JP7304051B2 - COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF - Google Patents
COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF Download PDFInfo
- Publication number
- JP7304051B2 JP7304051B2 JP2019009953A JP2019009953A JP7304051B2 JP 7304051 B2 JP7304051 B2 JP 7304051B2 JP 2019009953 A JP2019009953 A JP 2019009953A JP 2019009953 A JP2019009953 A JP 2019009953A JP 7304051 B2 JP7304051 B2 JP 7304051B2
- Authority
- JP
- Japan
- Prior art keywords
- kelp
- production
- aspergillus oryzae
- fermented product
- aspergillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
本発明は、褐藻類に属する海藻を麹菌で発酵させて得られる免疫調節用組成物及びその製造方法に関する。 TECHNICAL FIELD The present invention relates to an immunoregulatory composition obtained by fermenting seaweed belonging to brown algae with Aspergillus oryzae, and a method for producing the same.
近年、アレルギーや癌等の免疫に関わる疾患の患者数が増加している。その治療は長期間に及び、高額な医療費がかかること、また Quality of Life を低下させるだけでなく、最悪の場合は死に至らしめることから、その予防や多種の治療が必要とされる。このため、免疫調節作用を有する有用で安全な食品や医薬品が求められている。 In recent years, the number of patients with immune-related diseases such as allergies and cancers is increasing. Its treatment takes a long time, requires high medical costs, and not only lowers quality of life, but also leads to death in the worst case, so its prevention and various treatments are required. Therefore, there is a need for useful and safe foods and pharmaceuticals that have immunomodulatory activity.
一方、海藻は日本では古くから食材や薬として用いられてきた。海藻はミネラル、炭水化物を多く含むが、この炭水化物の大部分は海藻特有の多糖類であり、有用なものである。海藻が含む多糖類は、細胞壁に含まれて藻体を支える骨格多糖類、細胞間を充填する粘質多糖類、エネルギー貯蔵形態である貯蔵多糖類に特に分類される。特に粘質多糖類のフコイダンやアルギン酸は褐藻類特有の成分であり、利用の範囲は広く、工業、医療、食品等さまざまな分野に応用されている。さらに、生理活性としては抗凝固・抗血栓作用、抗炎症作用、抗ウイルス作用、コレステロール低下作用、高血圧低下作用等の報告がある。 On the other hand, seaweed has long been used as food and medicine in Japan. Seaweed contains many minerals and carbohydrates, and most of these carbohydrates are polysaccharides unique to seaweed and useful. Polysaccharides contained in seaweed are classified into skeletal polysaccharides that are contained in cell walls and support algal bodies, viscous polysaccharides that fill spaces between cells, and storage polysaccharides that are in the form of energy storage. In particular, fucoidan and alginic acid, which are viscous polysaccharides, are components peculiar to brown algae, and have a wide range of uses, and are applied in various fields such as industry, medicine, and food. Furthermore, as physiological activities, there are reports of anticoagulant/antithrombotic, anti-inflammatory, antiviral, cholesterol-lowering, and hypertension-lowering effects.
ここで、海藻の発酵物が免疫調節作用を有することを開示した文献として、例えば下記文献1には、海藻の納豆菌発酵物がマクロファージ産生能に基づく免疫賦活活性と抗腫瘍活性を有することが記載されている。 Here, as a document disclosing that a fermented product of seaweed has an immunomodulatory effect, for example, the following document 1 discloses that a fermented product of natto seaweed has an immunostimulatory activity and an antitumor activity based on the ability to produce macrophages. Are listed.
しかしながら、褐藻類に属する海藻の麹菌発酵物が免疫調節作用を有するという報告は、発明者が知る限り未だない。 However, as far as the inventor is aware, there has been no report that a koji mold fermented product of seaweed belonging to brown algae has an immunoregulatory effect.
したがって、本発明の目的は、IgE産生阻害活性、及びIL-12産生誘導活性を有する免疫調節用組成物を提供することにある。 Accordingly, an object of the present invention is to provide an immunoregulatory composition having IgE production inhibitory activity and IL-12 production-inducing activity.
上記目的を達成するため、本発明の1つは、褐藻類に属する海藻の麹菌発酵物を有効成分として含有する免疫調節用組成物を提供するものである。本発明の有効成分である褐藻類に属する海藻の麹菌発酵物は、IgE産生阻害活性、及びIL-12産生誘導活性を有するので、免疫調節作用を有する。 In order to achieve the above object, one of the present invention provides an immunoregulatory composition containing as an active ingredient an Aspergillus oryzae fermented product of seaweed belonging to brown algae. Aspergillus oryzae fermented seaweed belonging to brown algae, which is the active ingredient of the present invention, has IgE production inhibitory activity and IL-12 production-inducing activity, and thus has an immunoregulatory effect.
本発明の免疫調節用組成物は、IgE産生阻害活性及び/又はL-12産生誘導活性を有することが好ましい。 The immunomodulatory composition of the present invention preferably has IgE production inhibitory activity and/or L-12 production-inducing activity.
本発明の免疫調節用組成物における褐藻類に属する海藻は、マコンブ、ヒダカコンブ(ミツイシコンブ)、リシリコンブ、ナガコンブ、アラメ、及びワカメからなる群より選ばれた少なくとも1種であることが好ましい。 The seaweed belonging to brown algae in the immunomodulatory composition of the present invention is preferably at least one species selected from the group consisting of Laminaria japonica, Hidaka kelp (Mitsui kelp), Risilicon bu, Long kelp, Arame, and Wakame seaweed.
また、本発明のもう1つは、褐藻類に属する海藻を原料として含有する培養液に、麹菌を接種して好気性条件下で培養することにより、褐藻類に属する海藻の麹菌発酵物を得ることを特徴とする免疫調節用組成物の製造方法を提供するものである。これによれば、IgE産生阻害活性、及びIL-12産生誘導活性を有する免疫調節用組成物を得ることができる。 In another aspect of the present invention, a culture solution containing seaweed belonging to brown algae as a raw material is inoculated with koji mold and cultured under aerobic conditions to obtain an aspergillus fermentation product of seaweed belonging to brown algae. The present invention provides a method for producing an immunomodulatory composition characterized by: According to this, an immunoregulatory composition having IgE production inhibitory activity and IL-12 production-inducing activity can be obtained.
本発明の免疫調節用組成物の製造方法において、前記免疫調節用組成物はIgE産生阻害活性及び/又はIL-12産生誘導活性を有することが好ましい。 In the method for producing an immunoregulatory composition of the present invention, the immunoregulatory composition preferably has IgE production inhibitory activity and/or IL-12 production-inducing activity.
本発明の免疫調節用組成物の製造方法において、前記培養液中の前記褐藻類に属する海藻の固形分濃度を0.1~10.0質量%とすることが好ましい。これによれば、培養液の粘度等が発酵に適した状態となり、麹菌による発酵を良好に進行させることができる。 In the method for producing an immunomodulatory composition of the present invention, it is preferable that the solid content concentration of the seaweed belonging to the brown algae in the culture medium is 0.1 to 10.0% by mass. According to this, the viscosity and the like of the culture solution will be in a state suitable for fermentation, and the fermentation by the koji mold can proceed satisfactorily.
本発明の免疫調節用組成物の製造方法において、前記麹菌の培養を36~60時間行うことが好ましい。これによれば、IgE産生阻害活性、及びIL-12産生誘導活性を有する発酵物を効率よく得ることができる。 In the method for producing an immunomodulatory composition of the present invention, it is preferable to culture the koji mold for 36 to 60 hours. According to this, a fermented product having IgE production inhibitory activity and IL-12 production inducing activity can be obtained efficiently.
本発明の免疫調節用組成物の製造方法において、前記麹菌発酵物又はその乾燥物を、30~60℃の温水で処理して抽出物を得ることが好ましい。これによれば、IgE産生阻害活性、及びIL-12産生誘導活性を有する発酵物を効率よく得ることができる。 In the method for producing an immunomodulatory composition of the present invention, it is preferable to obtain an extract by treating the fermented product of Aspergillus oryzae or its dried product with warm water of 30 to 60°C. According to this, a fermented product having IgE production inhibitory activity and IL-12 production inducing activity can be obtained efficiently.
本発明の免疫調節用組成物の製造方法において、前記褐藻類に属する海藻として、マコンブ、ヒダカコンブ(ミツイシコンブ)、リシリコンブ、ナガコンブ、アラメ、及びワカメからなる群より選ばれた少なくとも1種を用いることが好ましい。 In the method for producing an immunomodulatory composition of the present invention, the seaweed belonging to the brown algae may be at least one selected from the group consisting of Laminaria japonicum, Hidaka kelp (Mitsui kelp), Risilicon bu, Long kelp, Arame, and Wakame seaweed. preferable.
本発明の有効成分である褐藻類に属する海藻の麹菌発酵物は、IgE産生阻害活性、及びIL-12産生誘導活性を有するので、免疫調節作用を有する。 Aspergillus oryzae fermented seaweed belonging to brown algae, which is the active ingredient of the present invention, has IgE production inhibitory activity and IL-12 production-inducing activity, and thus has an immunoregulatory effect.
本発明における褐藻類に属する海藻は、コンブ目コンブ科のマコンブ、ヒダカコンブ(ミツイシコンブ)、リシリコンブ、ナガコンブ、およびアラメ、コンブ目チガイソ科のワカメ、ヒバマタ目ホンダワラ科のヒジキ、ホンダワラ、およびアカモク、並びにナガマツモ目モズク科のモズク等が挙げられる。本発明においては、いずれの褐藻類に属する海藻も用いることができるが、容易かつ安価に入手でき、麹菌発酵物のIgE産生阻害効果やIL-12産生誘導活性が高いという点で褐藻類コンブ目に属する海藻のマコンブ、ヒダカコンブ(ミツイシコンブ)、リシリコンブ、ナガコンブ、アラメ、及びワカメからなる群から選ばれた少なくとも1種であることが好ましい。 Seaweeds belonging to brown algae in the present invention include Laminaria kelp, Hidaka kelp (Laminaria japonicum), Risiliconbu, Long kelp, and Arame, Wakame of Laminaria order Urticariaceae, Hijiki, Sargassum, and Akamoku of Fucus sargassum family Sargassum family, and Nagamatsumo Examples include mozuku of the order Mozuku family. In the present invention, seaweed belonging to any brown algae can be used. It is preferably at least one selected from the group consisting of seaweeds such as Laminaria kelp, Hidaka kelp (Mitsuishi kelp), Risilicon bu, Naga kelp, Arame, and Wakame seaweed belonging to .
原料として用いる褐藻類に属する海藻の形態は、特に限定されず、例えば、生、又は乾燥した褐藻類に属する海藻の粉砕物や粉末等が使用できる。 The form of seaweed belonging to brown algae used as a raw material is not particularly limited, and for example, pulverized or powdered raw or dried seaweed belonging to brown algae can be used.
本発明における麹菌としては、特に限定されないが、黄麹菌のアスペルギルス・オリゼ(Aspergillus oryzae)、及びアスペルギルス・ソーヤ(Aspergillus sojae)、アスペルギルス・ルーチェンシス(Aspergillus luchuensis)黒麹菌のアスペルギルス・ニガー(Aspergillus niger)、アスペルギルス・アワモリ(Aspergillus awamori)、及びアスペルギルス・サイトイ(Aspergillus saitoi)、並びに白麹菌のアスペルギルス・ウサミ(Aspergillus usamii)、及びアスペルギルス・カワチ(Aspergillus kawachii)等が挙げられる。この中でも特にアスペルギルス・オリゼ、アスペルギルス・ソーヤ、アスペルギルス・ルーチェンシスが好ましく使用される。 Aspergillus oryzae in the present invention is not particularly limited, but includes Aspergillus oryzae of yellow koji mold, Aspergillus sojae, Aspergillus luchuensis, Aspergillus niger of black koji mold, Aspergillus awamori, Aspergillus saitoi, white koji mold Aspergillus usamii, Aspergillus kawachii, and the like. Among these, Aspergillus oryzae, Aspergillus sojae and Aspergillus luchensis are particularly preferably used.
本発明における免疫調節用組成物の製造方法は、褐藻類に属する海藻を原料として含有する培養液に、麹菌を接種して好気性条件下で培養することにより、褐藻類に属する海藻の麹菌発酵物を得ることを特徴とする。 The method for producing an immunomodulatory composition according to the present invention comprises inoculating koji mold into a culture solution containing seaweed belonging to brown algae as a raw material and culturing the mixture under aerobic conditions, thereby fermenting koji mold of seaweed belonging to brown algae. Characterized by obtaining things.
培養液中には、褐藻類に属する海藻原料を固形分濃度で好ましくは0.1~10.0質量%、より好ましくは0.2~5.0質量%となるように添加し、更に必要に応じてその他の原料を添加して、好ましくは加熱滅菌することにより調製することができる。 In the culture solution, a seaweed raw material belonging to brown algae is added so that the solid content concentration is preferably 0.1 to 10.0% by mass, more preferably 0.2 to 5.0% by mass. It can be prepared by adding other raw materials according to the requirements, preferably by heat sterilization.
褐藻類に属する海藻原料の固形分濃度が上記範囲であれば、培養液の粘度等が発酵に適した状態となり、麹菌による発酵を良好に進行させることができる。褐藻類に属する海藻原料の固形分濃度が0.1質量%未満では、培養液の単位体積当たりの発酵物の収量が少なくなり、生産性が悪くなり、10.0質量%を超えると、培養液の撹拌がしにくくなることによって通気性が悪くなり、発酵の進行が低下する傾向がある。 If the solid content concentration of the seaweed raw material belonging to brown algae is within the above range, the viscosity and the like of the culture solution will be in a state suitable for fermentation, and fermentation by Aspergillus oryzae can proceed favorably. When the solid content concentration of the seaweed raw material belonging to brown algae is less than 0.1% by mass, the yield of the fermented product per unit volume of the culture solution decreases, resulting in poor productivity. It becomes difficult to agitate the liquid, resulting in poor air permeability, which tends to slow down the progress of fermentation.
培養液としては、麹菌の通常の培養に用いられるものでよく、例えば、ペプトン、イーストエクストラクト等のエキス類等を添加することができる。 The culture solution may be one used for ordinary culture of Aspergillus oryzae. For example, extracts such as peptone and yeast extract may be added.
また、加熱滅菌条件は、特に限定されないが、好ましくは65~130℃で5~50分程度の加熱滅菌が好ましく採用される。 The conditions for heat sterilization are not particularly limited, but heat sterilization at 65 to 130° C. for about 5 to 50 minutes is preferably employed.
なお、本発明においては、麹菌を用いることにより、褐藻類に属する海藻原料を酵素等によって分解することなく発酵させることができるが、更に発酵を促進させるため、海藻原料を酵素等によって分解して培地原料としてもよい。この場合の酵素としては、例えばセルラーゼ、ヘミセルラーゼ等を用いることができる。 In the present invention, by using koji mold, it is possible to ferment seaweed raw materials belonging to brown algae without decomposing them with enzymes or the like. It may also be used as a medium raw material. As the enzyme in this case, for example, cellulase, hemicellulase and the like can be used.
上記培養液に、好ましくは、予め前培養して活性化した麹菌を接種し、好気性条件下で、好ましくは20~37℃、より好ましくは25~30℃の温度下で、好ましくは36~60時間、より好ましくは48~52時間培養する。培養温度や時間が上記範囲未満ではIgE産生阻害活性やIL-12産生誘導活性が十分に上昇しない傾向があり、上記範囲を超えてもIgE産生阻害活性やIL-12産生誘導活性がそれ以上上昇せず、生産性が悪くなる傾向がある。 Preferably, pre-cultured and activated Aspergillus oryzae is inoculated into the above culture solution, and the temperature is preferably 20 to 37°C, more preferably 25 to 30°C, preferably 36 to 36°C, under aerobic conditions. Incubate for 60 hours, more preferably 48-52 hours. If the culture temperature or time is less than the above range, the IgE production inhibitory activity and IL-12 production-inducing activity tend not to increase sufficiently, and even if the above range is exceeded, the IgE production inhibitory activity and IL-12 production-inducing activity increase further. and productivity tends to be poor.
培養を好気性条件下とする方法としては、特に限定されないが、例えば振とう培養、通気撹拌培養、静置培養等が挙げられる。工業的に生産する場合には、通気撹拌培養が好ましく採用される。 The method of culturing under aerobic conditions is not particularly limited, but examples thereof include shaking culture, aeration-stirring culture, stationary culture, and the like. For industrial production, aerated agitation culture is preferably employed.
このようにして得られた麹菌の培養液は、そのまま、あるいは更に加工処理することにより、本発明の免疫調節用組成物の有効成分として利用することができる。なお、本発明において、麹菌発酵物とは、麹菌の培養液そのものだけでなく、その加工処理物も含むものとする。 The culture solution of Aspergillus oryzae thus obtained can be used as an active ingredient of the composition for immunoregulation of the present invention as it is or after further processing. In the present invention, the fermented product of Aspergillus oryzae includes not only the culture solution of Aspergillus oryzae itself but also its processed product.
培養液の加工処理方法としては、例えば、(1)培養液を濃縮して濃縮液としたり、(2)培養液をろ過、遠心分離等の手段で固液分離したり、(3)培養液又はその濃縮液を、例えば凍結乾燥、減圧乾燥、噴霧乾燥等の手段で乾燥し、必要により更に粉砕し、篩分け等を行って粉末化したり、(4)培養液を固液分離した上清を必要により濃縮し、乾燥させて粉末エキスとしたり、(5)培養液、その濃縮液、あるいは培養液の乾燥物に、抽出溶媒を添加して有効成分を抽出し、固液分離して抽出液を採取し、該抽出液を濃縮又は乾燥粉末化する方法等を採用することができる。 Examples of methods for processing the culture solution include (1) concentrating the culture solution to obtain a concentrated solution, (2) solid-liquid separation of the culture solution by means of filtration, centrifugation, or the like, and (3) culture solution. Alternatively, the concentrate is dried by means such as freeze drying, vacuum drying, spray drying, etc., and if necessary, it is further pulverized and powdered by sieving or the like, or (4) the supernatant obtained by solid-liquid separation of the culture solution. If necessary, concentrate and dry to obtain a powder extract, or (5) add an extraction solvent to the culture solution, its concentrate, or the dried product of the culture solution to extract the active ingredient, and then separate the solid and liquid to extract. A method of collecting a liquid and concentrating or drying and pulverizing the extract can be adopted.
培養液、その濃縮液、あるいは培養液の乾燥物に、抽出溶媒を添加して有効成分を抽出する場合、抽出溶媒としては、水、又は含水アルコールが好ましく用いられ、特に30~60℃の温水が好ましく、40~55℃の温水がさらに好ましく用いられる。温水の温度が30℃未満では、IgE産生阻害活性やIL-12産生誘導活性を高めにくく、60℃を超えると、IgE産生阻害活性やIL-12産生誘導活性が低下する傾向がある。 When extracting an active ingredient by adding an extraction solvent to the culture solution, its concentrate, or the dried product of the culture solution, water or hydrous alcohol is preferably used as the extraction solvent, particularly hot water at 30 to 60°C. is preferred, and hot water at 40 to 55°C is more preferred. When the temperature of the warm water is less than 30°C, it is difficult to increase the IgE production inhibitory activity and IL-12 production-inducing activity.
本発明の免疫調節用組成物は、上記のようにして得られた麹菌発酵物を有効成分として含有するものであればよい。本発明の免疫調節用組成物においては、本発明の目的を損なわない限り、他の成分を含有することに特に制限はない。例えば、賦形剤、崩壊剤、結合剤、滑沢剤、コーティング剤、着色剤、発色剤、矯味剤、着香剤、酸化防止剤、防腐剤、呈味剤、酸味剤、甘味剤、強化剤、ビタミン剤、膨張剤、増粘剤、界面活性剤等を添加することができる。 The immunoregulatory composition of the present invention may contain the koji mold fermented product obtained as described above as an active ingredient. There are no particular restrictions on the composition for immunoregulation of the present invention, as long as it does not impair the object of the present invention. For example, excipients, disintegrants, binders, lubricants, coating agents, coloring agents, coloring agents, flavoring agents, flavoring agents, antioxidants, preservatives, flavoring agents, acidulants, sweeteners, enhancers agents, vitamins, swelling agents, thickeners, surfactants and the like can be added.
本発明の免疫調節用組成物の投与形態は、特に限定されないが、経口投与組成物であることが好ましい。 Although the dosage form of the composition for immunoregulation of the present invention is not particularly limited, it is preferably an oral composition.
本発明の免疫調節用組成物の製品形態は、特に限定されない。経口投与組成物である場合の製品形態の例としては、液状(液剤)、シロップ状(シロップ剤)、粉末状(顆粒、細粒)、錠剤(錠剤、タブレット)、カプセル状(カプセル剤)、ソフトカプセル状(ソフトカプセル剤)、固形状、半液体状、クリーム状、ペースト状等が挙げられる。 The product form of the composition for immunoregulation of the present invention is not particularly limited. Examples of product forms for oral administration compositions include liquid (solution), syrup (syrup), powder (granules, fine granules), tablets (tablets, tablets), capsules (capsules), Examples include soft capsules (soft capsules), solids, semi-liquids, creams, pastes, and the like.
また、免疫調節用組成物は、例えば医薬品、医薬部外品、機能性食品、栄養補助食品、サプリメント、健康食品、動物用医薬品、動物用医薬部外品、動物用機能性食品、動物用栄養補助食品、動物用サプリメント、動物用健康食品等の製品自体あるいはその原料として使用することができる。 In addition, immunomodulatory compositions include, for example, pharmaceuticals, quasi-drugs, functional foods, dietary supplements, supplements, health foods, veterinary drugs, quasi-drugs for animals, functional foods for animals, and nutrition for animals. It can be used as a product itself or as a raw material for food supplements, animal supplements, animal health foods, and the like.
さらに、免疫調節用組成物は、IgE産生阻害活性やIL-12産生誘導活性を増加させる効果を付与する目的で、広く一般食品の原料としても使用できる。一般食品としては、例えば、パン、菓子、麺、畜肉又は魚肉の加工品、スープ、飲料、ふりかけ、調味料等が挙げられる。 Furthermore, the immunoregulatory composition can be widely used as a raw material for general foods for the purpose of imparting an effect of increasing IgE production inhibitory activity and IL-12 production-inducing activity. Common foods include, for example, bread, confectionery, noodles, processed meat or fish meat, soups, beverages, sprinkles, and seasonings.
本発明の免疫調節用組成物の有効投与量は、特に限定されず、服用者の年齢、体重、性別等によって適宜決定することができるが、通常、成人1日当たりの麹菌発酵物の服用量は、乾燥固形分量として3.5~60gが好ましく、10~50gがより好ましく、30~40gがさらに好ましい。本発明の免疫調節用組成物は、1日1回~数回に分け、又は任意の期間及び間隔で服用され得る。 The effective dose of the composition for immunoregulation of the present invention is not particularly limited, and can be appropriately determined according to the age, body weight, sex, etc. of the recipient. , the dry solid content is preferably 3.5 to 60 g, more preferably 10 to 50 g, even more preferably 30 to 40 g. The immunomodulatory composition of the present invention can be taken once to several times a day, or at any time and interval.
本発明の免疫調節用組成物は、後述する実施例に示されるように、IgE産生阻害活性及び/又はIL-12産生誘導活性を有するので、対象に投与することにより、その対象の免疫を調節することができる。ここで、「調節」とは、本明細書では、免疫応答を誘発し、増強し、刺激して、または支持することを指す。 The immunomodulatory composition of the present invention has IgE production inhibitory activity and/or IL-12 production-inducing activity, as shown in the examples below, and thus administration to a subject regulates the immunity of the subject. can do. As used herein, "modulation" refers to inducing, enhancing, stimulating or supporting an immune response.
免疫調節によって改善が期待される具体的な疾患等としては、例えば、アナフィラキシーショック、アレルギー性鼻炎、結膜炎、自己免疫性溶血性貧血、顆粒球減少症、血小板減少症、血清症、全身性エリテマトーデス(SLE)、関節リウマチ(RA)、糸球体腎炎、接触性皮膚炎、アレルギー性脳炎、移植拒絶反応等が挙げられる。その他、インフルエンザや風邪等にも改善が期待される。 Specific diseases expected to be improved by immunomodulation include, for example, anaphylactic shock, allergic rhinitis, conjunctivitis, autoimmune hemolytic anemia, granulocytopenia, thrombocytopenia, serosis, systemic lupus erythematosus ( SLE), rheumatoid arthritis (RA), glomerulonephritis, contact dermatitis, allergic encephalitis, transplant rejection, and the like. In addition, improvement is expected for influenza, colds, and the like.
以下、実施例を挙げて本発明を更に具体的に説明するが、これらの実施例は本発明を何ら限定するものではない。なお、特に断らない限り、含有率を示す%は質量%を表す。 EXAMPLES The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the present invention in any way. In addition, unless otherwise indicated, % which shows a content rate represents the mass %.
<実験例1>
各種微生物を用いて発酵させたヒダカコンブのIL-12産生量を定量した。
<Experimental example 1>
The amount of IL-12 produced by Hidaka kelp fermented with various microorganisms was quantified.
ここで、IL-12は、主にT細胞やナチュラルキラー(NK)細胞に作用して、その増殖促進、IFN-γ産生誘導作用、細胞傷害性の増強、さらにナイーブT細胞のTh1細胞への分化誘導等を引き起こす。そのため、IL-12 の産生量を増加させることで、細胞性免疫が亢進し、癌細胞にアポトーシスを誘導できるという報告がある。 Here, IL-12 mainly acts on T cells and natural killer (NK) cells, promotes their proliferation, induces IFN-γ production, enhances cytotoxicity, and further enhances the ability of naive T cells to Th1 cells. It induces differentiation and the like. Therefore, there is a report that increasing IL-12 production enhances cell-mediated immunity and induces apoptosis in cancer cells.
1.各種微生物によるヒダカコンブ発酵培養液の作製
ヒダカコンブを原料として、各種微生物の発酵培養液を作製した。発酵に用いた微生物には、納豆菌はバチルス・サブチリス ナットー、麹菌はアスペルギルス・オリゼを用いた。なお、これらの微生物は、いずれも市販されており、当業者が容易に入手できる微生物である。
(1)納豆菌の培養方法
-80℃中で冷凍保存していた納豆菌をSP培地を用いて30℃で1日振とう培養し、これを納豆菌前培養液とした。
1. Preparation of Hidaka Laminaria Fermentation Culture Solution Using Various Microorganisms Fermentation culture solutions of various microorganisms were prepared using Hidaka kelp as a raw material. Bacillus subtilis natto was used for the fermentation, and Aspergillus oryzae was used for the koji mold. All of these microorganisms are commercially available and readily available to those skilled in the art.
(1) Cultivation method of Bacillus natto Bacillus natto that had been frozen at −80° C. was cultured with shaking at 30° C. for one day using SP medium, and this was used as a pre-culture solution for Bacillus natto.
5%となるように蒸留水にヒダカコンブ粉末を添加し、ヒダカコンブ懸濁液を調製した。その懸濁液に121℃、15分間の加圧加熱滅菌を行い、十分に冷ましてから、ヒダカコンブ懸濁液の1%の納豆菌前培養液を添加した。その後、30℃で振とう培養を2日間行い、発酵培養液を得た。
(2)麹菌の培養方法
-80℃中で冷凍保存していた麹菌をPDB培地を用いて28℃で1日振とう培養し、これを麹菌前培養液とした。
Hidaka kelp powder was added to distilled water so as to be 5% to prepare a Hidaka kelp suspension. The suspension was subjected to autoclave sterilization at 121° C. for 15 minutes, and after sufficiently cooling, a 1% Bacillus natto preculture solution of the Hidaka kelp suspension was added. Thereafter, shaking culture was performed at 30°C for 2 days to obtain a fermentation broth.
(2) Cultivation method of Aspergillus oryzae Aspergillus oryzae that had been frozen at -80°C was cultured with shaking at 28°C for one day using PDB medium, and this was used as Aspergillus preculture solution.
1%となるように蒸留水にヒダカコンブ粉末を添加し、ヒダカコンブ懸濁液を調製した。その懸濁液に121℃、15分間の加圧加熱滅菌を行い、十分に冷ましてから、ヒダカコンブ懸濁液の1%の麹菌前培養液を添加した。その後、28℃で振とう培養を2日間行い、発酵培養液を得た。 Hidaka kelp powder was added to distilled water so as to be 1% to prepare a Hidaka kelp suspension. The suspension was subjected to autoclave heat sterilization at 121° C. for 15 minutes, and after cooling down sufficiently, a 1% Aspergillus oryzae preculture solution of Hidaka kelp suspension was added. After that, shaking culture was performed at 28° C. for 2 days to obtain a fermentation broth.
2.発酵物の作製
上記の各発酵培養液を-20℃で凍結し、次いで凍結乾燥を行った。この凍結乾燥物を10μg/mLとなるように蒸留水に懸濁し、50℃恒温槽中で1時間振とうした。これを遠心分離し、上清をヒダカコンブの発酵物とした。なお、対照には、ヒダカコンブ粉末を蒸留水と懸濁させた懸濁液を未発酵物として用いた。
2. Production of fermented product Each of the fermentation broths described above was frozen at -20°C and then freeze-dried. The freeze-dried product was suspended in distilled water to 10 μg/mL and shaken in a 50° C. constant temperature bath for 1 hour. This was centrifuged, and the supernatant was used as a fermented product of Hidaka kelp. As a control, an unfermented suspension was prepared by suspending Hidaka kelp powder with distilled water.
3.IL-12産生量の定量
上記の未発酵物又は発酵物と、RPMI-1640培地に希釈したBALB/Cマウス脾臓の細胞懸濁液(5×106cells/mL)をそれぞれ100μLずつ、96wellプレートに添加し、37℃で72時間インキュベートした。その後、培養上清を回収して遠心分離を行い、上清のみを回収し、IL-12産生量の定量に供した。
3. Quantitation of IL-12
IL-12産生量は、Quantikine(登録商標) ELISA Mouse IL-12 p70 Immunoassay を用いてキットのプロトコルに従い、定量した。 The amount of IL-12 produced was quantified using Quantikine (registered trademark) ELISA Mouse IL-12 p70 Immunoassay according to the protocol of the kit.
4.結果
図1に示すように、未発酵物のIL-12産生量は2.7pg/mL、納豆菌発酵物は検出限界以下、麹菌発酵物は58.7pg/mLであった。麹菌発酵物が顕著なIL-12産生量を示したことから、麹菌発酵ヒダカコンブを摂取することによる免疫調節作用を期待することができた。
4. Results As shown in FIG. 1, the amount of IL-12 produced in the unfermented product was 2.7 pg/mL, the natto fermented product was below the detection limit, and the koji mold fermented product was 58.7 pg/mL. Since the aspergillus fermented product showed a significant IL-12 production amount, it was expected that the aspergillus fermented hidaka kelp would have an immunoregulatory effect.
<実験例2>
麹菌を用いて発酵させたヒダカコンブ(以下、「麹菌発酵物」ともいう)のIgE産生阻害活性を測定した。
<Experimental example 2>
The IgE production inhibitory activity of Hidaka kelp fermented with Aspergillus oryzae (hereinafter also referred to as "Aspergillus fermented product") was measured.
ここで、IgEは、その抗体が産生されることによりI型アレルギーの主な原因となる。IgE抗体とはB細胞から産生される抗原特異的な抗体であり、マスト細胞表面のFcεRに結合して、体内に侵入してきた抗原と結合して、マスト細胞をさせることでマスト細胞からヒスタミン等の血管透過性の亢進や粘液分泌の促進を起こし、数分という単位で組織の発赤・膨張を誘導する。そのため、IgE抗体の産生を阻害することで、アレルギーの症状を緩和できると考えられている。 Here, IgE is the main cause of type I allergy due to the production of its antibodies. IgE antibody is an antigen-specific antibody produced from B cells, binds to FcεR on the surface of mast cells, binds to antigens that have invaded the body, and causes the mast cells to release histamine, etc. It causes increased vascular permeability and promotion of mucus secretion, and induces tissue redness and swelling in a matter of minutes. Therefore, it is believed that the symptoms of allergy can be alleviated by inhibiting the production of IgE antibodies.
1.麹菌発酵物の作製
麹菌としては A. oryzae 001(受託番号:NITE P-02748)、株式会社樋口松之助商店から購入した A. oryzae M-01、A. oryzae S-03、A. oryzae W-30、A. sojae No.9 、及び A. luchuensis SH-34を使用した。
1. Preparation of Aspergillus oryzae fermented product As aspergillus oryzae, A. oryzae 001 (accession number: NITE P-02748), A. oryzae M-01, A. oryzae S-03, and A. oryzae W-30 purchased from Higuchi Matsunosuke Shoten Co., Ltd. , A. sojae No. 9, and A. luchuensis SH-34 were used.
上記実験1と同様の方法で、各麹菌発酵物を得た。 Each Aspergillus fermented product was obtained in the same manner as in Experiment 1 above.
2.IgE産生阻害活性の測定
各麹菌発酵物をRPMI-1640培地で初濃度200μg/mL、600μg/mL、又は2000μg/mLに調整したものと、RPMI-1640培地に希釈したU266細胞懸濁液(2×105cells/mL)をそれぞれ300μLずつ、48wellプレートに添加し、37℃で24時間インキュベートした。その後、プレートシェイクを行い、次いで遠心分離を行った。上清を回収し、IgE産生阻害活性の測定に供した。
2. Measurement of IgE production inhibitory activity Each Aspergillus fermented product was adjusted to an initial concentration of 200 µg/mL, 600 µg/mL, or 2000 µg/mL in RPMI-1640 medium, and U266 cell suspension diluted in RPMI-1640 medium (2 ×10 5 cells/mL) was added to each 48-well plate at 300 μL and incubated at 37° C. for 24 hours. Thereafter, plate shaking was performed, followed by centrifugation. The supernatant was recovered and used for measurement of IgE production inhibitory activity.
IgE産生阻害活性は、Human IgE ELISA Quantitation Setを用いてキットのプロトコルに従い、定量した。IgE産生阻害率(%)は以下の式を用いて算出した。コントロールには抽出物の代わりに蒸留水を添加した。 The IgE production inhibitory activity was quantified using the Human IgE ELISA Quantitation Set according to the protocol of the kit. The IgE production inhibition rate (%) was calculated using the following formula. Distilled water was added to the control instead of the extract.
IgE産生阻害率(%)=(1-S/C)*100
S:発酵物添加区のIgE産生量、C:コントロールのIgE産生量
3.結果
図2に示すように、未発酵物のIgE産生阻害率は2.6%、A. oryzae 001発酵物(200μg/mL)は4.1%、A. oryzae M-01発酵物(200μg/mL)は6.6%、A. oryzae S-03発酵物(600μg/mL)は4.2%、A. oryzae W-30発酵物(200μg/mL)は19.4%、A. sojae No.9発酵物(2000μg/mL)は9.7%、及びA. luchuensis SH-34発酵物(600μg/mL)は4.1%であり、未発酵物と比較すると、いずれの発酵物にもIgE産生阻害活性の上昇が確認できた。
IgE production inhibition rate (%) = (1-S/C) * 100
S: IgE production amount in the fermented product addition group, C: IgE production amount in the control 3. Results As shown in FIG. 2, the IgE production inhibition rate of the unfermented product was 2.6%, the
このことから、麹菌発酵ヒダカコンブを摂取することによる免疫調節作用を期待することができた。 From this, it was possible to expect an immunoregulatory effect by ingesting Aspergillus fermented hidaka kelp.
<実験例3>
麹菌発酵ヒダカコンブのIL-12産生量を定量した。
<Experimental example 3>
The amount of IL-12 produced by Aspergillus fermented Hidaka kelp was quantified.
1.麹菌発酵ヒダカコンブの作製
上記実験1と同様の方法で、各麹菌発酵物を得た。
ただし、凍結乾燥物は10μg/mL、又は100μg/mLとなるように蒸留水に懸濁し、50℃恒温槽中で1時間振とうしたものを用いた。
1. Production of Aspergillus-Fermented Hidaka Kombu In the same manner as in Experiment 1 above, each Aspergillus-fermented product was obtained.
However, the freeze-dried product was suspended in distilled water to 10 μg/mL or 100 μg/mL, and was shaken in a 50° C. constant temperature bath for 1 hour before use.
2.IL-12産生量の定量
上記実験1と同様の方法で、各麹菌発酵物のIL-12産生量を定量した(A. sojae No.9発酵抽出物は除く)。
2. Quantification of IL-12 Production Amount IL-12 production of each Aspergillus fermented product was quantified in the same manner as in Experiment 1 above (excluding A. sojae No.9 fermented extract).
3.結果
図3に示すように、未発酵物のIL-12産生量は2.7pg/mL、A. oryzae 001発酵物(10μg/mL)は58.7pg/mL、A. oryzae M-01発酵物(100μg/mL)は8.9pg/mL、A. oryzae S-03発酵物(100μg/mL)は6.6pg/mL、A. oryzae W-30発酵物(100μg/mL)は31.4pg/mL、及びA. luchuensis SH-34発酵物(10μg/mL)は87pg/mLであり、未発出物と比較すると、いずれの発酵物にもIL-12産生量の上昇が確認できた。
3. Results As shown in FIG. 3, the amount of IL-12 produced in the unfermented product was 2.7 pg/mL, the
このことから、麹菌発酵ヒダカコンブを摂取することによる免疫調節作用を期待することができた。 From this, it was possible to expect an immunoregulatory effect by ingesting Aspergillus fermented hidaka kelp.
Claims (7)
The method for producing an immunomodulatory composition according to any one of claims 3 to 6 , wherein the fermented product of Aspergillus oryzae or its dried product is treated with warm water of 30 to 60°C to obtain an extract.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019009953A JP7304051B2 (en) | 2019-01-24 | 2019-01-24 | COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019009953A JP7304051B2 (en) | 2019-01-24 | 2019-01-24 | COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2020115785A JP2020115785A (en) | 2020-08-06 |
JP7304051B2 true JP7304051B2 (en) | 2023-07-06 |
Family
ID=71889073
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019009953A Active JP7304051B2 (en) | 2019-01-24 | 2019-01-24 | COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP7304051B2 (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006093267A1 (en) | 2005-03-04 | 2006-09-08 | Suntory Limited | Fermentation composition having immunomodulating effect |
JP2006320257A (en) | 2005-05-19 | 2006-11-30 | Grandhill Osaka:Kk | Seaweed fermented extract, method for producing the extract, and usage of the extract |
JP2018145161A (en) | 2017-03-09 | 2018-09-20 | 濱田 奈保子 | Composition for inhibiting elevation of blood pressure and method for producing the same |
JP2018186797A (en) | 2017-04-28 | 2018-11-29 | 有限会社ハマショク | Method for producing fermentation vinegar with nemacystus decipiens |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002226392A (en) * | 2001-01-31 | 2002-08-14 | Nissui Pharm Co Ltd | Immunostimulating composition |
JP5090014B2 (en) * | 2007-01-26 | 2012-12-05 | 洋佑 清水 | Fermented composition and food, beverage and pharmaceutical product using the composition |
EP2446754A4 (en) * | 2009-06-25 | 2014-01-22 | Kirin Holdings Kk | Fermentation product of a cereal-derived material and immunomodulator |
KR101948246B1 (en) * | 2012-06-07 | 2019-02-14 | 코웨이 주식회사 | Bead composition for a filter of water softener or water generator for skin, and water softener or water generator for skin comprising thereof |
CN106480147A (en) * | 2016-11-01 | 2017-03-08 | 福建农林大学 | A kind of method that red koji fermentation sea-tangle hydrolyzate prepares blood pressure lowering peptide |
-
2019
- 2019-01-24 JP JP2019009953A patent/JP7304051B2/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006093267A1 (en) | 2005-03-04 | 2006-09-08 | Suntory Limited | Fermentation composition having immunomodulating effect |
JP2006320257A (en) | 2005-05-19 | 2006-11-30 | Grandhill Osaka:Kk | Seaweed fermented extract, method for producing the extract, and usage of the extract |
JP2018145161A (en) | 2017-03-09 | 2018-09-20 | 濱田 奈保子 | Composition for inhibiting elevation of blood pressure and method for producing the same |
JP2018186797A (en) | 2017-04-28 | 2018-11-29 | 有限会社ハマショク | Method for producing fermentation vinegar with nemacystus decipiens |
Also Published As
Publication number | Publication date |
---|---|
JP2020115785A (en) | 2020-08-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2015004949A1 (en) | Novel lactobacillus and novel lactobacillus-containing medicine, food, beverage and feed | |
CN109125525B (en) | Composition for preventing and treating cardiovascular diseases and preparation method and application thereof | |
KR101690197B1 (en) | Method for producing fermented Sparassis crispa extracts with enhanced free amino acid content | |
JP2006320257A (en) | Seaweed fermented extract, method for producing the extract, and usage of the extract | |
AU2006219249A1 (en) | Fermented compositions having immunomodulatory actions | |
Zhu et al. | Anti-tumor and immunomodulatory activities of an exopolysaccharide from Rhizopus nigricans on CT26 tumor-bearing mice | |
JP2010047504A (en) | Atopic dermatitis mitigative | |
JP2018153182A (en) | Lactobacillus plantarum tci378, and use thereof in reducing fat and improving gastrointestinal function | |
JP5337535B2 (en) | NK activity enhancer | |
KR101829526B1 (en) | Composition for Immune Stimulation Comprising Fermented Cervi Parvum Cornu Extract or Fractions thereof | |
JP2015223106A (en) | Lactic acid bacterium having anti-allergic agent action, and use of the same | |
JPWO2007099997A1 (en) | Immunostimulator and method for producing the same | |
JP5206134B2 (en) | Immunomodulator and antiallergic agent containing the same | |
TWI732686B (en) | Yeast strain producing glutathione and method of producing glutathione using the same | |
KR101473475B1 (en) | Novel Lactobacillus sp. microorganism and the manufacturing method of a flavor improved jujube fermentation product using the same | |
JP6301522B1 (en) | Antihypertensive composition and method for producing the same | |
JP7304051B2 (en) | COMPOSITION FOR IMMUNOMODULATION AND METHOD FOR MANUFACTURE THEREOF | |
JP5081485B2 (en) | Anticancer agent and method for producing anticancer agent | |
KR20200103219A (en) | Composition for preventing or treating immune diseases comprising mixture of Microbiome | |
KR102244732B1 (en) | Probiotic acetic acid bacteria Acetobacter pasteurianus MGLV and its immunomodulatory effect | |
KR20120048208A (en) | A composition for skin protection including skin aging, anti-inflammatory comprising the extracts of lactarius hatsudake as an active ingredients | |
KR102098914B1 (en) | Composition of anti-inflammatory or anti-oxidation containing natto and kefir fermented component | |
CN110916188A (en) | Composition containing probiotics and konjak extract and preparation method and application thereof | |
JP2002249437A (en) | Health builder and composition thereof | |
JP6836298B1 (en) | Composition containing lactic acid bacteria and natto bacteria |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220120 |
|
RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20220120 |
|
A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20220324 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20220324 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230117 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230314 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20230314 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230613 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230619 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7304051 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |