JP5916288B2 - Process for producing innate immunity activator with enhanced innate immunity promoting action and royal jelly-derived innate immunity activator produced by the process - Google Patents

Description

  The present invention relates to a method for producing an innate immunity activator with enhanced innate immunity promoting action and a royal jelly-derived innate immunity activator produced by the method.

  Royal jelly (RJ) is a milky white substance made by mixing young bees in the bees with secretions of the hypopharyngeal gland and the greater vaginal gland. 12% by mass; avidin (MJRP1) having cell growth promoting action and cell death suppressing action (Patent Document 1), royal jelly main protein 3 (MRJP3) having immunosuppressive action, royalisin having antibacterial action, etc. ), Carbohydrates (content 10 mass%; fructose, glucose, sucrose, etc.) and lipids (content 5 to 7 mass%; bactericidal action, insulin action, anticancer action, nerve cell proliferation action (Non-patent Document 2) ), 10-hydroxy-2-decenoic acid with 3,10-hydroxydecanoic acid or trans-10-hydroxydeca-2 with immunoregulatory action It is mainly composed enoic acid (Non-patent Document 3), etc.).

  In addition, vitamins (vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, vitamin A, vitamin C, vitamin E, etc.), minerals (potassium, sodium, calcium, magnesium, copper, iron, zinc, phosphorus, etc.) , N-oxide derivatives of adenosine and adenosine-1-phosphate, which have a neuronal proliferative activity, substances similar to the human neurotransmitter acetylcholine, and minor components such as parotin, which is similar to the growth hormone parotin in human salivary glands Yes. On the other hand, honey is mostly composed of carbohydrates (78% by mass) and moisture (20% by mass), and its composition is greatly different from royal jelly.

  Royal jelly is, for example, an immune promoting action (Non-patent Documents 1 and 3), an anti-inflammatory action (Non-Patent Document 4), an antibacterial action (Non-Patent Documents 5 and 6), an anti-aging action, a growth promoting action, and prevention of menopause Treatment, anticancer action (Non-patent Documents 7 and 8), wound healing promoting action, blood flow increasing action, blood sugar lowering action, blood pressure lowering action (Non-Patent Document 9), serum cholesterol lowering action (Non-Patent Document 10), It is known to have various physiological effects such as an anti-hepatic disorder action (Non-Patent Document 11), an anti-atopic dermatitis action (Non-Patent Document 12), and a fatigue recovery action (Non-Patent Document 13). It is used for high health supplements and cosmetics. Recently, it has been found that protease degradation products of royal jelly protein have a strong antioxidant action (hydroxy radical scavenging action) (Non-patent Documents 14 and 2) and blood pressure lowering action (Non-patent Documents 15 and 16). It was.

  Royal jelly is a food that is fed to queen bees, queen bee larvae and young worker bee larvae, and the queen bee keeps eating royal jelly for 40 times longer life than worker bees. It is also known that Pope Pius XII, who became critically ill due to senility in 1954, has achieved an amazing recovery with the royal jelly administered by the doctor, Galeajii Rissie. It has not been revealed yet.

  On the other hand, regarding the natural immunity promoting action, those containing polysaccharides such as β-glucan derived from yeast cell wall and mechabufucoidan are known in the health food field.

  Regarding the processing and processing of royal jelly, royal jelly-derived food additives and pharmaceuticals having antioxidant and blood pressure lowering effects have been produced and developed by treating royal jelly with protease and the like (Patent Literature). 3-5).

  In recent years, the maintenance of immune function and health of the elderly who continue to increase with the aging of society has become an important issue for running a fulfilling life. In such an aging society, a drug that strengthens and maintains innate immunity that contributes to health maintenance is required, and further development is expected.

JP-A-6-113828 JP 2007-217358 A JP 2009-029772 A JP 2009-060896 A JP 2008-048729 A

Okamoto, I., Taniguchi, Y., Kunikata, T., Kohno, K., Iwaki, K., Ikeda, M., and Kurimoto, M. (2003) Life Sciences 73 (16), 2029-2045 Hattori, N., Nomoto, H., Fukumitsu, H., Mishima, S., and Furukawa, S. (2007) Biomed. Res. 28 (5), 261-266. Gasic, S., Vucevic, D., Vasilijic, S., Antunovic, M., Chinou, I., and Colic, M. (2007) Immunopharmacology and Immunotoxicology 29,521-536. Kohno, K., Okamoto, I., Sano, O., Arai, N., Iwaki, K., Ikeda, M., and Kurimoto, M. (2004) Biosci. Biotechnol. Biochem. 68 (1), 138 -145. de Souza, B.M., Konno, K., Cesar, L.M.M., Malaspina, O., and Palma, S.M. (2004) Peptides 25 (6), 919-928. Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990) J. Biol. Chem. 265 (19), 11333-11337. Toyoyuki Tamura, Akira Fujii, Noboru Kuboyama (1987) Journal of the Japanese Pharmacological Society 89 (2), 73-80. A study on the antitumor effects of Royal Jelly. Townsend, G.F., Morgan, J.F., Tolani, S., Hazlett, B., Morgan, H.J., And Shuel, R.W. (1960) Cancer Res. 20,503-510. Masataka Nomura, Naomi Washio, Yoshito Zama, Shingo Takatori, Shima Doi, Hiroki Kawasaki (2007) Pharmaceutical Journal 127 (11), 1877-1882. Guo, H., Saiga, A., Sato, M., Miyazawa, I., Shibata, M., and Morimatsu, F. (2007) J. Nutr. Sci. Vitaminol. (Tokyo) 53 (4), 345 -348. Kanbur, M., Eraslan, G., Beyaz, L., Silici, S., Liman, BC, Altinordulu, S., and Atasever, A. (2008) Exp. Toxicol. Pathol. 61 (2), 123- 132. Taniguchi, Y., Kohno, K., Inoue, S., Koya-Miyata, S., Okamoto, I., Arai, N., Iwaki, K., Ikeda, M., and Kurimoto, M. (2003) Int.Immunopharmacol.3 (9), 1313-1324. Kamakura, M., Mitani, N., Fukuda, T., and Fukushima, M. (2001) J. Nutr. Sci. Vitaminol. (Tokyo) 47 (6), 394-401. Guo, H., Kouzuma, Y., and Yonetani, M. (2008) Food Chemistry 113 (1), 238-245. Tokunaga, K., Yoshida, C., Suzuki, K., Maruyama, H., Futamura, Y., Araki, Y., and Mishima, S. (2004) Biol. Pharm. Bull. 27 (2), 189 -192. Takaki-Doi, S., Hashimoto, K., Yamamura, M., and Kamei, C. (2009) Acta. Med. Okayama 63 (1), 57-64.

  The present invention has been made in view of the above-mentioned background art, and the subject thereof is “a method for producing an innate immunity activator with enhanced innate immunity promoting action derived from royal jelly” which is excellent in a novel innate immunity promoting action. Is to provide. Another object of the present invention is to provide a royal jelly-derived innate immunity activator produced by the production method.

  As a result of intensive studies, the present inventor has unexpectedly found that royal jelly can be fermented with lactic acid bacteria despite the fact that royal jelly contains antibacterial substances such as royalisin and 10-hydroxydecenoic acid.

  In addition, the present applicant has already taken the time to complete about 10 minutes when a substance having an innate immunity promoting action is administered to a fully transformed insect larva in WO 2008/126905, WO 2009/157409, etc. It is disclosed that the following muscle contraction occurs (hereinafter abbreviated as “slowing muscle contraction”). And this slowing muscle contraction does not occur when lipopolysaccharide (LPS) causing false positives is administered in evaluating the presence and degree of innate immunity promoting action, and the method is simple, It has been confirmed that it is effective as a practical index for evaluating the presence and degree of innate immunity promoting action.

  And when the said evaluation was performed regarding the said "product obtained by fermenting royal jelly with lactic acid bacteria", it discovered that it showed slow muscular contraction and had an innate immunity promotion effect | action. Further, the inventors have found that the degree of the natural immunity promoting action of such a product is enhanced from the “natural immunity promoting action of royal jelly before fermentation”, and the present invention has been completed.

  That is, the present invention is a method for producing an innate immunity activator with enhanced innate immunity promoting action characterized by including a step of fermenting royal jelly with lactic acid bacteria.

The present invention also relates to a method for producing an innate immunity activator with enhanced innate immunity promoting action, characterized by comprising at least the following steps (1) to (3).
(1) Step of pre-culturing lactic acid bacteria (2) Step of fermenting royal jelly with lactic acid bacteria, and
(3) Process of sterilizing lactic acid bacteria

  Furthermore, the present invention is a royal jelly-derived innate immunity activator characterized by being produced by the above-mentioned “method for producing an innate immunity activator with enhanced innate immunity promoting action”.

The present invention also provides the above-mentioned royal jelly-derived innate immunity activator obtained by a screening method comprising at least the following steps (a) to (c).
(A) a step of administering a test substance to an organism having an innate immunity mechanism (b) a step of evaluating whether or not the test substance contracts muscles of the organism having the innate immunity mechanism (c) the innate immunity Selecting a substance evaluated to contract muscles of living organisms having a mechanism

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of "the natural immunity activation agent with which the natural immunity promotion effect derived from the royal jelly excellent in the natural immunity promotion effect | action was enhanced" can be provided, and it manufactures with this manufacturing method It is possible to provide a “royal jelly-derived innate immunity activator” which is excellent in action for promoting innate immunity.

It is a figure which shows the dose response curve of the innate immunity promoting effect of the yeast (beta) -glucan and the natural immunity activation agent (fermented royal jelly) derived from royal jelly. It is a figure which shows the natural immunity activation agent (fermented royal jelly) derived from royal jelly, and a dried royal jelly dose response curve.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the technical scope of the present invention.

  The method for producing an innate immunity activation agent with enhanced innate immunity promoting action derived from royal jelly of the present invention includes at least a step of fermenting royal jelly with lactic acid bacteria.

  The “royal jelly” (RJ) in the present invention refers to a substance produced by the reaction of different components secreted from the upper and lower pharyngeal glands of a young worker bee of honeybee. As will be described later, any royal jelly that can be fermented with lactic acid bacteria is not particularly limited and can be used in the present invention. Royal jelly can be fermented with lactic acid bacteria despite the inclusion of antimicrobial substances such as royalisin and 10-hydroxydecenoic acid. By using the method for producing an innate immunity activator with enhanced innate immunity promoting activity of the present invention, the innate immunity activator can be produced without the lactic acid bacteria being inhibited by the growth inhibitory substance contained in the royal jelly.

  Examples of the country of origin of royal jelly include Japan, the People's Republic of China, Taiwan, Thailand, Brazil, European countries, Oceania countries, and the United States, and royal jelly of any country of origin can be suitably used. Further, a plurality of royal jelly of the country of origin may be appropriately mixed and used. The royal jelly used for the fermentation is not particularly limited, but is preferably in a liquid state, and when using a lyophilized royal jelly, it is preferably dissolved in purified water, tap water, an appropriate buffer, or the like. The frozen royal jelly can be used after being thawed.

  The lactic acid bacterium in the present invention can be used without particular limitation as long as it can ferment royal jelly to produce an innate immunity activator with enhanced innate immunity promoting action. Specifically, lactic acid bacteria belonging to the genus Lactobacillus (genus Lactobacillus), the genus Lactococcus (genus Lactococcus), the genus Streptococcus (genus Streptococcus), the genus Leuconostoc (genus Leuconostoc), the genus Bifidobacterium (genus Bifidobacterium), etc. Is mentioned.

  Among these, Preferably, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus bulgaricus (Lactobacillus bacilli) Lactic acid bacteria belonging to the genus Lactobacillus, or lactic acid bacteria belonging to the genus Lactococcus such as Lactococcus lactis.

  Particularly preferred is Lactobacillus brevis, for example, Lactobacillus brevis NBRC12005, Lactobacillus brevis NBRC12520, Lactobacillus brevis NBRC3345, Lactobacillus brevis NBRC3960, Lactobacillus brevis NBRC13109, and Lactobacillus brevis NB110 NB.

  These lactic acid bacteria may be used alone, or two or more kinds of lactic acid bacteria may be used. Lactic acid bacteria can be cultured by methods such as stationary culture, shaking culture, stirring culture, aeration culture, and anaerobic culture. In addition, when lactic acid bacteria sink during static culture, the lactic acid bacteria may be dispersed by shaking, stirring, or the like.

  A method for producing an innate immunity activator with enhanced innate immunity promoting action when Lactobacillus brevis NBRC12005 (hereinafter sometimes abbreviated as “NBRC1205 strain”) is used as a lactic acid bacterium will be described below. The invention is not limited to the NBRC12005 strain. That is, the following method for producing an innate immunity activator is not limited to the NBRC12005 strain and can be applied to all lactic acid bacteria.

<Pre-culture process>
Prior to the following pre-culturing step, it is preferable to perform the pre-culturing step in order to increase the efficiency of fermentation by lactic acid bacteria, to prepare a large amount of lactic acid bacteria. Pre-culture is started by mixing the pre-culture solution and lactic acid bacteria. Pre-culture is not particularly limited as long as lactic acid bacteria can grow. For example, in the case of NBRC12005 strain, the medium composition of the pre-culture solution includes, for example, carbon source, nitrogen source, organic micronutrients, metals , Lipids etc. may be used alone or in combination of two or more. Preferably, medium components such as glucose, peptone, yeast extract, metals and the like can be mentioned.

  The NBRC12005 strain is inoculated in a culture medium for pre-culture, and the cells are pre-cultured. In the pre-culture of the NBRC12005 strain, the culture time is preferably from half a day to 4 days, and more preferably from 1 day to 2 days. The culture temperature is preferably 15 to 50 ° C., more preferably 25 to 40 ° C., and the initial pH is preferably 3 to 9, and more preferably 5 to 8 in advance. It is preferable to use the culture medium obtained in this way for the next preculture step.

<Pre-culture process>
When using the pre-culture solution after the completion of the culture in the pre-culture step, the NBRC12005 strain may be washed, or the pre-culture solution obtained by the pre-culture without washing is used as it is, Alternatively, the isolated NBRC12005 strain may be inoculated into the preculture solution. Moreover, when performing preculture after completion | finish of preculture, the preculture liquid and the lactic acid bacteria after completion | finish of preculture, or a culture solution are mixed. The mixing ratio in the case of mixing the culture solution after the end of the previous culture is preferably 0.001 to 0.5 parts by volume, more preferably 0.005 to 0 parts by volume with respect to 1 part by volume of the preculture. It is preferable to mix a culture solution corresponding to 2 parts by volume in view of good fermentation efficiency in the fermentation process which is the next process.

  In the preculture step, lactic acid bacteria are preferably cultured in a preculture solution containing glutamic acid or a salt thereof. When pre-culturing lactic acid bacteria, it is preferable to contain at least glutamic acid or a salt thereof in the pre-culture solution. Examples of the glutamate include potassium glutamate, sodium glutamate, magnesium glutamate, and calcium glutamate. Further, glutamic acid may be glutamic acid released from a protein or peptide by degrading “a protein or peptide containing glutamic acid” with a protease and / or peptidase.

  The preculture liquid may further contain a medium component. Examples of the medium component include a carbon source, a nitrogen source, organic micronutrients, metals, lipids and the like. These may contain single or 2 types or more. Preferred examples of the medium component include glucose, yeast extract, peptone, metals and the like.

  The glutamic acid concentration in the pre-culture step, that is, the glutamic acid concentration after mixing the pre-culture solution and the lactic acid bacteria or culture solution (culture solution of the lactic acid bacteria) obtained in the previous culture is 0.1 to 5 g / 100 mL. Preferably, 0.6 to 2 g / 100 mL is more preferable, and 0.8 to 1.5 g / 100 mL is particularly preferable. When the glutamic acid concentration in the pre-culture process is too low, the efficiency of fermentation in the fermentation process which is the next process may not be good.

  The preculture is started by mixing the preculture solution with the lactic acid bacteria or the culture solution obtained in the previous culture. Pre-culture conditions such as culture time, temperature, and pH are preferably pre-culture conditions that can convert glutamic acid into γ-aminobutyric acid, and may be the same as the culture conditions in the pre-culture described above. For example, the culture time is preferably from half a day to 4 days, and more preferably from 1 day to 2 days. The culture temperature is preferably 15-50 ° C, more preferably 25-40 ° C, and the initial pH is preferably 3-9, more preferably 5-8.

  In the pre-culture process, the lactic acid bacterium, which consumed at least 15 g / 100 mL of glutamic acid or its salt immediately after the start of the pre-culture process, increased the natural immunity promoting activity derived from the royal jelly of the present invention in the next process. It is preferable in that the agent can be obtained efficiently. A more preferable consumption rate is 20 g / 100 mL or more, and a more preferable consumption rate is 50 g / 100 mL or more. Depending on the concentration of glutamic acid or a salt thereof, the consumption rate of glutamic acid or a salt thereof in the pre-culture step can be adjusted as appropriate. A product obtained by consuming glutamic acid or a salt thereof at a consumption rate in the above range and converting it to γ-aminobutyric acid is preferable.

<Fermentation process>
When the lactic acid bacteria obtained by the pre-culture process are used for fermentation, the NBRC12005 strain may be washed with sterilized water, sterilized physiological saline, sterilized buffer, or the like. When lactic acid bacteria are washed, excess water may be removed by a glass filter, centrifugation, or the like, and the washed lactic acid bacteria may be suspended again in sterilized water, sterilized physiological saline, or sterilized buffer. Further, after the pre-culture is completed, the above-described washing is not performed, and the lactic acid bacterium obtained by separating the culture supernatant (separating cells) by a glass filter, centrifugation or the like may be used in the fermentation process. Good.

  Further, the necessary amount of lactic acid bacteria may be mixed with the fermentation solution at once, or the necessary amount of lactic acid bacteria may be divided into two or more times and mixed. Moreover, when using 2 or more types of lactic acid bacteria, each lactic acid bacteria may be mixed with a fermentation liquid simultaneously, and you may divide separately and may mix with a fermentation liquid.

  The mixing ratio of the lactic acid bacteria or the culture solution obtained in the pre-culture step and the fermentation solution is preferably 0.05 to 4 parts by volume of the culture solution with respect to 1 part by volume of the “lactic acid bacteria or the mixture of the culture solution and the fermentation solution”. 0.1 to 3 parts by volume is more preferable, and 0.2 to 1.5 parts by volume is particularly preferable.

  The fermentation process is carried out after the pre-culture process, and a fermented liquid containing royal jelly and preferably “glutamic acid or a salt thereof” and a lactic acid bacterium obtained in the pre-cultured liquid are mixed, and the natural immunity derived from the royal jelly of the present invention This is a step of producing an agent. The fermentation broth must contain at least royal jelly, but preferably contains glutamic acid or a salt thereof. Here, “mixing lactic acid bacteria” means not only the case where lactic acid bacteria obtained by separating the lactic acid bacteria obtained in the pre-culture step by an appropriate means (for example, centrifugation) are mixed with the fermentation broth, It also refers to the case where lactic acid bacteria are mixed with the fermentation broth in the state of the culture broth.

  The solid content concentration of the royal jelly when the lactic acid bacteria obtained in the fermentation broth and the pre-culture step or the culture solution is mixed is preferably 1 to 20 g / 100 mL, more preferably 3 to 15 g / 100 mL, and particularly preferably 4 to 10 g / 100 mL.

  Moreover, it is preferable that a fermented liquor contains glutamic acid or its salt in the point which enhances an innate immunity promotion effect | action, and glutamic acid potassium, glutamic acid sodium, magnesium glutamic acid, and calcium glutamate are illustrated as glutamate. The glutamic acid (salt) contained may be glutamic acid (salt) released from the protein or peptide by degrading the “protein or peptide containing glutamic acid” with a protease and / or peptidase.

  The glutamic acid concentration in the fermentation step, that is, the glutamic acid concentration after mixing the fermentation broth and the lactic acid bacteria obtained in the pre-culture step or the culture solution (culture solution of the lactic acid bacteria) is preferably 0.1 to 10 g / 100 mL, Especially preferably, it is 1-5 g / 100 mL.

  The fermentation broth may further contain a medium component. Examples of the medium component include a carbon source, a nitrogen source, organic micronutrients, metals, lipids and the like. They are used alone or in combination of two or more. Preferred examples of the medium component include yeast extract, peptone, metals and the like.

  Fermentation is started by mixing the fermented liquor and the lactic acid bacteria or the broth obtained in the pre-culture step. Fermentation conditions such as fermentation time, temperature, and pH are not particularly limited, but the number of fermentation days is preferably from half a day to 10 days, and more preferably from 2 days to 6 days. The fermentation temperature is preferably 15 to 50 ° C, more preferably 25 to 40 ° C. The initial pH of the fermentation is preferably 3-9, more preferably 5-8. Moreover, although not limited, stationary fermentation is preferable.

  In the present invention, “fermentation” means that the intended “royal jelly-derived innate immune activator” is produced by mixing lactic acid bacteria and fermentation liquor. It doesn't matter if it grows or doesn't.

<Sterilization process>
The “method for producing an innate immunity activator with enhanced innate immunity promoting action” in the present invention preferably further includes a step of sterilizing lactic acid bacteria. Examples of the sterilization method include a method of sterilization by heating, a method of sterilization using a chemical, a method of sterilizing lactic acid bacteria by filtration, a method of sterilizing lactic acid bacteria using centrifugation, and the like. Alternatively, two or more types may be combined. Preferably, it is a method of sterilizing lactic acid bacteria by heating, and the heating temperature at that time is preferably 60 ° C. or more, more preferably 80 to 100 ° C., particularly preferably 90 to 100 ° C. If the heating temperature is too low, lactic acid bacteria may not be sterilized to the extent that there is no problem as an innate immune activator with enhanced innate immunity promoting action, and if the heating temperature is too high, the innate immunity obtained In some cases, the activator is decomposed, the higher order structure is changed, or the like is lost.

<Purification process>
The “method for producing an innate immunity activation agent with enhanced innate immunity promoting effect” of the present invention preferably has a purification step. Examples of purification methods include gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, normal phase chromatography, ultrafiltration, electrophoresis, etc., and these can be used alone or in combination. It is preferable to carry out purification. When purifying by chromatography or the like, it is also preferable to use a material obtained by collecting the supernatant in advance by centrifugation.

  Gel filtration chromatography has a carrier for gel filtration chromatography that can separate proteins of various molecular weights, and examples thereof include crosslinked agarose resin and silica particles. Examples of ion exchange groups used in ion exchange chromatography include anion exchangers and cation exchangers. Examples of anion exchangers include diethylaminoethyl group (DEAE group), quaternary aminoethyl group (QAE). ) And the like. Examples of the cation exchanger include a carboxymethyl (CM) group and a sulfopropyl (SP) group. For purification of the innate immunity activator of the present invention, an anion exchanger having a diethylaminoethyl group or the like is preferable.

Examples of the carrier used for 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 an octadecyl group (C18), and a carrier in which C30, C8, C4 and the like having a different alkyl group length from the octadecyl group are bonded.
Examples of the carrier used for normal phase chromatography include silica gel, a carrier having a cyanopropyl group, a functional group having a diol structure, an aminopropyl group, a polyamine, and the like.

  As a result of purification, at least a polysaccharide was obtained as an active ingredient. Accordingly, the present invention is also the above-mentioned royal jelly-derived innate immunity activator containing a polysaccharide (as an active ingredient). Preferably, the royal jelly-derived innate immunity activator containing an acidic polysaccharide (as an active ingredient). Of the inclusions, only polysaccharides or acidic polysaccharides may have an innate immunity promoting effect. In that case, although not limited, the “royal jelly-derived innate immunity activator” of the present invention is “a royal jelly-derived innate immunity activator which is a polysaccharide or an acidic polysaccharide”.

<Use / use of innate immune activator, fermented royal jelly composition>
The royal jelly-derived innate immunity activator of the present invention can be used as an agent for activating human innate immunity (promoting action). The “royal jelly-derived innate immunity activator” of the present invention is desirably taken and administered. Moreover, it can be used not only as a medicine taken by humans but also in a food. It can also be administered to livestock. You may mix | blend with livestock feed as a supplement, a dietary supplement, a pharmaceutical etc.

  The active ingredient may be used after being isolated. The dosage form is not particularly limited, and examples thereof include powders, powders, granules, tablets, capsules, pills, suppositories, and liquids. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer, etc.

  The royal jelly-derived innate immunity activator of the present invention, a purified product thereof, or an isolated active ingredient can be made into a fermented royal jelly composition by containing various components. Examples of such various components include sugars, lipids, emulsifiers, thickeners, seasonings, fragrances, acidity regulators, preservatives, fruit juices, fragrances, nutritional ingredients, and the like, as long as the effects of the present invention are not impaired. Can be used. Moreover, these various components may be used independently and may be used in mixture of 2 or more types.

  Examples of the sugar 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 sourness adjusters 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 fruit juices 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. Examples of nutritional components include vitamins such as vitamin D; minerals such as calcium, magnesium, iron, manganese, and zinc;

  The “royal jelly-derived innate immune activator” obtained by the production method of the present invention, a purified product thereof, an isolated active ingredient, or a fermented royal jelly composition can be contained in food. 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 Etc. These foods are also useful as 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 those food shapes.

<Evaluation of innate immunity promoting action>
The presence / absence / intensity of the innate immunity promoting action is preferably evaluated by the method described in WO 2008/126905 and WO 2009/157409 (method outlined below). The above-mentioned “agent derived from royal jelly produced by a production method comprising a step of fermenting royal jelly with lactic acid bacteria” is an innate immunity activator having an innate immunity promoting action, It was found and confirmed by the method described in the international publication pamphlet (method outlined below).

  Further, when the royal jelly is fermented with lactic acid bacteria by the method described in the above international publication pamphlet (the method described below), it has been found that the natural immunity promoting action is enhanced over the royal jelly as a raw material. Can provide the “method for producing an innate immunity activator with enhanced innate immunity promoting effect”.

  That is, when a sample is administered to a fully transformed insect larva such as a silkworm, when a substance having an innate immunity promoting action is administered to the completely transformed insect larva, it takes about 10 minutes to complete. Muscle contraction (hereinafter abbreviated as “slowing muscle contraction”) occurs. This slowing muscle contraction does not occur when lipopolysaccharide (LPS), which is a cause of false positives when evaluating the innate immunity promoting action, is used, so it is practical for evaluating the innate immunity promoting action. It can be used as a general indicator.

The royal jelly-derived innate immunity activator of the present invention is preferably obtained by a screening method including at least the following steps (a) to (c), and further includes other steps as necessary. But you can.
(A) a step of administering a test substance to an organism having an innate immunity mechanism (b) a step of evaluating whether or not the test substance contracts muscles of the organism having the innate immunity mechanism (c) the innate immunity Selecting a substance evaluated to contract muscles of living organisms having a mechanism

  It is preferable to evaluate the magnitude of the innate immunity promoting activity based on the magnitude of the slowing muscle contraction activity when administered to an organism having an innate immunity mechanism because the evaluation of the magnitude of the innate immunity promoting action is accurate. .

  The “organism having an innate immune mechanism” is preferably an organism belonging to insects. The “insects” refers to a class of arthropods from the greater submaxillary subfamily, consisting of four subclasses of caterpillars, flying beetles, ungual insects, and moth insects. The “organism belonging to insects” is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably a larva from the viewpoint of convenience of handling. The larva is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include lepidopterous (including moths and butterflies), Coleoptera (including beetles) larvae, and the like. The larva is preferably a large larva from the viewpoint of ease of administration of the test substance. The “large larva” refers to a larva having a body length of 1 cm or more. As the larvae, for example, silkworms (larvae of Bombyx mori) and erysan larvae are preferable.

  As the organism having an innate immune mechanism, it is preferable to use an organism that can easily measure the degree of muscle contraction by the test substance in the step (b), and a silkworm is particularly suitable also in this respect. Moreover, as a silkworm, it is also preferable to use the decapitation muscle specimen of a silkworm, for example as shown in the Example mentioned later. The use of silkworm decapitation muscle specimens is advantageous in that it eliminates the input of signals from the center.

  The administration method of the test substance to the organism having the innate immune mechanism is not particularly limited and can be appropriately selected depending on the purpose. For example, oral administration, intraperitoneal administration, injection into blood, enteral Injection to feed, addition to feed (food), and the like. The dose of the test substance to the organism having the innate immunity mechanism is not particularly limited and can be appropriately selected according to the purpose.

  It is evaluated whether or not the test substance contracts the muscle of an organism having an innate immune mechanism (step (b)). As a method for evaluating whether or not the test substance contracts muscles of an organism having the innate immunity mechanism, for example, the degree of contraction of muscles of the organism having the innate immunity mechanism is determined as a Contract value value (C value). (E.g. Ishii K., Hamamoto H., Kamimura M., Sekimizu K., J. Biol. Chem. Jan. 25; 283 (4): 2185-91 (2008) )reference).

  Specifically, the body length of the organism having the innate immune mechanism before and after administration of the test substance is measured, and “body length before administration” − “body length after administration” is expressed as “body length before administration”. The muscle contraction value (C value) divided by “is obtained. The greater the degree of muscle contraction, the greater the C value. If there is no muscle contraction, the C value is zero. Conversely, if the muscle relaxes, the C value becomes negative (minus). It is appropriate to monitor the process of muscle contraction by an appropriate method, for example, using a transducer connected to a recorder, and measure the C value when the degree of muscle contraction is maximized. The time required for muscle contraction varies depending on the type and amount of the administered test substance, and therefore is appropriately selected.

  When the muscle contraction value (C value) is a positive (plus) value, the test substance is evaluated as having an innate immunity promoting action. Moreover, it can be evaluated that the greater the muscle contraction value (C value), the greater the test substance has a natural immunity promoting effect. If the muscular contraction value (C value) is larger than the royal jelly before fermentation by fermenting the royal jelly with lactic acid bacteria, it can be evaluated that the natural immunity promoting action is enhanced.

  The mechanism by which the innate immunity promoting effect can be evaluated by the magnitude of slowing muscle contraction when administered to larvae of fully transformed insects is as follows. That is, when a substance that promotes the innate immune function (peptidoglycan, β-glucan, etc.) enters the body of an organism having an innate immune system, it binds to the receptor of the immunocompetent cell, and as a result, reactive oxygen species Is produced in the case of larvae of fully transformed insects, resulting in activation of BmPP, a paralytic peptide, and the activated BmPP acts on muscle cells to promote muscle contraction.

  Muscle contraction also occurs when other neurotransmitters act, but in that case muscle contraction is a fast reaction that ends within a few seconds after administration of the sample, whereas the innate immune system is activated The muscle contraction in this case is a slow muscle contraction that requires about 10 minutes to complete the contraction, so that both can be clearly distinguished.

  Moreover, this evaluation method has an advantage that the pharmacokinetics can be reflected as compared with the evaluation method using immunocompetent cells such as macrophages. Further, when evaluation is performed using cultured cells, it is not affected by “LPS, which is considered to have a possibility of innate immunity promoting action and is evaluated as a false positive substance and is problematic” (LPS is innate immune function activity) There is also an advantage that it is not evaluated that there is a chemical action.

  Therefore, the innate immunity promoting action of a composition that may contain various components that have not been refined can be evaluated, which is a particularly suitable index for the present invention. Therefore, as a result of evaluation using this as an index, the production method of the present invention has excellent innate immunity through a step of selecting a substance evaluated to contract muscles of an organism having an innate immune mechanism (step (c)). It was found as a method for producing an activator.

  When the test substance is used for evaluation by the steps (a) to (b), and then in the step (c), the muscle of the organism having the innate immune mechanism is contracted from various test substances. By selecting an evaluated substance, an innate immunity activator can be screened easily and efficiently.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example and an examination example, this invention is not limited to a following example. Hereinafter, simply “%” means “% by mass” unless otherwise specified.

[Preparation of innate immune activator]
Example 1
<Pre-culture process>
Into a 300 mL Erlenmeyer flask with baffle, put 100 mL of 1% glucose, 1% yeast extract liquid medium (pH 7.2) and 1 mL of 10% manganese yeast solution, put a silico stopper (registered trademark), 121 ° C And then autoclaved for 15 minutes to prepare a liquid medium.
50 μL of NBRC12005 strain stocked with glycerol was added to the liquid medium, and static culture was performed at 30 ° C. for 1 day. Next, 3 mL of the culture solution of NBRC12005 strain was added to the liquid medium prepared in the same manner as described above, and static culture was performed at 30 ° C. for 1 day, and NBRC1205 strain was cultured in advance.

<Pre-culture process>
A 5 L MBS minijar fermenter was charged with 3 L of 1% glucose and 1% yeast extract liquid medium (pH 7.2) and 1 mL of 10% manganese yeast solution, and autoclaved at 121 ° C. for 20 minutes. . 75 mL of 40% sodium glutamate monohydrate solution autoclaved (at 121 ° C. for 15 minutes) was added to prepare a preculture solution.
Next, 90 mL of the culture solution obtained by the pre-culture was added and pre-cultured for 1 day. The culture temperature was 30 ° C., and static culture was performed without aeration.

<Fermentation process>
A 10 L MBS jar fermenter was charged with 3 L of 2% yeast extract and 60 mL of 10% manganese yeast solution and autoclaved at 121 ° C. for 20 minutes.
Next, after adjusting natural raw royal jelly (solid content concentration 34%) to pH 6.7 with 1N NaOH, 1.2 liters of royal jelly diluent diluted with purified water 1.6 times and autoclave sterilization (121 ° C. (15 minutes) was added to a fermentation broth consisting of 0.6 L of 40% sodium glutamate monohydrate solution, and 1.2 L of the culture broth obtained by the pre-culture was added and fermentation was performed for 5 days. Fermentation temperature was 30 degreeC and stationary fermentation was performed without aeration. However, stirring was performed once a day during fermentation. If there was an insoluble precipitate in the 10% manganese yeast solution, the solution was left at room temperature for 15 minutes, and the supernatant was used as a 10% manganese yeast solution.

  After completion of the fermentation for 5 days, sterilization was performed by heating at 105 ° C. for 30 minutes, and the “royal jelly-derived innate immunity activator” of the present invention was obtained by lyophilization. This lyophilized innate immunity activator had a γ-aminobutyric acid content of 27.4%.

[Measurement of innate immunity promoting effect]
(1) Preparation of silkworm muscle specimen As described below, innate immunity activity was evaluated using measurement of silkworm slow muscle contraction. The slow muscle contraction was measured according to the method described in Ishii K., Hamamoto H., Kamimura M., Sekimizu K., J. Biol. Chem. Jan. 25; 283 (4): 2185-91 (2008). went.
In other words, the dissimilar scissors of the lower part of the eye-shaped crest of the silkworm (weight 5 g) of the fifth day of the fifth infancy that was bred with the antibiotic-containing artificial diet (Silk Mate 2S, Nippon Agricultural Industry) from the first day of the fifth age And the intestinal tract and the silk gland were removed with tweezers. Next, using a semicircular anatomical needle, a sewing thread having a length of about 20 cm was passed through the slightly lower side from the cut surface, and both ends of the thread were tied to form a ring. A silk thread sample was prepared by binding a 20 cm sewing thread to the tail leg of the silkworm, passing a 27 g clip through the thread, and tying both ends of the thread. The prepared specimen was suspended from a stand for 1 to 2 hours and stabilized.

(2) Attaching the muscle specimen to the transducer terminal After calibrating the transducer terminal and fixing the terminal horizontally to the stand, attach a 27g clip to one end of the terminal and remove the clip to the other end. The silkworm muscle specimen was attached and the balance of both ends was adjusted.

(3) Silkworm muscle contraction test A suspension of an innate immune activator at a concentration of 200 mg / mL was prepared with an autoclaved 0.9% NaCl aqueous solution. Next, suspensions diluted to 1/4, 1/16, 1/64, 1/256 using 0.9% NaCl aqueous solution were prepared. These suspensions were taken into a 1 mL tuberculin syringe and injected into a silkworm muscle specimen attached to a transducer terminal by 0.05 mL using a 27 gauge injection needle. After 6 minutes, the length of the muscle sample before and after muscle contraction was measured with a ruler, and the muscle contraction value was calculated as follows.
C value (muscle contraction value) = ([length before contraction] − [length after contraction]) / [length before contraction]

  Air, yeast β-glucan (Sigma, G5011) and mechabufucoidan (RIKEN vitamin) were used as positive controls for muscle contraction, and 0.9% NaCl aqueous solution was used as a negative control.

(4) Data analysis The logarithm of the weight (mg) of the sample administered to each muscle specimen is plotted on the horizontal axis and the C value (muscle contraction value) is plotted on the vertical axis, and Kaleidagraph version 4.0 (Synergy Software) is used. Then, a non-linear regression curve fitting was performed to create a dose response curve. From the obtained dose-response curve, the weight (mg) of the sample giving C value (muscle contraction value) = 0.15 was determined (this is defined as one activity unit (unit)), and the activity (U / Mg) was calculated. These results are shown in Table 1 and FIG.

Comparative Example 1
The royal jelly freeze-dried product used as a raw material in Example 1 was evaluated in the same manner as in Example 1. The results are shown in Table 1 and FIG.

Reference example 1
The yeast β-glucan was manufactured by Oriental Yeast Co., and the mechabufucoidan was manufactured by Riken Vitamin Co., and the innate immunity promoting action was measured in the same manner as in Example 1 and Comparative Example 1. The results are shown in Table 1 and FIG.

Comparative Example 2
Vegetables (mini tomato, spinach, Japanese radish, ginger) were freeze-dried and pulverized in a mortar. Then, 5 mL of distilled water was added to 1 g of each sample, and centrifuged at 8000 rpm for 5 minutes. Distilled water (3 mL) was added to the precipitate, and heat treatment was performed at 121 ° C. for 20 minutes using an autoclave. This was centrifuged at 8000 rpm for 10 minutes at room temperature to obtain a supernatant, which was used as a hot water extract. The hot water extract sample is diluted with 0.9% NaCl so that the dilution rate is 0.001 to 1% by mass, 0.05 mL is injected into the body cavity of a decapitation muscle specimen of silkworm, and the length is increased 6 minutes later. The innate immunity promoting action was measured in the same manner as in Example 1 (Table 1). Here, the “dilution rate” is the mass% of the hot water extract sample with respect to the whole liquid diluted with 0.9% NaCl aqueous solution.

  The administration of the innate immunity activator of the present invention was inferior to yeast-derived β-glucan and mechabufucoidan, but had an innate immunity promoting action. And the effect was 100 times or more superior to the freeze-dried royal jelly which is a raw material, and the effect of this invention has been confirmed. Furthermore, it turned out that it has a natural immunity promotion effect higher than the various vegetables compared.

  In addition, administration of the royal jelly-derived innate immunity activator of the present invention caused muscle contraction in a concentration-dependent manner similar to yeast-derived β-glucan, and its specific activity was 0.50 U / mg (FIG. 1, Table). 1). From the comparison with the freeze-dried royal jelly which is a raw material of the innate immunity activator of the present invention, the innate immunity promoting action is enhanced by 175 times depending on the fermentation treatment (FIG. 2, Table 1). We were able to confirm the "effect by".

  When a 0.9% NaCl aqueous solution as a sample solvent was administered, muscle contraction did not occur (muscle contraction value = 0.00), and contraction occurred in air as a positive control (muscle contraction value = 0.43). ). In yeast β-glucan and mekabufucoidan, contraction occurred in a concentration-dependent manner, and the specific activities calculated from the dose response curve were 77 U / mg and 2.0 U / mg, respectively.

Example 2
[Separation of active ingredients of innate immunity activator]
(1) Preparation of Hot Water Extraction Solution 50 mg of 0.9% NaCl aqueous solution was added to 10 g of the powder of “royal jelly-derived innate immunity activator” obtained in Example 1 to prepare a 200 mg / mL suspension. . Next, it heated at 121 degreeC for 20 minute (s) using the autoclave. After cooling the suspension, 48 mL of supernatant (hot water extract fraction) was obtained by centrifugation at 8000 rpm for 5 minutes at room temperature using a high-speed centrifuge (Hitachi Koki CR-21, rotor R10A2). Next, 16 mL of this centrifugation supernatant was dispensed, 32 mL each of 99.5% ethanol was added and stirred well, and 1.5 g (dry weight) of white precipitate (by dry weight) was centrifuged at 8000 rpm for 5 minutes at room temperature. Ethanol precipitation fraction) was obtained. 400 mg of the precipitate was dissolved in 8 mL of 10 mM Tris-HCl buffer (pH 7.9), and the supernatant was recovered by centrifugation at 15000 rpm for 5 minutes. This was used as a starting material for anion exchange chromatography.

(2) Separation of polysaccharides by anion exchange chromatography 8 mL of an aqueous sample solution was applied to a column packed with 20 mL of DEAE-cellulose (Whatman, DE52) equilibrated with 10 mM Tris-HCl buffer (pH 7.9). After applying and washing the column with 100 mL of 10 mM Tris-HCl buffer (pH 7.9), the adsorbed fraction was eluted with 10 mL of 10 mM Tris-HCl buffer (pH 7.9) containing 0.4 M NaCl, 5 mL each was fractionated into 42 fractions.

  For each fraction, the total sugar amount was quantified by the phenol-sulfuric acid method, and the innate immunity promoting action was measured. Table 2 shows the results of the sugar content and muscle contraction value (C value) (innate immunity promoting action) of the anion exchange chromatography fraction.

(3) Sugar determination by phenol-sulfuric acid method 0.1 mL of 5 (w / v)% phenol aqueous solution was added to 0.1 mL of each fraction diluted 10-fold with 10 mM Tris-HCl buffer (pH 7.9). After stirring with a vortex mixer for 2 seconds, 0.5 mL of concentrated sulfuric acid was added and stirred, and reacted at room temperature for 20 minutes. 0.2 mL of each reaction solution was dispensed into a 96-well microplate, and the absorbance at 490 nm was measured with a plate reader (MTP-300, Corona Electric). A calibration curve was prepared using D-glucose as a standard sample.

(4) Concentration of fractions After adding 6.0 mL of 99.5% ethanol to 3.0 mL of each fraction and stirring well, the mixture was centrifuged at 8000 rpm for 5 minutes at room temperature to collect the precipitate. To the dried precipitate, 0.5 mL of 0.9% NaCl aqueous solution was added and dissolved to prepare a 6-fold concentrated solution.

(5) Measurement of muscle contraction value (C value) of fraction fraction concentrate The silkworm muscle specimen was injected with 0.2 mL each of the 6-fold concentrate concentrate. The length of the muscle sample is measured using a ruler, and the muscle contraction value (C value) = ([length before contraction] − [length after contraction]) / [length before contraction]) is calculated. (Table 2). Air was used as a positive control for silkworm muscle contraction, and 0.9% NaCl aqueous solution was used as a negative control.

  In DEAE-cellulose column chromatography, the polysaccharide contained in the hot water extract of the innate immune activator was separated into two peaks centering on fractions 6 and 27. The sugar recovery rate was 58%.

  Both sugar peak 1 (fractions 4-6) and peak 2 (fractions 26-30) showed muscle contraction activity (innate immunity promoting action). At this time, even when 0.9% NaCl aqueous solution as a sample solvent was administered, silkworm muscle contraction did not occur (muscle contraction value = 0.00), but contraction occurred in the positive control air (muscle contraction). Shrinkage value = 0.38).

  As shown in Table 1 and FIGS. 1 and 2, an innate immunity activator having a significantly higher innate immunity promoting action than the lyophilized royal jelly as a starting material was obtained by fermentation treatment using lactic acid bacteria. From Table 2, there were two types of active ingredients. Moreover, it was a polysaccharide and it was estimated that it was an acidic polysaccharide.

  The innate immunity activator in the present invention is highly safe because it is derived from natural royal jelly, and can be widely used as an innate immunity enhancer as an additive for functional foods, foods, pharmaceuticals and the like.

Claims (3)

A method for producing an innate immunity activator comprising the following acidic polysaccharide as an active ingredient, wherein the innate immunity promoting action is enhanced 100 times or more compared to unfermented royal jelly,
(A) a step of fermenting royal jelly with lactic acid bacteria Lactobacillus brevis , and
(B) supernatant of hot water extract of contained in the lactic acid bacterium fermented product "the lactic acid bacterium fermented product, when fractionated using anion exchange chromatography, two two that will be separated as a peak step Ru are contained in the acidic polysaccharide "as an active ingredient,
Natural method for producing immunostimulatory agent you comprising a. The method for producing an innate immunity activation agent with enhanced innate immunity promoting activity according to claim 1, comprising at least the following steps (1) to ( 5 ).
(1) Step of pre-culturing lactic acid bacteria (2) Step of fermenting royal jelly with lactic acid bacteria, and
(3) Process of sterilizing lactic acid bacteria
(4) Separating and purifying the acidic polysaccharide
(5) A step of containing the acidic polysaccharide obtained in step (4) as an active ingredient   The method for producing an innate immunity activator according to claim 2, wherein the step (3) of sterilizing lactic acid bacteria is a step of sterilizing lactic acid bacteria by heating.

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