to the total protein mass in the royal jelly is at most 44% by weight. % Is preferable. This protein can be quantified by high performance liquid chromatography using a GPC column.
That is, the degree of the effect of royal jelly can be determined by the amount of the indicator substance. Furthermore, immediately after collection of royal jelly, it is stored over time, and when this indicator substance is quantified, the content of this indicator substance monotonically increases, especially at high temperatures of 40 ° C. and 50 ° C. Since the various biological activities of royal jelly are similarly reduced, a molecular weight of about 10
It can be seen that low molecular weight proteins of less than 000 are indicators of royal jelly freshness.

(2) Analysis of indicator substance of royal jelly freshness by high performance liquid chromatography The low molecular weight protein having a molecular weight of less than about 10,000 in the royal jelly of the present invention is characterized in that it can be identified by high performance liquid chromatography. The above indicator substances are
Identification and identification even by gel filtration using high performance liquid chromatography
It can be quantified. Therefore, such analysis results can be used for quality control and for discrimination / evaluation of effects. Such gel filtration analysis may be carried out according to a generally known method. As such a preferable example, for example, using TSK gel G3000SW manufactured by Tosoh Corporation as a column, 0.3 M sodium chloride, 0 M .0
0.1 M phosphate buffer containing 5% sodium azide (pH
7.0) was used as a developing solution, the flow rate was set to 0.3 ml / min, the column temperature was set to 35 ° C., and as a result of detection by absorbance at 280 nm, the amount of the indicator protein for freshness can be determined. Under these analytical conditions, the indicator protein appears as a peak at a retention time of 35-45 minutes. Also, from the results of gel filtration analysis of known molecular weight, the molecular weight of the indicator substance,
It was determined to be about 10,000.

(3) A molecular weight of about 10 which is an indicator of freshness
Separation and Purification of Low-Molecular-Weight Proteins of Less than 000 Low-molecular-weight proteins having a molecular weight of less than about 10,000 in the royal jelly of the present invention can be separated and purified by an ultrafiltration membrane.
Raw royal jelly was dissolved at 3.0% by weight in 10 mM Tris-HCl buffer (pH 7.0) and UF 30,000 (Minip
(Late 30; ultrafiltration), 8 times concentration and desalting 5 times to separate low molecular weight proteins having a molecular weight of less than about 10,000. In addition, from the result of gel filtration analysis of known molecular weight, the protein was determined to be a low molecular weight protein having a molecular weight of less than about 10,000.

(4) Method for qualitative determination of low-molecular-weight protein having a molecular weight of less than about 10,000 in royal jelly by electrophoresis The low-molecular-weight protein having a molecular weight of less than about 10,000 in the royal jelly of the present invention is composed of proteins by tricine-SDS polyacrylamide gel electrophoresis. And the molecular weight of proteins. This electrophoresis method
Schagger et al. (Schagger, H. et al., Anal. Biochem.,
166, 368-379 (1987)). Although not particularly limited, a preferred method is to electrophorese a water-soluble royal jelly protein (3% royal jelly aqueous solution (W / V)) on a polyacrylamide gel (10% uniform) with an electrode solution containing tricine at a current of 20 mA. And staining with Coomassie Brilliant Blue to identify the protein. The molecular weight of the protein of the present invention in such electrophoresis was determined to be less than about 10,000 when a peptide marker kit (Pharmacia Biotech) was used as a molecular weight marker.

[0011]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited only to such examples.

Example 1 A molecular weight of about 10,000 which is an index of freshness of royal jelly under various storage temperature conditions.
TSK manufactured by Tosoh Corporation
Using gel G3000SW as a column, 0.1 M containing 0.3 M sodium chloride, 0.05% sodium azide.
M phosphate buffer (pH 7.0) was used as a developing solution, and the flow rate was 0.1 mL.
GCP with 3 ml / min and column temperature set at 35 ° C.
Was used to measure the content. That is, royal jelly is 4
℃, 20 ℃, 40 ℃ and 50 ℃, 0 days (starting fresh royal jelly content of low molecular weight protein less than about 10,000 molecular weight / total protein mass in royal jelly (%)), after 1 day After 3 days, 5 days, and 7 days, the content of low molecular weight proteins having a molecular weight of less than about 10,000 in the royal jelly was measured. As shown in Table 1,
When stored at 4 ° C., low-molecular-weight proteins having a molecular weight of less than about 10,000 were not degraded for proteins having a molecular weight of about 10,000 or more, and low-molecular-weight proteins having a molecular weight of less than about 10,000 did not increase until 7 days later. Also, 20 ° C
Under storage conditions, the low molecular weight proteins having a molecular weight of less than about 10,000 increased slightly after 5 and 7 days to 45.2%. On the other hand, under the conditions of high temperature storage at 40 ° C. and 50 ° C., 1
Until ７7 days, low molecular weight proteins with a molecular weight of less than about 10,000 increase over time to 63.8%
And 74.1%. That is, this molecular weight is about 100
It is understood that a low molecular weight protein of less than 00 can serve as an indicator of freshness of royal jelly.

[0013]

[Table 1]

<Example 2> Using the mouse anti-fatigue test as an index, the physiological activity test of the royal jelly of Example 1 stored at 50 ° C. over time was performed. That is, dd as an experimental animal
Y mice, 5 weeks old, male, 10 animals per group were used. As a method of administering royal jelly, a lyophilized product of royal jelly was mixed with a powdered feed (CE-2) so that the lyophilized product became 5% on a raw conversion basis, and allowed to freely ingest for 2 weeks. (Calculated intake is 5g / K
g) As an anti-fatigue test, a flow layer for measuring the momentum of the Kyoto University Matsumoto mouse was used. The water flow rate of the aqueous layer was 8 L / min, and the mice were allowed to swim. The point at which breathing could not be carried out for 7 seconds or longer was taken as the swimming time. As a grouping, 1 group is a control (normal diet), 2 groups are a group receiving fresh royal jelly, 3 groups
The groups were royal jelly-treated groups stored at 50 ° C. for 1 day, 4
The groups were royal jelly-treated groups stored at 50 ° C. for 3 days, 5
The group was a royal jelly-administered group stored at 50 ° C for 5 days, and the 6th group was a royal jelly-administered group stored at 50 ° C for 7 days.
As for the selection of mice, some mice were good at swimming and some were poor at swimming, and those with poor swimming hardly increased their swimming time even after repeated swimming. Therefore, only those swimming for 40 minutes or more in the swimming time before administration were selected and subjected to the experiment. As a schedule for the experiment, first, the mice were allowed to swim once a week. Preliminary swimming was performed in the first week, and swimming time before administration was measured in the second week. The swimming time was measured two weeks after the start of administration on the fourth week. The results are shown in Table 2 as the average swimming time ratio (average of the swimming time ratio after administration when the swimming time before administration is 1). From this, it can be seen that the lower the content of the low molecular weight protein having a molecular weight of less than about 10,000 which is an indicator of the freshness of royal jelly, the longer the swimming time. Therefore, it was confirmed that the protein content had a very good correlation with the anti-fatigue activity, one of the pharmacological activities of royal jelly.

[0015]

[Table 2]

[0016]

According to the present invention, an indicator substance for clarifying the freshness of royal jelly can be provided.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C07K 14/435 G01N 27/26 301A (72) Inventor Hirotaka Miyazaki 560 Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Prefecture Pola Totsuka F-term in the laboratory (reference) 4B041 LC10 LD06 LK14 4H045 AA20 AA30 CA51 EA50 FA71 GA10 HA04

Claims (4)

[Claims] 1. A molecular weight of about 10,000 in royal jelly
An indicator of royal jelly freshness, characterized by using a low molecular weight protein of less than as an indicator. 2. The indicator substance of royal jelly freshness according to claim 1, wherein the low molecular weight protein having a molecular weight of less than about 10,000 has the following properties. 1) TSK Gel G3000 manufactured by Tosoh Corporation
Using SW (7.5 × 30cm) as a column, 0.3M
Sodium chloride, containing 0.05% sodium azide.
A 1 M phosphate buffer (pH 7.0) was used as a developing solution, the flow rate was set to 0.3 ml / min, and the column temperature was set to 35 ° C.
A low molecular weight protein having a peak at a retention time of 35 to 45 minutes in high performance liquid chromatography detected at an absorbance of 80 nm and having a molecular weight of less than about 10,000. 2) A low molecular weight protein having a molecular weight of less than about 10,000 as measured by tricine-SDS polyacrylamide gel electrophoresis. 3. The indicator substance of royal jelly freshness according to claim 1, wherein the indicator substance shows freshness of royal jelly. 4. A low-molecular-weight protein having a molecular weight of less than about 10,000, which is a freshness indicator substance according to any one of claims 1 to 3, and accounts for 44% by weight or less of the total protein in royal jelly. An indicator of royal jelly freshness, characterized by the following: