JP5424677B2 - Method for producing royal jelly having blood pressure lowering action - Google Patents

Description

  The present invention relates to a method for producing a royal jelly having a blood pressure lowering action, and a royal jelly composition and a blood pressure lowering agent containing the royal jelly obtained by the method.

  Royal jelly is a milky white paste-like substance secreted by the pharyngeal glands of young worker bees, and its components are abundant in amino acids including essential amino acids and are composed of high-quality proteins. In addition, it contains trace components such as vitamins, minerals and carbohydrates. Examples of vitamins include vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, vitamin A, vitamin C, and vitamin E, which are effective in promoting growth and preventing aging. Minerals include potassium, magnesium, calcium, copper, iron, phosphorus and the like. Examples of carbohydrates include glucose and fructose. Furthermore, acetylcholine-like substances, organic acids (such as 10-hydroxydecenoic acid), fats, and protein-like antibacterial substances such as leuricin are included.

  As described above, royal jelly contains various nutritional components and has many known effects such as antibacterial action, immune enhancement action, anti-inflammatory action, anti-aging action, prevention / treatment action for menopause, and anticancer action. ing.

  On the other hand, the number of patients with hypertension among lifestyle-related diseases is estimated to be 20 million in Japan over 30 years old, and high blood pressure has an aspect called current national disease. Hypertension is a risk factor for various chronic diseases, and is known to induce vascular disorders such as cerebrovascular disorders and myocardial infarction. As the population ages more and more in the future, the number of hypertensives is expected to increase, and improvement of hypertension or prevention of the onset is an issue (Non-patent Document 1).

Angiotensin converting enzyme (hereinafter sometimes abbreviated as ACE) converts angiotensin I into angiotensin II, which is a blood pressure increasing substance, and degrades bradykinin that has a blood pressure lowering action, thereby increasing blood pressure. Therefore, if there is a substance that inhibits ACE (ACE inhibitor), blood pressure can be lowered, and it is expected to be effective in preventing or treating hypertension.
As an ACE inhibitor that lowers blood pressure by suppressing ACE activity, for example, captopril has been developed and used as a pharmaceutical product.

  On the other hand, in the food field, milk casein (Non-patent document 2), wakame protein (patent document 1, non-patent document 3), bonito (non-patent document 4, non-patent document 5), sardine (non-patent document 6), etc. Numerous foods containing ACE inhibitors such as enzymatic degradation products, fermented milk (Non-patent Document 7), and Bunaharitake extract (Non-patent Document 8) are commercially available.

Attempts have been made to obtain ACE inhibitors by hydrolyzing royal jelly using the digestive enzymes of animals such as trypsin and proteolytic enzymes derived from Bacillus subtilis (Patent Document 2, Patent Document 3, Non-Patent Document) Reference 9). Furthermore, peptides such as isoleucine-tyrosine, valine-tyrosine, and isoleucine-valine-tyrosine are known as components involved in lowering blood pressure when royal jelly is hydrolyzed using a proteolytic enzyme derived from Bacillus subtilis (non-patented). Reference 10). However, trypsin used to digest royal jelly is an animal-derived digestive enzyme, and royal jelly hydrolyzed with trypsin has a problem that it is difficult to completely rule out contamination of animal-derived infectious substances, making it safer. An ACE inhibitor derived from royal jelly is desired. Furthermore, royal jelly hydrolyzed with a proteolytic enzyme derived from Bacillus subtilis has a problem of low ACE inhibitory activity, and an ACE inhibitor having higher ACE inhibitory activity is desired.
In addition, it is described that an ACE inhibitor is produced by allowing a proteolytic enzyme derived from Bacillus stearothermophilus to act at an optimal temperature of 70 ° C. on royal jelly (Patent Document 4). However, in this method, when the reaction time is lengthened, the ACE inhibitory activity sharply decreases and is not practical.
As described above, different proteolytic enzymes are used for various food materials. This is probably because the protein contained in the food material is different, and the optimal proteolytic enzyme for each protein is selected.

JP 2002-138100 A JP-A-4-279597 JP 2006-28065 A JP 2005-6533 A

Hiroyuki Fujita et al., Pharmacology and Treatment Vol.25 (8), 147 (2155) -151 (2159) (1977) Maruyama S., Agr. Biol. Chem., 51, 1581 (1987) Health Sciences, 20, 82-90 (2004) Yokoyama K., Biosci. Biotech. Biochem., 56, 1541 (1992) Fujita H. et al., J. Food Sci., 65, 564 (2000) Eiji Seki et al., Journal of Japanese Society for Agricultural Chemistry, Vol. 69 (8), 1013-1020 (1995) Yamamoto N. et al., J Dairy Sci, 77, 917-922 (1994) Yuji Sakamoto et al., Applied Pharmacology 61 (4/5), 221-229 (2001) Hiroe Maruyama et al., Japan Society for Food Science and Technology, Volume 50 (7), 310-315 (2003) Tokunaga K. et al., Bio. Pharm. Bull., 27 (2), 189-192 (2004)

  An object of the present invention is to provide a new technique capable of obtaining a royal jelly having a blood pressure lowering action, particularly an angiotensin converting enzyme inhibitory action.

  As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by allowing a protease derived from Bacillus stearothermophilus to act on royal jelly, and the present invention has been completed. .

That is, the present invention
1. a method for producing a royal jelly having a blood pressure lowering action, which comprises a step of allowing a proteolytic enzyme derived from Bacillus stearothermophilus to act on royal jelly,
2. A method for producing the royal jelly, wherein the blood pressure lowering action is an angiotensin converting enzyme inhibitory action,
3. The method for producing any of the above royal jelly, wherein the royal jelly is a substantially fully digested royal jelly,
4. The method for producing any one of the above-mentioned royal jelly, wherein the temperature at which the proteolytic enzyme derived from Bacillus stearothermophilus is allowed to act on the royal jelly is 40 to 60 ° C.,
5. A royal jelly composition containing the royal jelly obtained by any one of the above production methods, and
6. An antihypertensive agent containing royal jelly obtained by any one of the above production methods,
Consists of.

  According to the present invention, a royal jelly having an antihypertensive action, particularly an angiotensin converting enzyme inhibitory action, can be produced by a safer and simpler method, and a royal jelly composition and an antihypertensive agent can be produced using the royal jelly having the antihypertensive action. Can be provided.

  The royal jelly of the present invention is produced by the action of a proteolytic enzyme derived from Bacillus stearothermophilus.

  Examples of the country of origin of royal jelly include Japan, China, Taiwan, Thailand, Brazil, European countries, Oceania countries, the United States, etc. Royal jelly of any country of origin may be used. Further, a plurality of royal jelly of the country of origin may be appropriately mixed and used. The royal jelly is preferably a liquid, and when lyophilized royal jelly is used, it can be used after being dissolved in purified water, tap water, or an appropriate buffer. The frozen royal jelly can be used after being thawed.

  Further, the royal jelly may be one to which processing such as heating, centrifugation, alcohol extraction, filtration, freeze drying or hot air drying, and various nutrients are added.

  The proteolytic enzyme derived from Bacillus stearothermophilus that acts on royal jelly is obtained by culturing Bacillus stearothermophilus, and may be a culture solution or a purified product. The proteolytic enzyme derived from Bacillus stearothermophilus may be obtained by gene recombination, and may be further modified with, for example, sugar or polyethylene glycol.

  A proteolytic enzyme derived from Bacillus stearothermophilus may be allowed to act alone, and further, a proteolytic enzyme derived from Bacillus stearothermophilus and another proteolytic enzyme may be used in combination to act on royal jelly. In addition, the required amount of the proteolytic enzyme may be allowed to act on the royal jelly at once, or the required amount may be divided into two or more times to act on the royal jelly. In addition, when two or more types of proteolytic enzymes are allowed to act on royal jelly in combination with proteolytic enzymes derived from Bacillus stearothermophilus and other proteolytic enzymes, each proteolytic enzyme acts simultaneously with royal jelly. You may make it act separately and may make it act with a royal jelly.

  The solid content concentration of the royal jelly after mixing with the proteolytic enzyme derived from Bacillus stearothermophilus can be 0.1-30 W / W%, preferably 1-15 W / W%, more preferably 4-10 W / W% can be mentioned. Here, for example, 10 W / W% means that 10 g of a dried product is obtained when 100 g of royal jelly is dried by freeze drying or the like.

  The treatment conditions of royal jelly with a proteolytic enzyme derived from Bacillus stearothermophilus, such as the treatment time, pH, and temperature, are not particularly limited as long as the target antihypertensive substance is produced. It can be appropriately set in consideration of stability and stability and reactivity of a protease derived from Bacillus stearothermophilus. In order to obtain a royal jelly having a high blood pressure lowering action, it is preferable that the royal jelly is substantially completely digested by a proteolytic enzyme derived from Bacillus stearothermophilus to sufficiently produce a blood pressure lowering substance. Here, substantially complete digestion means that a protein contained in royal jelly is hydrolyzed by a proteolytic enzyme and hydrolyzed into a fragment that is no longer substantially hydrolyzed by the enzyme. Under such conditions, a proteolytic enzyme derived from Bacillus stearothermophilus is allowed to act on royal jelly under various conditions, and the blood pressure-lowering action, preferably ACE inhibitory action, of the obtained royal jelly is measured by a known method, resulting in a high action. It can be determined by selecting the resulting conditions.

  The time for the action of the protease derived from Bacillus stearothermophilus and royal jelly is not particularly limited as long as the target antihypertensive substance is produced, but it is several hours to 10 days, preferably 1 hour to 48 hours. Is preferred. Long-term reactions had little effect on the production of hypotensive substances.

  The pH at the time of allowing the proteolytic enzyme derived from Bacillus stearothermophilus to act on the royal jelly is not particularly limited as long as it is a pH at which the target antihypertensive substance is produced, but pH 3-11, preferably pH 5-11, Preferably pH 7-10 is suitable.

  The temperature at which the proteolytic enzyme derived from Bacillus stearothermophilus acts on the royal jelly is not particularly limited as long as it is a temperature at which a blood pressure lowering substance is produced, but is 30 ° C to 80 ° C, preferably 40 ° C to 60 ° C, More preferably, 40 ° C to 50 ° C, and still more preferably 40 ° C. In general, the enzyme reaction is performed at the optimum reaction temperature of the enzyme used. However, when proteolytic enzyme derived from Bacillus stearothermophilus is allowed to act on royal jelly at the optimal reaction temperature of 70 ° C., the activity of the blood pressure lowering substance rapidly decreases when the reaction time is prolonged (patent document) 4) Poor operability and inappropriate. If the reaction temperature is too low, the production efficiency of the blood pressure lowering substance is poor. Furthermore, by making it react at high temperature, the cost for the heating at the time of manufacture becomes high. Further, when the reaction is carried out at a high temperature, there is a problem that royal jelly is colored.

  While the proteolytic enzyme derived from Bacillus stearothermophilus is allowed to react with the royal jelly, it may be left still, or may be shaken or stirred.

  The production of the royal jelly of the present invention generally includes a step of inactivating the above-mentioned proteolytic enzyme derived from Bacillus stearothermophilus. The method for inactivating the protease derived from Bacillus stearothermophilus may be to inactivate the protease derived from Bacillus stearothermophilus to the extent that there is no problem as a food. Examples of the inactivation method include a method of inactivation by heating, a method of inactivation using a chemical agent, and a method of removing proteolytic enzymes derived from Bacillus stearothermophilus by filtration, and these methods can be used alone. It is also possible to combine two or more types. Preferably, the proteolytic enzyme derived from Bacillus stearothermophilus is inactivated by heating. It is appropriate to heat at a heating temperature of 60 ° C or higher, preferably 80 ° C or higher, more preferably 90 ° C or higher. Furthermore, when a food sterilization step is required, it may be combined with a step of inactivating the proteolytic enzyme derived from the present stearothermophilus.

  Further, when a protease derived from Bacillus stearothermophilus is allowed to act on royal jelly, a stabilizer or a reaction accelerator for a protease derived from Bacillus stearothermophilus may be added.

  The royal jelly produced by the action of a proteolytic enzyme derived from Bacillus stearothermophilus is a royal jelly having a blood pressure lowering action, preferably an ACE inhibitory action. The royal jelly is preferably a royal jelly that has been substantially completely digested with a proteolytic enzyme derived from Bacillus stearothermophilus.

  The ACE inhibitor may be purified by treating the royal jelly of the present invention with various types of chromatography. Examples of the purification method include gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, normal phase chromatography, ultrafiltration, electrophoresis and the like. These can be used for purification alone or in combination.

  Gel filtration chromatography includes a carrier for gel filtration chromatography that can separate proteins having various molecular weights, and a carrier for gel filtration chromatography that can separate proteins having a molecular weight of about 10,000 or less is preferable. Examples of ion exchange groups used in ion exchange chromatography include anion exchangers and cation exchangers. Examples of the anion exchanger include a diethylaminoethyl group (DEAE group) and a quaternary aminoethyl group (QAE group). Examples of the cation exchanger include a carboxymethyl group (CM group) and a sulfopropyl group (SP group). Examples of the carrier used in the hydrophobic chromatography include a carrier having a butyl group (Butyl group), an ethyl group (Ethyl group), and a phenyl group (Phenyl group) bound thereto. Examples of the carrier used for the reverse phase chromatography include octadecyl group (C18), and a carrier to which C30, C8, C4, etc. having different alkyl group length from octadecyl group are bonded. Examples of the carrier used for normal phase chromatography include silica gel, as well as a carrier bonded with a cyanopropyl group, a functional group having a diol structure, an aminopropyl group, a polyamine, and the like.

  The royal jelly of the present invention can be used as a blood pressure lowering agent by performing purification using the above-described chromatography or the like. Moreover, it can provide as a royal jelly composition by adding various components to the blood pressure-lowering royal jelly of this invention or its refined product. Examples of the various components include foods, sugars, lipids, emulsifiers, thickeners, seasonings, fragrances, sourness adjusters, preservatives, fruit juices, fragrances, various nutritional components, and the like, and do not impair the effects of the present invention. Can be used in Moreover, various components may be used independently and may be used in mixture of 2 or more types. Examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose and the like. Examples of emulsifiers include sucrose fatty acid esters, glycerin fatty acid esters, and lecithin. Examples of the thickener include carrageenan, gum arabic, xanthan gum, guar gum, pectin, locust bean gum thickener starch, gellan gum and the like. Examples of the sourness adjuster include citric acid, lactic acid, malic acid, fumaric acid, gluconic acid, tartaric acid and the like. Examples of the preservative include benzoic acid and its salt, sorbic acid and its salt, paraben, sodium sulfite, pectin degradation product, glycine and the like. Examples of the fruit juice include tomato juice, plum juice, apple juice, lemon juice, orange juice, and berry juice. Examples of the fragrances include spices such as herbs and spices, fruit fragrances, and fragrances such as vanilla. In addition, other preferable nutrients include vitamins such as vitamin D and minerals such as calcium, magnesium, iron, manganese, and zinc.

  The royal jelly, royal jelly composition or antihypertensive agent of the present invention can also be used as a food prepared by adding and blending them. Specific examples of such foods include, for example, beverages, confectionery, candy, gum, bread, livestock meat products, dairy products, retort foods, instant foods, frozen foods, jelly-like foods, beekeeping products, pickles, seasonings And so on. These foods are also useful as so-called health foods, functional foods, foods for specified health use, functional nutritional foods, dietary supplements, supplements and the like. Moreover, granule, powder, tablet, capsule, chewable, drink, jelly, paste, grain etc. can be mentioned as the shape as those foods.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

  Screening for a proteolytic enzyme suitable for treatment to obtain a royal jelly having a blood pressure lowering effect was performed. Table 1 shows the proteolytic enzymes used in the tests and their origins. The blood pressure lowering effect was measured using ACE inhibitory activity as an index.

  Specifically, first, about 50 mL of purified water was added to 10 g of 30 W / W% royal jelly, pH was adjusted by adding HCl or NaOH, purified water was added to 60 mL, and a royal jelly dilution was prepared. Add the proteolytic enzymes shown in Table 1 and react at 40 ° C for 14 hours, then heat in boiling water for 30 minutes to inactivate the proteolytic enzymes, allow to cool, and centrifuge at 3,000 rpm for 10 minutes. I got Qing. The supernatant was measured for ACE inhibitory activity. ACE inhibitory activity was performed by the method shown below. The pH of the royal jelly diluent is pH 7.0 for the proteolytic enzymes shown in (1) and (4) of Table 1, and the proteolytic enzymes shown in (2), (3) and (5) of Table 1. Was adjusted to pH 3.0.

  The amount of proteolytic enzyme added was 532 mg of protease S “Amano” G per 100 mL of royal jelly diluent, and the other proteolytic enzymes were measured for activity according to the following [Proteolytic enzyme activity measurement method] shown below. It was added to the royal jelly diluent so as to have the same activity (ΔA660) as Amano G.

[Method for measuring ACE inhibitory activity]
<Preparation of borate buffer>
450 mM 50 mM Na ₂ B ₄ O ₇ and 550 mL 220 mM H ₃ BO ₄ were mixed to prepare a borate buffer solution having a pH of 8.3.
<Sample preparation>
The supernatant was diluted 10 times with borate buffer (pH 8.3) to prepare a sample.
<Preparation of substrate solution>
After dissolving 170 mg of Bz-Gly-His-Leu · H ₂ O (molecular weight: 447.49, manufactured by Peptide Laboratories) and 1.78 g of NaCl in about 30 mL of borate buffer, adjust the pH to 8.3 with 1N NaOH and borate buffer The volume was made up to 50 mL with the solution to obtain a substrate solution.
<Preparation of ACE solution>
Angiotensin converting enzyme (from rabbit lung, Sigma) was dissolved in borate buffer to make a 60 mU / mL solution as an ACE solution.
<Method for measuring ACE inhibitory activity>
(1) Measurement of inhibitory activity 30 μL of the sample and 250 μL of the substrate solution were mixed and allowed to stand at 37 ° C. for 5 minutes. After leaving for 5 minutes, 100 μL of ACE solution was added, and after reacting at 37 ° C. for 30 minutes, 250 μL of 1N HCl was added to stop the reaction. After stopping the reaction, 1.5 mL of ethyl acetate was added and stirred vigorously for 1 minute. Next, the mixture was centrifuged at 3,000 rpm for 10 minutes, and 0.5 mL of the upper ethyl acetate layer was collected and evaporated to dryness with a centrifugal evaporator (40 ° C., 1.5 hours). After evaporating to dryness, 2 mL of purified water was added and dissolved, and the absorbance at 228 nm was measured. The absorbance at this time is A.
(2) Blank measurement 30 μL of the sample and 250 μL of the substrate solution were mixed and left at 37 ° C. for 35 minutes. After standing for 35 minutes, 250 μL of 1N HCl was added, and then 100 μL of ACE solution was added. Further, 1.5 mL of ethyl acetate was added and stirred vigorously for 1 minute. Next, the mixture was centrifuged at 3,000 rpm for 10 minutes, and 0.5 mL of the upper ethyl acetate layer was collected and evaporated to dryness with a centrifugal evaporator (40 ° C., 1.5 hours). After evaporating to dryness, 2 mL of purified water was added and dissolved, and the absorbance at 228 nm was measured. The absorbance at this time is B.
(3) Measurement of control (1) In the measurement of inhibitory activity, the same operation was performed using a borate buffer instead of the sample, and the absorbance at 228 nm was measured. The absorbance at this time is C. Further, in the above (2) blank measurement, the same operation was performed using a borate buffer instead of the sample, and the absorbance at 228 nm was measured. The absorbance at this time is D.
(4) Calculation The ACE inhibitory activity was determined by the following formula.
ACE residual activity (%) = (A−B) × 100 / (C−D)
ACE inhibitory activity (%) = 100-ACE residual activity

[Proteolytic enzyme activity measurement method]
(1) Preparation of Substrate Solution Preparation of substrate solution A: 0.6 g of casein (Hamalstein formulation) was suspended in 75 mL of 100 mM lactic acid buffer (pH 3.0) and dissolved by heating in boiling water. After standing to cool, the pH was adjusted to 3.0, and the volume was adjusted to 100 mL to obtain substrate solution A.
Preparation of substrate solution B: 0.6 g of casein (Hamalstein formulation) was suspended in 75 mL of 100 mM bis-trispropane buffer (pH 7.0) and dissolved by heating in boiling water. After standing to cool, the pH was adjusted to 7.0, and the volume was adjusted to 100 mL to obtain substrate solution B.
In addition, the substrate solution A was used for the proteolytic enzyme activity measurement of (2), (3) and (5) shown in Table 1, and the proteolytic enzyme activity measurement of (1) and (4) shown in Table 1 was used. Used substrate solution B. Furthermore, when proteolytic enzyme activity is high and dilution is required, 100 mM lactate buffer (pH 3.0) is used for the proteolytic enzyme activity measurement of (2), (3) and (5) shown in Table 1, The proteolytic enzyme activities (1) and (4) shown in Table 1 were diluted using 100 mM bis-trispropane buffer (pH 7.0).
(2) Measurement of proteolytic enzyme activity
After 2 mL of the substrate solution was allowed to stand at 40 ° C. for 5 minutes, 0.4 mL of the sample solution was added, and after reacting at 40 ° C. for 30 minutes, 2 mL of 0.44 M trichloroacetic acid solution was added to stop the reaction. The reaction was stopped for 10 minutes at room temperature, followed by filtration with No. 2 filter paper (Advantech Toyo). To 0.5 mL of the filtrate, 1.25 mL of a 0.55 M Na ₂ CO ₃ solution and 0.25 mL of a phenol reagent diluted 3-fold with purified water were added, and the mixture was allowed to stand at 37 ° C. for 30 minutes, and then the absorbance at 660 nm was measured. The absorbance at this time is A.
(3) Blank measurement
The measurement was performed in the same manner as in the above (2) measurement of proteolytic enzyme activity except that 0.4 mL of the sample solution was added after adding 2 mL of 0.44 M trichloroacetic acid solution. The absorbance at 660 nm is B.
(4) ΔA660 was determined by the following formula. In the following formula, D means the dilution rate.
ΔA660 = (A−B) × D

  Table 2 shows the ACE inhibitory activity of royal jelly treated with each proteolytic enzyme. Among the proteolytic enzymes tested, royal jelly treated with protease S “Amano” G showed good ACE inhibitory activity.

  Proteolytic enzyme activity of protease S “Amano” G added with 532 mg per 100 mL of royal jelly diluent is the value of “A-B” when diluted 100-fold with 100 mM bis-trispropane buffer (pH 7.0). Was 0.25 (ΔA660 = 25).

  The IC50 of protease S “Amano” G and protease N “Amano” G (manufactured by Amano Enzyme, Inc., derived from Bacillus subtilis) was compared.

  About 50 mL of purified water was added to 10 g of 30 W / W% royal jelly, and HCl or NaOH was added to adjust to pH 7.0, and then purified water was added to 60 mL to prepare a royal jelly dilution. Protease S “Amano” G was added at 532 mg per 100 mL of royal jelly diluent, reacted at 40 ° C. for 14 hours, heated in boiling water for 30 minutes to inactivate the proteolytic enzyme, allowed to cool, lyophilized, A lyophilized product was obtained. Protease N “Amano” G was also measured for proteolytic enzyme activity according to [Proteolytic enzyme activity measurement method] shown in Example 1, and the same protease activity (ΔA660) added to the royal jelly diluent was used. The same operation as in Amano G was performed to prepare a freeze-dried product. The activity of protease N “Amano” G was measured using the substrate solution B shown in [Proteolytic enzyme activity measurement method].

  The obtained lyophilized product was dissolved in a borate buffer and IC50 was measured. IC50 was measured according to [Method for measuring ACE inhibitory activity] in Example 1.

  The results are shown in Table 3. As shown in Table 3, the freeze-dried product of protease S “Amano” G-treated royal jelly had a lower IC50 value and higher ACE inhibitory activity. IC50 shown in Table 3 indicates the concentration of the sample.

  The freeze-dried product of protease S “Amano” G-treated royal jelly obtained in Example 2 was subjected to a single-dose blood pressure lowering medicinal effect test. Spontaneously raised spontaneously hypertensive rats (SHR) for 3 weeks, lyophilized product was suspended in purified water to 2.5 mg / mL in 14-week-old SHR (6 mice per group, male), 10 mL per kg body weight (25 mg / kg). As a result, the average blood pressure in the lyophilized product administration group 6 hours after administration was 174.8 mmHg. On the other hand, the average blood pressure in the subject group was 197.3 mmHg, and the blood pressure-lowering effect was significantly (p <0.05) by administering the lyophilized product. The subject group was administered 10 mL of purified water.

  About 20 mL of purified water was added to 10 g of 30 W / W% royal jelly, and HCl or NaOH was added to adjust the pH to 8.0, and then purified water was added to 35 mL to prepare a royal jelly dilution. Protease S “Amano” G was added at 200 mg per 100 mL of royal jelly diluent, reacted at 30 ° C, 40 ° C, 50 ° C, 60 ° C for 1, 2, 4, 7, 24, 48 hours, then in boiling water for 30 minutes. After heating, the proteolytic enzyme was inactivated, allowed to cool, and then diluted 43-fold with borate buffer, and the inhibition rate of ACE was measured. The inhibition rate of ACE was measured according to [Method for measuring ACE inhibitory activity] in Example 1. Table 4 shows the transition of the inhibition rate of ACE.

  As shown in Table 4, the inhibition rate of ACE was good by carrying out the reaction at 40 ° C. for 7 hours or more. In addition, the inhibition rate of ACE was good by carrying out the reaction for 1 hour at 50 ° C and for 2 hours at 60 ° C. These ACE inhibition rates were equivalent to those obtained by reacting at 70 ° C. for 4 hours. In addition, when the reaction temperature was 40 to 60 ° C., a slight decrease in ACE inhibitory activity was observed even if the reaction time was lengthened, but as shown in JP 2005-6533 (Patent Document 4). No significant decrease in activity was observed.

  Usually, the temperature at which the enzyme is allowed to act is generally the optimum reaction temperature from the viewpoint of efficiency. Enzymes derived from thermophilic bacteria, such as this protease S “Amano” G, are thought to show a sharp decrease in activity due to the high optimum reaction temperature. Therefore, it is considered that the action of the proteolytic enzyme on the royal jelly at a temperature lower than the optimum temperature as in this example is peculiar to the proteolytic enzyme derived from thermophilic bacteria.

  The royal jelly having a blood pressure lowering effect obtained by the present invention can be suitably used for food and drink as a royal jelly composition or a blood pressure lowering agent, and can be particularly suitably used for health foods and the like.

Claims (3)

Royal jelly to look including the step of reacting Bacillus stearothermophilus (Bacillus stearothermophilus) derived proteolytic enzymes, wherein the process is characterized by being performed at a temperature of 30 to 40 ° C., royal jelly having a hypotensive effect Manufacturing method. 2. The method for producing a royal jelly having a blood pressure lowering action according to claim 1, wherein the step is performed at a temperature of 40 ° C. 3. The method for producing a royal jelly according to claim 1, wherein the blood pressure lowering action is an angiotensin converting enzyme inhibitory action.