JP2007084533A - Immune response-modulating composition and food with the same as active ingredient - Google Patents
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本発明は、免疫応答調節組成物及び該免疫応答調節組成物を有効成分として含有する食品に関し、詳しくは卵白オボムコイド等の免疫調節機能を有するタンパク質と、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導することができる免疫調節機能を有する乳酸菌との組み合わせによる非特異的な免疫応答調節機能の相互効果を利用した免疫応答調節組成物、並びに該免疫応答調節組成物を有効成分として含有する食品等に関する。 The present invention relates to an immune response-modulating composition and a food containing the immune response-modulating composition as an active ingredient, and more specifically, induces a protein having an immunomodulating function such as egg white ovomucoid and a Th1-type cytokine and / or a Th2-type cytokine. The present invention relates to an immune response-modulating composition that utilizes the mutual effect of non-specific immune response-modulating functions in combination with lactic acid bacteria having an immune-modulating function, and foods that contain the immune response-modulating composition as active ingredients .
生体の免疫反応において、種々の免疫担当細胞から産生されるサイトカインは免疫応答の方向性を制御している。この免疫応答制御において中心的な役割を担っているのが、ヘルパーＴ細胞（Ｔｈ）であり、産生するサイトカインのパターンによって、Ｔｈ１とＴｈ２のサブセットに分類されている。Ｔｈ１タイプ細胞は、主にインターロイキン２（ＩＬ−２）、インターロイキン１２（ＩＬ−１２）、インターフェロンγ（ＩＦＮ−γ）、腫瘍壊死因子（ＴＮＦ−α）等を産生し、マクロファージやナチュラルキラー細胞を活性化することで、主にウイルス、バクテリア等に対する感染防御などの細胞性免疫に関与することが知られている。他方、Ｔｈ２タイプ細胞は、主にインターロイキン４（ＩＬ−４）、インターロイキン５（ＩＬ−５）、インターロイキン６（ＩＬ−６）、インターロイキン１０（ＩＬ−１０）、インターロイキン１３（ＩＬ−１３）等を産生し、寄生虫に対する感染防御やＢ細胞からのＩｇＥ等の抗体産生などの体液性免疫に関与することが知られている。特に、Ｔｈ２細胞はアレルギー反応に関与するＩｇＥ抗体の産生を誘導するサイトカインを産生し、またアレルギーではＴｈ１とＴｈ２のバランスが崩れ、Ｔｈ２優位の状態にあるとされている。 In the immune response of a living body, cytokines produced from various immunocompetent cells control the direction of the immune response. Helper T cells (Th) play a central role in controlling the immune response, and are classified into Th1 and Th2 subsets according to the pattern of cytokines produced. Th1-type cells mainly produce interleukin 2 (IL-2), interleukin 12 (IL-12), interferon γ (IFN-γ), tumor necrosis factor (TNF-α), macrophages and natural killer It is known that by activating cells, it is mainly involved in cellular immunity such as defense against infections against viruses and bacteria. On the other hand, Th2-type cells are mainly interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 13 (IL -13) and the like, and are known to be involved in humoral immunity such as infection protection against parasites and production of antibodies such as IgE from B cells. In particular, Th2 cells produce cytokines that induce the production of IgE antibodies involved in allergic reactions, and in allergies, the balance between Th1 and Th2 is lost, and it is said that Th2 is dominant.
Ｔｈ１タイプのサイトカインであるＩＬ−１２はＴｈ２細胞の分化を抑え、またＩＦＮ−γはＴｈ２細胞の活性化を抑制することによりアレルギー反応を抑制する。Ｔｈ１タイプの免疫応答は、その異常亢進に起因して、細胞性免疫反応を誘導、活性化し、慢性関節リウマチ、Ｉ型糖尿病、橋本甲状腺炎、重症筋無力症、多発性硬化症等の臓器特異的自己免疫疾患の誘発、増悪に深く関与している。また、臓器移植に伴う拒絶反応は、やはりＴｈ１タイプの細胞性免疫反応が深く関わっている。これらの自己免疫疾患や移植後の拒絶反応を予防又は治療するためにはＴｈ１タイプの免疫応答を制御することが重要であると考えられている。他方、Ｔｈ２タイプのサイトカインであるＩＬ−４はＢ細胞に対してＩｇＥ抗体の産生を誘導するとともに、肥満細胞の活性化及び増殖を誘導する作用を有している。このように、Ｔｈ２タイプの免疫応答は、その異常亢進に起因して、即時型アレルギー反応、遅延型アレルギー反応などアレルギー性炎症反応を誘導・活性化し、蕁麻疹、食物アレルギー、アナフィラキシーショック、好酸球増加症候群、喘息、アレルギー性鼻炎、アレルギー性結膜炎、アトピー性皮膚炎等種々のアレルギー性疾患の誘発、増悪に深く関与している。また、全身性エリテマトーデス等の抗体産生或いは液性免疫が異常に亢進した病態にある全身性自己免疫疾患もやはりＴｈ２タイプの免疫応答の異常亢進が深く関わっている。これらのアレルギー性疾患を予防又は治療するためにはＴｈ２タイプの免疫応答を制御することが重要であると考えられている。 IL-12, a Th1-type cytokine, suppresses Th2 cell differentiation, and IFN-γ suppresses allergic reaction by suppressing Th2 cell activation. Th1-type immune response induces and activates cellular immune response due to its abnormal increase, organ-specific such as rheumatoid arthritis, type I diabetes, Hashimoto thyroiditis, myasthenia gravis, multiple sclerosis Is deeply involved in the induction and exacerbation of experimental autoimmune diseases. In addition, the rejection associated with organ transplantation is also closely related to the Th1-type cellular immune response. In order to prevent or treat these autoimmune diseases and rejection after transplantation, it is considered important to control the Th1-type immune response. On the other hand, IL-4, a Th2-type cytokine, induces the production of IgE antibodies against B cells and has the effect of inducing mast cell activation and proliferation. In this way, the Th2-type immune response induces and activates allergic inflammatory reactions such as immediate allergic reaction and delayed allergic reaction due to its abnormal enhancement, urticaria, food allergy, anaphylactic shock, eosinic acid He is deeply involved in the induction and exacerbation of various allergic diseases such as bulbar syndrome, asthma, allergic rhinitis, allergic conjunctivitis and atopic dermatitis. In addition, systemic autoimmune diseases such as systemic lupus erythematosus, which are in a pathological state in which humoral immunity is abnormally enhanced, are also deeply related to abnormally enhanced Th2-type immune responses. In order to prevent or treat these allergic diseases, it is considered important to control a Th2-type immune response.
ところで、乳酸菌の中には免疫系のバランスを調節し抗アレルギー作用を有するものがあることが知られている。例えば、乳酸菌の免疫調節機能に関して、乳製品製造に適した乳酸菌である、ラクトバチルス属菌中ラクトバチルス アシドフィルスＴＭＣ０３５６菌株（ＦＥＲＭ Ｐ−１９２３２）の生菌、死菌又は菌体処理物の何れかを有効成分とする、腸内生残性に優れ、腸管上皮に炎症反応を起こすことなく、マクロファージ細胞から炎症性及び抗炎症性サイトカインをバランスよく誘導し、生体でのＩｇＥ抗体産生を抑制する抗アレルギー作用を有する免疫機能調節剤が知られている（例えば、特許文献１参照）。 By the way, it is known that some lactic acid bacteria have an antiallergic action by regulating the balance of the immune system. For example, regarding the immunoregulatory function of lactic acid bacteria, any of live, killed or treated cells of Lactobacillus acidophilus TMC0356 strain (FERM P-19232) in Lactobacillus is a lactic acid bacterium suitable for dairy production. Anti-allergy that is an active ingredient, has excellent intestinal survival, induces inflammatory and anti-inflammatory cytokines from macrophage cells in a well-balanced manner without causing an inflammatory reaction in the intestinal epithelium, and suppresses IgE antibody production in vivo An immune function regulator having an action is known (for example, see Patent Document 1).
一方、卵白タンパク質には他のタンパク質に対する抗体応答抑制作用があり、中でもオボムコイドは食物アレルゲンの他にもダニ等の抗原に対する抗体応答抑制作用を有することが、本発明者らによって明らかにされている（例えば、特許文献２参照）。また、本発明者らは、乳酸菌及びオボムコイドの相互効果に関して検討し、インビボでの結果は用いた乳酸菌及びオボムコイドの相互効果が認められなかったことを報告している（例えば、非特許文献１参照）。 On the other hand, the present inventors have shown that egg white protein has an antibody response suppressing action against other proteins, and in particular, ovomucoid has an antibody response suppressing action against antigens such as mites in addition to food allergens. (For example, refer to Patent Document 2). In addition, the present inventors examined the mutual effect of lactic acid bacteria and ovomucoid, and reported that the in vivo results did not show the mutual effect of the used lactic acid bacteria and ovomucoid (see, for example, Non-Patent Document 1). ).
その他、 その表面にサイトカイン誘導物質（リステリア溶血素、サルモネラの鞭毛タンパク質フラジェリン）を融合タンパク質（シグナル配列とサイトカイン誘導物質遺伝子と膜アンカー配列とを順次結合したＤＮＡの発現産物）として発現させたラクトバチルス・カゼイ等の乳酸菌の菌体を経口用サイトカイン誘導剤として用い、生体にＩＦＮ−γ、ＩＬ−８等のサイトカインの産生を促進することができ、感染症、自己免疫疾患、炎症、腫瘍などの各種疾患の治療に有用な経口用サイトカイン誘導剤が提案されている（例えば、特許文献３参照）。 In addition, Lactobacillus expressed cytokine fusion substance (Listeria hemolysin, Salmonella flagellar protein flagellin) on its surface as a fusion protein (DNA expression product that sequentially binds signal sequence, cytokine inducer gene and membrane anchor sequence) -Using lactic acid bacteria such as casei as an oral cytokine inducer, it is possible to promote the production of cytokines such as IFN-γ and IL-8 in the body, such as infectious diseases, autoimmune diseases, inflammation, tumors, etc. An oral cytokine inducer useful for the treatment of various diseases has been proposed (see, for example, Patent Document 3).
幼小児で多く発症する食物アレルギーにおけるアレルゲン除去や対症療法は、幼小児の発育、成長に支障をきたすばかりでなく、精神的な苦痛及び経済的負担を伴う。また、幼小児の成長に伴って食物アレルギーがダニアレルギーや喘息等のアレルギー性疾患の引き金になるとも言われており、アレルギーになりにくい体質への改善が望まれている。本発明の課題は、食物アレルギー等の各種免疫疾患の治療・予防に有用な免疫応答調節組成物や、該免疫応答調節組成物を食品に添加した免疫応答調節用の食品を提供することにある。 Allergen removal and symptomatic treatment in food allergies that often occur in young children not only interfere with the growth and growth of young children, but also involve mental pain and economic burden. It is also said that food allergies trigger allergic diseases such as mite allergies and asthma with the growth of young children, and improvements to a constitution that is unlikely to cause allergies are desired. An object of the present invention is to provide an immune response modulating composition useful for the treatment and prevention of various immune diseases such as food allergies, and a food for immune response modulation in which the immune response modulating composition is added to food. .
本発明者らは、前述のように、乳酸菌及びオボムコイドの相互効果に関して検討し、インビボにおける乳酸菌及びオボムコイドの相互効果が認められなかったことを報告しているが、乳酸菌及びオボムコイドの相互効果に関して、さらに乳酸菌の種類や、オボムコイドの分解物及び修飾物などの組み合わせの検討を進めるために、先ずラクトバチルス菌、ビフィドバクテリウム菌、エンテロコッカス菌に関してサイトカイン産生また抗体産生を評価し、抗アレルギー作用を有する菌株をいくつか見い出した。これら抗アレルギー作用を有する乳酸菌と、オボムコイド等の免疫調節機能を有するタンパク質との組み合わせにより、相乗的な免疫調節機能を有する免疫応答調節組成物が得られるとの知見を得た。本発明はかかる知見に依拠するものである。 As described above, the present inventors examined the mutual effect of lactic acid bacteria and ovomucoid, and reported that the mutual effect of lactic acid bacteria and ovomucoid was not recognized in vivo, but regarding the mutual effect of lactic acid bacteria and ovomucoid, In order to further investigate the types of lactic acid bacteria and the combination of ovomucoid degradation products and modifications, we first evaluated cytokine production and antibody production for Lactobacillus, Bifidobacterium, and Enterococcus, and had antiallergic effects. Several strains were found. The inventors have found that an immune response-modulating composition having a synergistic immunomodulating function can be obtained by combining these lactic acid bacteria having an antiallergic action and a protein having an immunomodulating function such as ovomucoid. The present invention relies on such knowledge.
すなわち本発明は、（１）免疫調節機能を有する、タンパク質、その分解物及びそれらの化学修飾体からなる群から選ばれる少なくとも１種の物質と、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導し、かつ、前記化学物質と協働して免疫調節機能を向上させることができる乳酸菌とを含有する免疫応答調節組成物や、（２）免疫調節機能を有するタンパク質が、卵白オボムコイドであることを特徴とする上記（１）記載の免疫応答調節組成物や、（３）乳酸菌が、Ｔｈ１タイプサイトカインであるＩＬ−１２及びＩＦＮ−γ、並びにＴｈ２タイプサイトカインであるＩＬ−１０を誘導する乳酸菌であることを特徴とする免疫応答調節組成物に関する。 That is, the present invention induces (1) at least one substance selected from the group consisting of a protein, a degradation product thereof, and a chemical modification thereof having an immunoregulatory function, and a Th1-type cytokine and / or a Th2-type cytokine. And the immune response regulating composition containing lactic acid bacteria capable of improving the immunoregulatory function in cooperation with the chemical substance, and (2) the protein having the immunoregulatory function is egg white ovomucoid. And (3) the lactic acid bacterium is a lactic acid bacterium that induces IL-12 and IFN-γ that are Th1-type cytokines, and IL-10 that is a Th2-type cytokine. The present invention relates to a composition for regulating immune response.
また本発明は、（４）Ｔｈ１タイプサイトカインであるＩＬ−１２及びＩＦＮ−γ、並びにＴｈ２タイプサイトカインであるＩＬ−１０を誘導する乳酸菌が、Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ)であることを特徴とする上記（３）記載の免疫応答調節組成物や、（５）上記（１）〜（４）のいずれか記載の免疫応答調節組成物を有効成分として含有する食品や、（６）Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ) を含有する免疫応答調節剤や、（７）Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ) を添加した食品に関する。 The present invention also relates to (4) Lactobacillus salivarius ssp. Salicinius (JCM1042), Enterococcus faecium (FERM P), which induces IL-12 and IFN-γ, which are Th1 type cytokines, and IL-10, a Th2 type cytokine. -20637) or Enterococcus faecium (JCM5804T), or the immune response modulating composition according to (3) above, or (5) the immune response modulating composition according to any one of (1) to (4) above Or (6) Lactobacillus salivarius ssp. Salicinius (JCM1042), Enterococcus faecium (FERM P-20737) or Enterococcus faecium (JCM5804T), or (7) Lactobacillus salivarius ssp Salicinius (JCM1042), Enterococcus faecium (FERM P-20737) or Enterococcu It relates to foods with added s faecium (JCM5804T).
本発明によると、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導することができる免疫調節機能を有する乳酸菌を含有する免疫応答調節剤や、該免疫応答調節剤を有効成分とする非特異的な免疫応答調節機能を有する食品を得ることができる。また、卵白オボムコイド等の免疫調節機能を有するタンパク質と、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導することができる免疫調節機能を有する乳酸菌との組み合わせにより、非特異的な免疫応答調節機能を有する免疫応答調節組成物や、該免疫応答調節組成物を有効成分として含有する非特異的な免疫応答調節機能を有する食品を得ることができる。 According to the present invention, an immune response regulator containing a lactic acid bacterium having an immunoregulatory function capable of inducing a Th1-type cytokine and / or a Th2-type cytokine, and nonspecific immunity comprising the immune response regulator as an active ingredient A food product having a response regulating function can be obtained. In addition, it has a non-specific immune response regulation function by combining a protein having an immune regulation function such as egg white ovomucoid and a lactic acid bacterium having an immune regulation function capable of inducing a Th1-type cytokine and / or a Th2-type cytokine. It is possible to obtain an immune response modulating composition or a food having a non-specific immune response modulating function containing the immune response modulating composition as an active ingredient.
本発明の免疫応答調節組成物としては、免疫調節機能を有するタンパク質、免疫調節機能を有するその分解物、及び免疫調節機能を有するそれらの化学修飾体からなる群から選ばれる少なくとも１種の物質（以下、これらを総称して「免疫調節機能を有する本件タンパク質等」ということがある）と、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導し、かつ、前記化学物質と協働して免疫調節機能を向上させることができる乳酸菌とを含有するものであれば特に制限されず、上記「免疫応答調節組成物」には、免疫応答抑制組成物、免疫応答賦活組成物等が含まれ、また「免疫調節機能」には、免疫応答抑制機能、免疫応答賦活機能等が含まれる。 The immune response modulating composition of the present invention includes at least one substance selected from the group consisting of a protein having an immunomodulating function, a degradation product thereof having an immunomodulating function, and a chemical modification thereof having an immunomodulating function ( Hereinafter, these may be collectively referred to as “the present protein having an immunoregulatory function”), and a Th1-type cytokine and / or a Th2-type cytokine are induced, and the immunoregulatory function in cooperation with the chemical substance. There is no particular limitation as long as it contains lactic acid bacteria capable of improving immunity, and the above-mentioned “immune response modulating composition” includes an immune response suppressing composition, an immune response stimulating composition, and the like. The “regulatory function” includes an immune response suppression function, an immune response activation function, and the like.
上記免疫調節機能を有するタンパク質としては、免疫応答抑制機能、免疫応答賦活機能等を有するタンパク質であれば特に制限されないが、免疫応答抑制機能を有するタンパク質としては、卵類、牛乳類、肉類、魚類、甲殻類及び軟体動物類、穀類、豆類及びナッツ類、果実類、野菜類、ビール酵母若しくはゼラチンなどアレルギーを引き起こす食品に含まれるアレルゲン、特に乳アレルゲンの主要成分としてのαｓ１カゼインや、β−ラクトグロブリンや、卵白アレルゲン成分としてはオボアルブミンとオボムコイドや、小麦アレルゲンの主要成分としてグリアジンや、そばの主要タンパク質である分子量２４ｋＤａと７６ｋＤａのタンパク質や、落花生の主要タンパク質であるＡｒａ ｈ１、中でも卵白オボムコイドを好適に例示することができる。また、免疫応答賦活機能等を有するタンパク質としては、ラクトフェリン、インターフェロン誘導シグナル伝達活性を有するタンパク質（特開２００４−１７３６７９）、膜局在型プロテインチロシンキナーゼ（特開平１０−３１３８６８）、ホエーのプロテアーゼ処理物等を挙げることができる。 The protein having the above-mentioned immunoregulatory function is not particularly limited as long as it has an immune response suppressing function, an immune response activation function, and the like, but as a protein having an immune response suppressing function, eggs, milk, meat, fish Allergens in foods that cause allergies such as crustaceans and mollusks, cereals, beans and nuts, fruits, vegetables, brewer's yeast or gelatin, especially αs1 casein as a major component of milk allergens, and β-lacto Globulin and egg white allergen components are ovalbumin and ovomucoid, wheat allergens are mainly gliadin, buckwheat major proteins with molecular weights of 24 kDa and 76 kDa, peanut major protein Ara h1, especially egg white ovomucoid. Suitable examples It can be. Examples of the protein having an immune response activation function include lactoferrin, a protein having interferon-induced signaling activity (JP 2004-173679), a membrane-localized protein tyrosine kinase (JP 10-313868), and protease treatment of whey. And the like.
上記免疫調節機能を有するその分解物としては、トリプシン、Ｖ８プロテアーゼ等プロテアーゼによる分解物を例示することができ、また免疫調節機能を有するそれらの化学修飾体としては、グルコシル化及びリン酸化などの酵素による修飾物、アセチル化、エステル化、アミド化などの化学修飾物、加熱、酸化、酸加水分解、アルカリ処理、脱糖などの処理物を挙げることができる。 Examples of the degradation product having an immunomodulating function include degradation products by proteases such as trypsin and V8 protease, and those chemically modified products having an immunomodulating function include enzymes such as glucosylation and phosphorylation. And modified products by chemical modification, chemical modification products such as acetylation, esterification, and amidation, and processed products such as heating, oxidation, acid hydrolysis, alkali treatment, and desugaring.
上記Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導しうる乳酸菌としては、生菌や死菌（断片も含む）の状態で、Ｔｈ１タイプサイトカイン及びＴｈ２タイプサイトカインを誘導しうる乳酸菌や、Ｔｈ１タイプサイトカインを誘導しうる乳酸菌が好ましく、Ｔｈ１タイプサイトカイン及びＴｈ２タイプサイトカインを誘導しうる乳酸菌がより好ましい。ここで、Ｔｈ１タイプサイトカインとしては、ＩＬ−２、ＩＬ−１２、ＩＦＮ−γ、ＴＮＦ−α等を挙げることができるが、中でも、ＩＬ−１２やＩＦＮ−γを好適に例示することができ、またＴｈ２タイプサイトカインとしては、ＩＬ−４、ＩＬ−５、ＩＬ−６、ＩＬ−１０、ＩＬ−１３等を挙げることができるが、中でも、ＩＬ−６やＩＬ−１０を好適に例示することができる。例えば、Ｔｈ１タイプサイトカイン及び／又はＴｈ２タイプサイトカインを誘導しうる乳酸菌の選択基準としては、マクロファージ系細胞株Ｊ７７４．１細胞を用いた評価において、ＬＰＳ １μｇ／ｍｌでの刺激による場合と同等のＩＬ−１２ｐ４０あるいはＩＬ−６産生量を示す乳酸菌を選択するのが好ましい。また、マウス脾臓細胞を用いた評価において、５００ｐｇ／ｍｌ以上好ましくは２０００ｐｇ／ｍｌ以上のＩＬ−１２ｐ７０産生量、あるいは１０ｎｇ／ｍｌ以上好ましくは３０ｎｇ／ｍｌ以上のＩＦＮ−γ産生量を示す乳酸菌を選択するのが好ましい。このような免疫調節機能を有する乳酸菌は、ラクトバチラス属、ビフィドバクテリウム属、エンテロコッカス属、ラクトコッカス属、ストレプトコッカス属、などの乳酸菌群からも選ぶことができ、特に好適に、Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ)を具体的に例示することができる。 As the lactic acid bacterium capable of inducing the Th1-type cytokine and / or Th2-type cytokine, the lactic acid bacterium capable of inducing the Th1-type cytokine and Th2-type cytokine in the state of live bacteria or dead bacteria (including fragments), and the Th1-type cytokine Inducible lactic acid bacteria are preferred, and lactic acid bacteria capable of inducing Th1-type cytokines and Th2-type cytokines are more preferred. Here, examples of the Th1-type cytokine include IL-2, IL-12, IFN-γ, TNF-α, etc. Among them, IL-12 and IFN-γ can be preferably exemplified. Examples of the Th2-type cytokine include IL-4, IL-5, IL-6, IL-10, and IL-13. Among them, IL-6 and IL-10 are preferably exemplified. it can. For example, as a selection criterion for lactic acid bacteria capable of inducing a Th1-type cytokine and / or a Th2-type cytokine, in the evaluation using the macrophage cell line J774.1 cell, the same IL− as in the case of stimulation with LPS 1 μg / ml It is preferable to select lactic acid bacteria that show 12p40 or IL-6 production. In addition, in the evaluation using mouse spleen cells, lactic acid bacteria having an IL-12p70 production amount of 500 pg / ml or more, preferably 2000 pg / ml or more, or an IFN-γ production amount of 10 ng / ml or more, preferably 30 ng / ml or more are selected. It is preferable to do this. Lactic acid bacteria having such an immunoregulatory function can be selected from a group of lactic acid bacteria such as Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Streptococcus, and the like, and particularly preferably, Lactobacillus salivarius ssp. Salicinius. (JCM1042), Enterococcus faecium (FERM P-20737) or Enterococcus faecium (JCM5804T) can be specifically exemplified.
また、前記免疫調節機能を有する本件タンパク質等と協働して免疫調節機能を向上させることができる乳酸菌は、例えば、供試乳酸菌と、オボムコイド等の免疫調節機能を有する本件タンパク質等とをマウスに連続的に経口投与し、アレルゲン等の抗原で免疫した後、血清中の抗原特異的ＩｇＥ抗体、ＩｇＧ抗体、ＩｇＧ１抗体及びＩｇＧ２ａ抗体をＥＬＩＳＡにより測定し、これら抗体の内１以上の抗体産生を、供試乳酸菌単独及び本件タンパク質等単独の場合よりも有意に抑制する乳酸菌を選択することにより得ることができる。 In addition, lactic acid bacteria capable of improving the immunoregulatory function in cooperation with the present protein having the immunomodulating function include, for example, a test lactic acid bacterium and the present protein having an immunomodulating function such as ovomucoid in a mouse. Continuously orally administered and immunized with an antigen such as allergen, antigen-specific IgE antibody, IgG antibody, IgG1 antibody and IgG2a antibody in the serum were measured by ELISA, and production of one or more of these antibodies was It can be obtained by selecting a lactic acid bacterium that significantly suppresses the test lactic acid bacterium alone and the present protein alone.
本発明の免疫応答調節組成物は、そのまま用いて免疫応答抑制剤や免疫応答賦活剤等の免疫応答調節剤として、あるいは食品に添加して免疫応答抑制用や免疫応答賦活剤用の免疫応答調節用食品として、主に経口摂取される。免疫応答調節剤としての摂取形態、例えば粉末、顆粒、錠剤、カプセル剤、シロップ剤、懸濁液等の剤型は特に制限されず、また、摂取量等も年齢や症状、疾患の程度により適宜決定することができる。さらに、免疫応答抑制組成物は食経験がある食品素材を利用することから、免疫応答抑制組成物中に含まれる食物アレルゲンに対するアレルギーを有する人を除いて、生体に悪影響を与えないという利点を有する。この場合の投与量については、基本的にはアレルゲンタンパク質に対する免疫応答を抑制することができる量であればよく、例えば、１日あたり、５〜５００ｍｇ／ｋｇ体重の投与が適当である。また、経口投与する場合、薬学的に許容される通常の担体、結合剤、安定化剤、賦形剤、希釈剤、ｐＨ緩衝剤、崩壊剤、可溶化剤、溶解補助剤、等張剤などの各種調剤用配合成分を添加することができる。 The immune response modulating composition of the present invention can be used as it is as an immune response modifier such as an immune response suppressor or immune response enhancer, or added to food to control immune responses for suppressing immune responses or immune response enhancers. As a food, it is mainly taken orally. The intake form as an immune response modifier, such as powder, granules, tablets, capsules, syrups, suspensions, etc., is not particularly limited, and the intake amount is appropriately determined depending on age, symptoms, and degree of disease. Can be determined. Furthermore, since the immune response suppressing composition uses food materials that have dietary experience, it has the advantage that it does not adversely affect the living body, except for those who are allergic to food allergens contained in the immune response suppressing composition. . The dose in this case may be basically an amount that can suppress the immune response to the allergen protein. For example, administration of 5 to 500 mg / kg body weight per day is appropriate. When administered orally, conventional pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegrating agents, solubilizers, solubilizers, isotonic agents, etc. The various compounding ingredients for preparation can be added.
上記のように、本発明の免疫応答調節組成物、好ましくは本発明の免疫応答抑制組成物は、食品に配合しても使用されるが、かかる本発明の免疫応答調節組成物を有効成分として含有する食品としては、例えば、上記免疫応答抑制剤を添加した食品を例示することができ、ここで、食品としてはヨーグルト、ドリンクヨーグルト、ジュース、牛乳、豆乳、酒類、コーヒー、紅茶、煎茶、ウーロン茶、スポーツ飲料等の各種飲料や、プリン、クッキー、パン、ケーキ、ゼリー、煎餅などの焼き菓子、羊羹などの和菓子、冷菓、チューインガム等のパン・菓子類や、うどん、そば等の麺類や、かまぼこ、ハム、魚肉ソーセージ等の魚肉練り製品や、みそ、しょう油、ドレッシング、マヨネーズ、甘味料等の調味類や、チーズ、バター等の乳製品や、豆腐、こんにゃく、その他佃煮、餃子、コロッケ、サラダ等の各種総菜などを挙げることができる。食品中の免疫応答抑制組成物の含量については、上記と同様に、例えばアレルゲンタンパク質に対する免疫応答を抑制することができる量であればよい。 As described above, the immune response-modulating composition of the present invention, preferably the immune response-suppressing composition of the present invention, can be used even when blended with foods. Examples of the food to be contained include foods to which the above immune response inhibitor is added, and examples of the foods include yogurt, drink yogurt, juice, milk, soy milk, alcoholic beverages, coffee, tea, sencha, oolong tea. , Various drinks such as sports drinks, baked confectionery such as pudding, cookies, bread, cakes, jelly and rice crackers, Japanese confectionery such as sheep crab, confectionery such as frozen confectionery, chewing gum, noodles such as udon and soba , Fish paste products such as ham and fish sausage, seasonings such as miso, soy sauce, dressing, mayonnaise, sweeteners, and dairy products such as cheese and butter Tofu, mention may be made of konjac, and other boiled, dumplings, croquettes, and various side dishes of salad. About the content of the immune response suppression composition in foodstuffs, it is sufficient if it is the quantity which can suppress the immune response with respect to allergen protein like the above, for example.
次に本発明は、Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ) を含有する免疫応答調節剤や、Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）又はEnterococcus faecium(ＪＣＭ５８０４Ｔ) を添加した食品にも関し、これらの内、Lactobacillus salivarius ssp. Salicinius(ＪＣＭ１０４２)及びEnterococcus faecium(ＪＣＭ５８０４Ｔ)は独立行政法人理化学研究所バイオリソースセンター（ＪＣＭ）から入手することができる。また、Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７）は、平成１７年（西暦２００５年）８月２４日（受領日）付で独立行政法人産業技術総合研究所 特許生物寄託センター（〒３０５−５４６６ 日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託されている。 Next, the present invention relates to an immune response modulator containing Lactobacillus salivarius ssp. Salicinius (JCM1042), Enterococcus faecium (FERM P-20737) or Enterococcus faecium (JCM5804T), Lactobacillus salivarius ssp. FERM P-20637) or Enterococcus faecium (JCM5804T), and among these, Lactobacillus salivarius ssp. Salicinius (JCM1042) and Enterococcus faecium (JCM5804T) are from the RIKEN BioResource Center (JCM). It can be obtained. In addition, Enterococcus faecium (FERM P-20737) is a patent biological deposit center (National Institute of Advanced Industrial Science and Technology) on August 24, 2005 (date of receipt) (Ibaraki, Japan 305-5466, Japan). It is entrusted to Tsukuba City, East 1-chome, 1-chome, 1-Chuo 6).
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
［Ｊ７７４．１細胞を用いた評価］
（１）乳酸菌の培養
ラクトバチルス属（Lactobacillus）乳酸菌であるL.gasseri１９菌株（Ｌ１〜Ｌ１９）、L.acidophilus１５株（Ｌ２０〜Ｌ３４）、L.johnsonii５菌株（Ｌ３５〜Ｌ３９）、L.salivarius ssp. Salicinius１０菌株（Ｌ４０〜Ｌ４９）、L.reuteri１菌株（Ｌ５０）、L.casei ssp. alactosus１菌株（Ｌ５１）、L. rhamnosus１菌株（Ｌ５２）、L.amylovorus４菌株（Ｌ５３〜Ｌ５６）、L. crispatus２菌株（Ｌ５７〜Ｌ５８）の計５８菌株（Ｌ１〜Ｌ５８）を供試した。これらの乳酸菌は、ＡＰＴ−ｂｒｏｔｈ培地（Difco Laboratories）で３７℃・４０時間培養を行った。
[Evaluation using J774.1 cells]
(1) Culture of lactic acid bacteria L. gasseri 19 strains (L1-L19), L. acidophilus 15 strains (L20-L34), L. johnsonii 5 strains (L35-L39), L. salivarius ssp. Salicinius 10 strain (L40-L49), L. reuteri 1 strain (L50), L. casei ssp. Alactosus 1 strain (L51), L. rhamnosus 1 strain (L52), L. amylovorus 4 strain (L53-L56), L. crispatus 2 strain ( A total of 58 strains (L1 to L58) of L57 to L58) were used. These lactic acid bacteria were cultured in APT-broth medium (Difco Laboratories) at 37 ° C. for 40 hours.
（２）Ｊ７７４．１細胞の培養
マウスのmonocyte-macrophage cell line Ｊ７７４．１はＡＴＣＣより購入した。ＦＣＳを１０％加えたＲＰＭＩ１６４０培地（Sigma）を用い、３７℃・５％ＣＯ２条件下でＪ７７４．１細胞の培養を行った。細胞の継代は３日おきに、また、培地交換は継代して４８時間後に行った。
(2) Culture of J774.1 cells Mouse monocyte-macrophage cell line J774.1 was purchased from ATCC. J774.1 cells were cultured using RPMI 1640 medium (Sigma) supplemented with 10% FCS under conditions of 37 ° C. and 5% CO 2 . Cells were passaged every 3 days, and medium was changed 48 hours after passage.
（３）サイトカインの測定
Ｊ７７４．１細胞を５×１０5個／ｍｌに調製し、４８ウェル培養プレートに０．５ｍｌ／ウェルずつ播種した。１時間プレインキュベートした後、乳酸菌（湿重量１、１０ｍｇ／ｍｌ）を５０μｌ／ウェル添加した。乳酸菌は生理食塩水で調製し、死菌は１００℃・５０分加熱したものを用いた。２４時間培養後、上清を回収した。そして、サイトカインＩＬ−１２ｐ４０及びＩＬ−６の測定は、Ｍｏｕｓｅ ＢＤ Ｏｐｔ ＥＩＡ ＥＬＩＳＡ ｓｅｔ（BD Pharmingen）を用いて行った。
(3) Measurement of cytokines J774.1 cells were prepared at 5 × 10 5 cells / ml and seeded at 0.5 ml / well in a 48-well culture plate. After preincubation for 1 hour, lactic acid bacteria (wet weight 1, 10 mg / ml) were added at 50 μl / well. Lactic acid bacteria were prepared in physiological saline, and dead bacteria were heated at 100 ° C. for 50 minutes. After culturing for 24 hours, the supernatant was collected. And measurement of cytokine IL-12p40 and IL-6 was performed using Mouse BD Opt EIA ELISA set (BD Pharmingen).
（４）結果
各乳酸菌によるＩＬ−１２ｐ４０の産生量を図１（生菌）及び図３（死菌）に、ＩＬ−６の産生量を図２（生菌）及び図４（死菌）に示す。生菌では、ＩＬ−１２ｐ４０誘導能の高い菌が６菌株、ＩＬ−６誘導能の高い菌が１３菌株認められた。一方、死菌では、ＩＬ−１２ｐ４０誘導能の高い菌が３菌株、ＩＬ−６誘導能の高い菌が２１菌株認められた。
(4) Results The production amount of IL-12p40 by each lactic acid bacterium is shown in FIG. 1 (live bacteria) and FIG. 3 (dead bacteria), and the production amount of IL-6 is shown in FIG. 2 (live bacteria) and FIG. 4 (dead bacteria). Show. Among viable bacteria, 6 strains having high IL-12p40-inducing ability and 13 strains having high IL-6-inducing ability were observed. On the other hand, among the killed bacteria, 3 strains having high IL-12p40 inducing ability and 21 strains having high IL-6 inducing ability were observed.
（５）考察
Ｊ７７４．１細胞においてサイトカイン産生に影響を与え、ＩＬ−１２ｐ４０又はＩＬ−６を誘導している菌株は、生体におけるサイトカイン産生に対しても影響を与える可能性が高いと考えられる。そこで、ＩＬ−１２ｐ４０及びＩＬ−６誘導能の高い菌、ＩＬ−１２ｐ４０又はＩＬ−６誘導能のどちらかが高い菌など特徴的なサイトカイン誘導能を示した乳酸菌、また菌種を考慮して選択した乳酸菌の８菌株（Ｌ２、Ｌ１６、Ｌ３２、Ｌ３４、Ｌ３９、Ｌ４１、Ｌ５２、Ｌ５６）に関して、マウス脾臓細胞を用いた評価を行うこととした。なお、Ｌ２はL. gasseri（ＪＣＭ１０１７）、Ｌ１６はL. gasseriの一菌株、Ｌ３２はL. acidophilusの一菌株、Ｌ３４はL. acidophilusの一菌株、Ｌ３９はL. johnsoniiの一菌株、Ｌ４１はL. salivarius ssp. salicinius（ＪＣＭ１０４２）、Ｌ５２はL. rhamnosus（ＪＣＭ１１３６Ｔ）、Ｌ５６はL. amylovorusの一菌株を示す。
(5) Discussion It is considered that a strain that affects cytokine production in J774.1 cells and induces IL-12p40 or IL-6 is also highly likely to affect cytokine production in vivo. Therefore, a lactic acid bacterium having a characteristic cytokine-inducing ability such as a bacterium having a high ability to induce IL-12p40 and IL-6, a bacterium having a high ability to induce IL-12p40 or IL-6, and a bacterium species are selected. The eight lactic acid bacteria strains (L2, L16, L32, L34, L39, L41, L52, and L56) were evaluated using mouse spleen cells. L2 is L. gasseri (JCM1017), L16 is a strain of L. gasseri, L32 is a strain of L. acidophilus, L34 is a strain of L. acidophilus, L39 is a strain of L. johnsonii, and L41 is L. salivarius ssp. salicinius (JCM1042), L52 represents L. rhamnosus (JCM1136T), and L56 represents a strain of L. amylovorus.
［マウス脾臓細胞を用いた評価］
（１）乳酸菌の培養
Ｊ７７４．１細胞を用いた評価で選択した乳酸菌８菌株（Ｌ２、Ｌ１６、Ｌ３２、Ｌ３４、Ｌ３９、Ｌ４１、Ｌ５２、Ｌ５６）について、評価を行った。これらの乳酸菌は、ＡＰＴ−ｂｒｏｔｈ培地（Difco Laboratories）で３７℃・１８時間培養を行った。
[Evaluation using mouse spleen cells]
(1) Culture of Lactic Acid Bacteria Eight lactic acid bacteria strains (L2, L16, L32, L34, L39, L41, L52, and L56) selected by evaluation using J774.1 cells were evaluated. These lactic acid bacteria were cultured in APT-broth medium (Difco Laboratories) at 37 ° C. for 18 hours.
（２）脾臓細胞の調製
マウスから採取した脾臓をセルストレイナー（Falcon2350）で濾過した後、ＲＰＭＩ１６４０培地（Sigma）１０ｍｌで懸濁し、遠心した（１９０×ｇ・１０分）。沈殿した細胞に溶血バッファー（０．１５Ｍ ＮＨ４Ｃｌ、１０ｍＭ ＫＨＣＯ３、０．１ｍＭ Ｎａ２ＥＤＴＡ：ｐＨ７．２）を２ｍｌ添加し、５分静置した。ＲＰＭＩ培地を１０ｍｌ添加し、２回洗浄を行った後、ＦＣＳを１０％加えたＲＰＭＩ培地を用いて６×１０6個／ｍｌに調製した。
(2) Preparation of spleen cells The spleen collected from the mice was filtered with a cell strainer (Falcon 2350), suspended in 10 ml of RPMI 1640 medium (Sigma), and centrifuged (190 × g · 10 minutes). 2 ml of hemolysis buffer (0.15M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA: pH 7.2) was added to the precipitated cells, and the mixture was allowed to stand for 5 minutes. After adding 10 ml of RPMI medium and washing twice, it was adjusted to 6 × 10 6 cells / ml using RPMI medium supplemented with 10% FCS.
（３）サイトカインの測定
脾臓細胞を４８ウェル培養プレートに０．５ｍｌ／ウェルずつ播種した。１時間プレインキュベートした後、乳酸菌（湿重量５、５０、５００μｇ／ｍｌ）を５０μｌ／ウェル添加した。乳酸菌は生理食塩水で調製し、死菌は１００℃・５０分加熱したものを用いた。２４時間及び７２時間培養後、上清を回収した。そして、サイトカインＩＬ−１２ｐ７０、ＩＦＮ−γ及びＩＬ−１０の測定は、Ｍｏｕｓｅ ＢＤ Ｏｐｔ ＥＩＡ ＥＬＩＳＡ ｓｅｔ（BD Pharmingen）を用いて行った。
(3) Measurement of cytokines Spleen cells were seeded in a 48-well culture plate at 0.5 ml / well. After preincubation for 1 hour, lactic acid bacteria (wet weight 5, 50, 500 μg / ml) were added at 50 μl / well. Lactic acid bacteria were prepared in physiological saline, and dead bacteria were heated at 100 ° C. for 50 minutes. After culturing for 24 hours and 72 hours, the supernatant was collected. The cytokines IL-12p70, IFN-γ and IL-10 were measured using a Mouse BD Opt EIA ELISA set (BD Pharmingen).
（４）結果
各乳酸菌によるＩＬ−１２ｐ７０の産生量を図５、ＩＦＮ−γの産生量を図６、ＩＬ−１０の産生量を図７に示す。Ｌ１６及びＬ４１は、生菌、死菌ともに、ＩＬ−１２ｐ７０、ＩＦＮ−γ及びＩＬ−１０を強く誘導し、Ｔｈ１タイプサイトカイン誘導能が高いことが示唆された。また、他の菌株では、Ｌ３４は生菌でＩＦＮ−γを、Ｌ５２は生菌及び死菌でＩＬ−１２ｐ７０を強く誘導していた。
(4) Results FIG. 5 shows the amount of IL-12p70 produced by each lactic acid bacterium, FIG. 6 shows the amount of IFN-γ produced, and FIG. 7 shows the amount of IL-10 produced. L16 and L41 strongly induced IL-12p70, IFN-γ, and IL-10 in both live and dead bacteria, suggesting that Th1 type cytokine induction ability is high. In other strains, L34 was viable and IFN-γ, and L52 was viable and dead, and IL-12p70 was strongly induced.
（５）考察
マウス脾臓細胞においてＴｈ１タイプサイトカイン産生に影響を与え、ＩＬ−１２ｐ７０及びＩＦＮ−γを誘導している菌株は、生体においてもＴｈ１タイプサイトカインを誘導する可能性が高いと考えられる。そこで、生菌、死菌ともにＴｈ１タイプサイトカイン誘導能が高かったＬ４１に関して、マウスへの投与試験を行うこととした。
(5) Discussion It is considered that a strain that affects Th1-type cytokine production in mouse spleen cells and induces IL-12p70 and IFN-γ is highly likely to induce Th1-type cytokine even in the living body. Therefore, it was decided to conduct a test for administration of L41, which had a high Th1-type cytokine-inducing ability for both live and dead bacteria.
［マウスへの投与試験による評価］
（１）乳酸菌の培養
脾臓細胞を用いた評価でＴｈ１タイプサイトカイン誘導能の高かった乳酸菌Ｌ４１について、評価を行った。Ｌ４１は、ＡＰＴ−ｂｒｏｔｈ培地（Difco Laboratories）で３７℃・１８時間培養を行った。
[Evaluation by administration test to mice]
(1) Culture of lactic acid bacteria Lactic acid bacteria L41, which was highly evaluated for Th1-type cytokines by evaluation using spleen cells, was evaluated. L41 was cultured in an APT-broth medium (Difco Laboratories) at 37 ° C. for 18 hours.
（２）マウスへの投与及び免疫
投与試験は、１試験区あたりマウス１０匹を用いた。経口投与は生理食塩水又は乳酸菌（湿重量１０ｍｇ／ｍｌ）を１匹あたり２００μｌずつ７日連続で行った。乳酸菌は生理食塩水で調製し、死菌は１００℃・５０分加熱したものを用いた。また、免疫は１週目及び３週目に、生理食塩水で１ｍｇ／ｍｌに調製したオボアルブミン（ＯＶＡ）とＡｌｕｍ（４０〜５０ｍｇ／ｍｌ 水酸化アルミニウムゲル）を等量混合した溶液を１匹あたり１００μｌ腹腔内に注射した。
(2) Administration to mice and immunization In the administration test, 10 mice were used per test group. Oral administration was performed for 7 consecutive days with 200 μl of physiological saline or lactic acid bacteria (wet weight 10 mg / ml) per animal. Lactic acid bacteria were prepared in physiological saline, and dead bacteria were heated at 100 ° C. for 50 minutes. In addition, immunization was carried out on the 1st and 3rd weeks by using one solution in which equal amounts of ovalbumin (OVA) and Alum (40-50 mg / ml aluminum hydroxide gel) prepared to 1 mg / ml with physiological saline were mixed. Per 100 μl intraperitoneally.
（３）抗体の測定
４週目に採血を行い、血清中のＯＶＡ特異的ＩｇＥ抗体、ＩｇＧ抗体、ＩｇＧ１抗体及びＩｇＧ２ａ抗体をＥＬＩＳＡにより測定した。
(3) Measurement of antibody Blood was collected at 4 weeks, and OVA-specific IgE antibody, IgG antibody, IgG1 antibody, and IgG2a antibody in the serum were measured by ELISA.
（４）結果
ＯＶＡ特異的ＩｇＥ抗体量を図８、ＩｇＧ抗体量、ＩｇＧ１抗体量及びＩｇＧ２ａ抗体量を図９に示す。Ｌ４１の生菌及び死菌は、ＯＶＡ特異的ＩｇＥ抗体量を有意に抑制していた（＜０．０５）。一方、ＯＶＡ特異的ＩｇＧ抗体の産生に対する影響はなかったが、ＩｇＧ１抗体の産生を抑制、ＩｇＧ２ａ抗体の産生を誘導する傾向が認められた。
(4) Results FIG. 8 shows the amount of OVA-specific IgE antibody, and FIG. 9 shows the amounts of IgG antibody, IgG1 and IgG2a. L41 live and dead bacteria significantly suppressed the amount of OVA-specific IgE antibody (<0.05). On the other hand, although there was no influence on the production of OVA-specific IgG antibody, there was a tendency to suppress the production of IgG1 antibody and induce the production of IgG2a antibody.
（５）考察
マウスへの投与試験では、Ｌ４１は、ＩｇＥ抗体の産生を抑制したが、ＩｇＧ抗体など他の抗体の産生は抑制しなかった。また、Ｌ４１は、ＩｇＧ１抗体の産生を抑制し、ＩｇＧ２ａ抗体の産生を誘導する傾向が認められたことから、ＩｇＧ１抗体及びＩｇＧ２ａ抗体産生に関与するＴｈ２タイプサイトカイン（ＩＬ−４）が抑制され、Ｔｈ１タイプサイトカイン（ＩＦＮ-γ）が誘導されていると考えられた。したがって、Ｌ４１は、他の抗体反応に大きな影響を与えることなくＩｇＥ抗体が関与するアレルギー作用を抑制し、またＴｈ１細胞の分化を誘導して免疫バランスを調節する可能性があると考えられた。
(5) Discussion In the administration test to mice, L41 suppressed the production of IgE antibody, but did not suppress the production of other antibodies such as IgG antibody. In addition, since L41 suppressed the production of IgG1 antibody and induced the production of IgG2a antibody, Th2 type cytokine (IL-4) involved in the production of IgG1 antibody and IgG2a antibody was suppressed, and Th1 It was thought that type cytokine (IFN-γ) was induced. Therefore, it was considered that L41 may suppress allergic effects involving IgE antibodies without significantly affecting other antibody responses, and may induce differentiation of Th1 cells to regulate immune balance.
［マウス脾臓細胞を用いた評価］
（１）乳酸菌の培養
ビフィドバクテリウム属（Bifidobacterium）乳酸菌であるB.bifidum３菌株（Ｂ１〜Ｂ３）、B.breve３菌株（Ｂ４〜Ｂ６）、B. adolescentis４菌株（Ｂ７〜Ｂ１０）、B.thermophilum２菌株（Ｂ１１〜Ｂ１２）、B.infantis２菌株（Ｂ１３〜Ｂ１４）、B.longum４菌株（Ｂ１５〜Ｂ１８）、B.pseudocatenulatum１菌株（Ｂ１９）、B.pseudolongum２菌株（Ｂ２０〜Ｂ２１）、B.suis１菌株（Ｂ２２）、B.catenulatum１菌株（Ｂ２３）、B.angulatum１菌株（Ｂ２４）、B.gallicum１菌株（Ｂ２５）の計２５菌株（Ｂ１〜Ｂ２５）を供試した。これらの乳酸菌は、ＭＲＳ−ｂｒｏｔｈ培地（Difco Laboratories）で３５℃・４０時間培養を行った。
[Evaluation using mouse spleen cells]
(1) Culture of lactic acid bacteria B. bifidum 3 strains (B1-B3), B. breve 3 strains (B4-B6), B. adolescentis 4 strains (B7-B10), B. thermophilum 2 Strain (B11-B12), B.infantis2 strain (B13-B14), B.longum4 strain (B15-B18), B.pseudocatenulatum1 strain (B19), B.pseudolongum2 strain (B20-B21), B.suis1 strain ( A total of 25 strains (B1 to B25) of B22), B. catenulatum 1 strain (B23), B. angulatum 1 strain (B24), and B. gallicum 1 strain (B25) were used. These lactic acid bacteria were cultured in MRS-broth medium (Difco Laboratories) at 35 ° C. for 40 hours.
（２）脾臓細胞の調製
マウスから採取した脾臓をセルストレイナー（Falcon2350）で濾過した後、ＲＰＭＩ１６４０培地（Sigma）１０ｍｌで懸濁し、遠心した（１９０×ｇ・１０分）。沈殿した細胞に溶血バッファー（０．１５Ｍ ＮＨ４Ｃｌ、１０ｍＭ ＫＨＣＯ３、０．１ｍＭ Ｎａ２ＥＤＴＡ：ｐＨ７．２）を２ｍｌ添加し、５分静置した。ＲＰＭＩ培地を１０ｍｌ添加し、２回洗浄を行った後、ＦＣＳを１０％加えたＲＰＭＩ培地を用いて６×１０6個／ｍｌに調製した。
(2) Preparation of spleen cells The spleen collected from the mice was filtered with a cell strainer (Falcon 2350), suspended in 10 ml of RPMI 1640 medium (Sigma), and centrifuged (190 × g · 10 minutes). 2 ml of hemolysis buffer (0.15M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA: pH 7.2) was added to the precipitated cells, and the mixture was allowed to stand for 5 minutes. After adding 10 ml of RPMI medium and washing twice, it was adjusted to 6 × 10 6 cells / ml using RPMI medium supplemented with 10% FCS.
（３）サイトカインの測定
脾臓細胞を４８ウェル培養プレートに０．５ｍｌ／ウェルずつ播種した。１時間プレインキュベートした後、乳酸菌（湿重量５、５０、５００μｇ／ｍｌ）を５０μｌ／ウェル添加した。乳酸菌は生理食塩水で調製し、死菌は１００℃・５０分加熱したものを用いた。２４時間及び７２時間培養後、上清を回収した。そして、サイトカインＩＬ−１２ｐ７０、ＩＦＮ-γ及びＩＬ−１０の測定は、Ｍｏｕｓｅ ＢＤ Ｏｐｔ ＥＩＡ ＥＬＩＳＡ ｓｅｔ（BD Pharmingen）を用いて行った。
(3) Measurement of cytokines Spleen cells were seeded in a 48-well culture plate at 0.5 ml / well. After preincubation for 1 hour, lactic acid bacteria (wet weight 5, 50, 500 μg / ml) were added at 50 μl / well. Lactic acid bacteria were prepared in physiological saline, and dead bacteria were heated at 100 ° C. for 50 minutes. After culturing for 24 hours and 72 hours, the supernatant was collected. And the measurement of cytokine IL-12p70, IFN-gamma, and IL-10 was performed using Mouse BD Opt EIA ELISA set (BD Pharmingen).
（４）結果
各乳酸菌によるＩＬ−１２ｐ７０の産生量を図１０、ＩＦＮ-γの産生量を図１１、ＩＬ−１０の産生量を図１２に示す。Ｂ１６の生菌はＩＦＮ-γを、死菌はＩＬ−１２ｐ７０を強く誘導していた。また、Ｂ２０の死菌はＩＬ−１２ｐ７０を、Ｂ２２の生菌はＩＦＮ-γ及びＩＬ−１０を、Ｂ２４の生菌はＩＦＮ-γを誘導していた。なお、Ｂ１６はB.longumの一菌株、Ｂ２０はB. pseudolongumの一菌株、Ｂ２２はB. suisの一菌株、Ｂ２４はB. angulatum（ＪＣＭ７０９６Ｔ）を示す。
(4) Results The production amount of IL-12p70 by each lactic acid bacterium is shown in FIG. 10, the production amount of IFN-γ is shown in FIG. 11, and the production amount of IL-10 is shown in FIG. B16 live bacteria strongly induced IFN-γ, and dead bacteria strongly induced IL-12p70. Moreover, the dead bacteria of B20 induced IL-12p70, the live bacteria of B22 induced IFN-γ and IL-10, and the live bacteria of B24 induced IFN-γ. B16 is a strain of B. longum, B20 is a strain of B. pseudolongum, B22 is a strain of B. suis, and B24 is B. angulatum (JCM7096T).
（５）考察
マウス脾臓細胞においてＴｈ１タイプサイトカイン産生に影響を与え、ＩＬ−１２ｐ７０及びＩＦＮ−γを誘導している菌株は、生体においてもＴｈ１タイプサイトカインを誘導する可能性が高いと考えられる。したがって、Ｂ１６の生菌及び死菌、Ｂ２０の死菌などは、Ｔｈ１細胞の分化を誘導して免疫バランスを調節する可能性があると考えられた。
(5) Discussion It is considered that a strain that affects Th1-type cytokine production in mouse spleen cells and induces IL-12p70 and IFN-γ is highly likely to induce Th1-type cytokine even in the living body. Therefore, it was considered that viable and dead bacteria of B16, dead bacteria of B20, and the like may induce differentiation of Th1 cells and regulate immune balance.
［マウス脾臓細胞を用いた評価］
（１）乳酸菌の培養
エンテロコッカス属（Enterococcus）乳酸菌であるE.faecalis８菌株（Ｅ１〜Ｅ５及びＥ１３〜Ｅ１５）、E.faecium８菌株（Ｅ６〜Ｅ１２及びＥ１６）の計１６菌株を供試した。これらの乳酸菌は、ＳＣＤ−ｂｒｏｔｈ培地（Difco Laboratories）で３５℃・１８時間培養を行った。
[Evaluation using mouse spleen cells]
(1) Culture of Lactic Acid Bacteria A total of 16 strains of E. faecalis 8 strains (E1 to E5 and E13 to E15) and E. faecium 8 strains (E6 to E12 and E16), which are Enterococcus lactic acid bacteria, were tested. These lactic acid bacteria were cultured in SCD-broth medium (Difco Laboratories) at 35 ° C. for 18 hours.
（２）脾臓細胞の調製
マウスから採取した脾臓をセルストレイナー（Falcon2350）で濾過した後、ＲＰＭＩ１６４０培地（Sigma）１０ｍｌで懸濁し、遠心した（１９０×ｇ・１０分）。沈殿した細胞に溶血バッファー（０．１５Ｍ ＮＨ４Ｃｌ、１０ｍＭ ＫＨＣＯ３、０．１ｍＭ Ｎａ２ＥＤＴＡ：ｐＨ７．２）を２ｍｌ添加し、５分静置した。ＲＰＭＩ培地を１０ｍｌ添加し、２回洗浄を行った後、ＦＣＳを１０％加えたＲＰＭＩ培地を用いて６×１０6個／ｍｌに調製した。
(2) Preparation of spleen cells The spleen collected from the mice was filtered with a cell strainer (Falcon 2350), suspended in 10 ml of RPMI 1640 medium (Sigma), and centrifuged (190 × g · 10 minutes). 2 ml of hemolysis buffer (0.15M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA: pH 7.2) was added to the precipitated cells, and the mixture was allowed to stand for 5 minutes. After adding 10 ml of RPMI medium and washing twice, it was adjusted to 6 × 10 6 cells / ml using RPMI medium supplemented with 10% FCS.
（３）サイトカインの測定
脾臓細胞を４８ウェル培養プレートに０．５ｍｌ／ウェルずつ播種した。１時間プレインキュベートした後、乳酸菌（湿重量５、５０、５００μｇ／ｍｌ）を５０μｌ／ウェル添加した。乳酸菌は生理食塩水で調製し、１００℃・５０分加熱したものを用いた。２４時間及び７２時間培養後、上清を回収した。そして、サイトカインＩＬ−１２ｐ７０、ＩＦＮ-γ及びＩＬ−１０の測定は、Ｍｏｕｓｅ ＢＤ Ｏｐｔ ＥＩＡ ＥＬＩＳＡ ｓｅｔ（BD Pharmingen）を用いて行った。
(3) Measurement of cytokines Spleen cells were seeded in a 48-well culture plate at 0.5 ml / well. After preincubation for 1 hour, lactic acid bacteria (wet weight 5, 50, 500 μg / ml) were added at 50 μl / well. Lactic acid bacteria were prepared with physiological saline and heated at 100 ° C. for 50 minutes. After culturing for 24 hours and 72 hours, the supernatant was collected. And the measurement of cytokine IL-12p70, IFN-gamma, and IL-10 was performed using Mouse BD Opt EIA ELISA set (BD Pharmingen).
（４）結果
各乳酸菌によるＩＬ−１２ｐ７０の産生量を図１３、ＩＦＮ-γの産生量を図１４、ＩＬ−１０の産生量を図１５に示す。Ｅ１２は、ＩＬ−１２ｐ７０、ＩＦＮ-γ及びＩＬ−１０を強く誘導し、Ｔｈ１タイプサイトカイン誘導能が高いことが示唆された。また、Ｅ１０はＩＬ−１２ｐ７０、ＩＦＮ-γを、Ｅ１４及びＥ１６はＩＦＮ−γを誘導していた。なお、Ｅ１０はE. faeciumの一菌株、Ｅ１２はE. faecium（ＦＥＲＭ Ｐ−２０６３７）、Ｅ１４はE. faecalis（ＪＣＭ５８０３Ｔ）、Ｅ１６はE. faecium（ＪＣＭ５８０４Ｔ）を示す。
(4) Results FIG. 13 shows the amount of IL-12p70 produced by each lactic acid bacterium, FIG. 14 shows the amount of IFN-γ produced, and FIG. 15 shows the amount of IL-10 produced. E12 strongly induced IL-12p70, IFN-γ and IL-10, suggesting that Th1 type cytokine induction ability is high. E10 induced IL-12p70 and IFN-γ, and E14 and E16 induced IFN-γ. E10 represents one strain of E. faecium, E12 represents E. faecium (FERM P-20637), E14 represents E. faecalis (JCM5803T), and E16 represents E. faecium (JCM5804T).
（５）考察
マウス脾臓細胞においてＴｈ１タイプサイトカイン産生に影響を与え、ＩＬ−１２ｐ７０及びＩＦＮ−γを誘導している菌株は、生体においてもＴｈ１タイプサイトカインを誘導する可能性が高いと考えられる。そこで、Ｔｈ１タイプサイトカイン誘導能が高かったＥ１２及びＥ１６に関して、マウスへの投与試験を行うこととした。
(5) Discussion It is considered that a strain that affects Th1-type cytokine production in mouse spleen cells and induces IL-12p70 and IFN-γ is highly likely to induce Th1-type cytokine even in the living body. Therefore, with regard to E12 and E16, which had a high Th1-type cytokine induction ability, it was decided to conduct administration tests to mice.
［マウスへの投与試験による評価］
（１）乳酸菌の培養
脾臓細胞を用いた評価でＴｈ１タイプサイトカイン誘導能の高かった乳酸菌Ｅ１２及びＥ１６について、評価を行った。ＳＣＤ−ｂｒｏｔｈ培地（Difco Laboratories）で３５℃・１８時間培養を行った。
[Evaluation by administration test to mice]
(1) Culture of lactic acid bacteria Lactic acid bacteria E12 and E16, which were highly Th1-type cytokine-inducible by evaluation using spleen cells, were evaluated. Culturing was performed at 35 ° C. for 18 hours in an SCD-broth medium (Difco Laboratories).
（２）マウスへの投与及び免疫
投与試験は、１試験区あたりマウス１０匹を用いた。経口投与は生理食塩水又は乳酸菌（湿重量１０ｍｇ／ｍｌ）を１匹あたり２００μｌずつ７日連続で行った。乳酸菌は生理食塩水で調製し、１００℃・５０分加熱したものを用いた。また、免疫は１週目及び３週目に、生理食塩水で１ｍｇ／ｍｌに調製した ＯＶＡとＡｌｕｍ（４０〜５０ｍｇ／ｍｌ 水酸化アルミニウムゲル）を等量混合した溶液を１匹あたり１００μｌ腹腔内に注射
した。
(2) Administration to mice and immunization In the administration test, 10 mice were used per test group. Oral administration was performed for 7 consecutive days with 200 μl of physiological saline or lactic acid bacteria (wet weight 10 mg / ml) per animal. Lactic acid bacteria were prepared with physiological saline and heated at 100 ° C. for 50 minutes. Immunization was carried out in the first and third weeks by intraperitoneal injection of a solution of equal amounts of OVA and Alum (40-50 mg / ml aluminum hydroxide gel) prepared to 1 mg / ml with physiological saline. Injected.
（３）抗体の測定
４週目に採血を行い、血清中のＯＶＡ特異的ＩｇＥ抗体、ＩｇＧ抗体をＥＬＩＳＡにより測定した。
(3) Measurement of antibody Blood was collected on the 4th week, and OVA-specific IgE antibody and IgG antibody in the serum were measured by ELISA.
（４）結果
ＯＶＡ特異的ＩｇＥ抗体量を図１６、ＩｇＧ抗体量を図１７に示す。Ｅ１２及びＥ１６は、ＯＶＡ特異的ＩｇＥ抗体量を有意に抑制していた（＜０．０５）。一方、ＯＶＡ特異的ＩｇＧ抗体の産生に対する影響はなかった。したがって、Ｅ１２及びＥ１６は、他の抗体反応に大きな影響を与えることなくＩｇＥ抗体が関与するアレルギー作用を抑制する可能性があると考えられた。
(4) Results FIG. 16 shows the amount of OVA-specific IgE antibody, and FIG. 17 shows the amount of IgG antibody. E12 and E16 significantly suppressed the amount of OVA-specific IgE antibody (<0.05). On the other hand, there was no effect on the production of OVA-specific IgG antibodies. Therefore, E12 and E16 were thought to have the potential to suppress allergic effects involving IgE antibodies without significantly affecting other antibody responses.
（５）考察
マウスへの投与試験では、Ｅ１２及びＥ１６は、ＩｇＥ抗体の産生を抑制したが、ＩｇＧ抗体など他の抗体の産生は抑制しなかった。したがって、Ｅ１２及びＥ１６は、他の抗体反応に大きな影響を与えることなくＩｇＥ抗体が関与するアレルギー作用を抑制する可能性があると考えられた。
(5) Discussion In the administration test to mice, E12 and E16 suppressed the production of IgE antibody, but did not inhibit the production of other antibodies such as IgG antibody. Therefore, E12 and E16 were thought to have the potential to suppress allergic effects involving IgE antibodies without significantly affecting other antibody responses.
［マウスへの投与試験による評価］
Ｅ１２（Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７））は、ＳＣＤ−ｂｒｏｔｈ培地（Difco Laboratories）で３５℃・１８時間培養を行った。
[Evaluation by administration test to mice]
E12 (Enterococcus faecium (FERM P-20737)) was cultured in an SCD-broth medium (Difco Laboratories) at 35 ° C. for 18 hours.
（２）卵白の調製
鶏卵の卵白を凍結乾燥した。
(2) Preparation of egg white Egg white of chicken eggs was freeze-dried.
（３）β−ラクトグロブリン（以下β−ＬＧ）の調製
新鮮な牛乳よりAschaffenburgとDrewryの方法に従い、粗β−ＬＧ画分を得た。この粗画分をさらにＤＥＡＥ ６５０Ｓ（ＴＯＳＯＨ）を用いたイオン交換クロマトグラフィーにより精製した。移動相には５０ｍＭイミダゾール−塩酸緩衝液（ｐＨ６．４）を用い、ＮａＣｌの０から０．５Ｍのリニアグラジェントによりβ−ＬＧを分画し、透析による脱塩後、凍結乾燥を行った。
(3) Preparation of β-lactoglobulin (hereinafter β-LG) A crude β-LG fraction was obtained from fresh milk according to the method of Aschaffenburg and Drewry. This crude fraction was further purified by ion exchange chromatography using DEAE 650S (TOSOH). As a mobile phase, 50 mM imidazole-hydrochloric acid buffer (pH 6.4) was used, β-LG was fractionated with a linear gradient of NaCl from 0 to 0.5 M, desalted by dialysis, and then lyophilized.
（４）マウスへの投与及び免疫
投与試験は、１試験区あたりマウス８匹を用いた。経口投与は、下記４試験区（表１）で実施した。
乳酸菌及び卵白は７日間連続で投与した。卵白及び乳酸菌は生理食塩水で調製し、乳酸菌は調製後１００℃・５０分加熱したものを用いた。また、免疫は投与開始から１週目及び３週目に、１匹あたりβ−ＬＧ５０μｇを 水酸化アルミニウムゲル（４０〜５０ｍｇ／ｍｌ）とともに腹腔内に注射した。
(4) Administration to mice and immunization In the administration test, 8 mice were used per test group. Oral administration was carried out in the following 4 test sections (Table 1).
Lactic acid bacteria and egg white were administered for 7 consecutive days. Egg whites and lactic acid bacteria were prepared with physiological saline, and lactic acid bacteria were heated at 100 ° C. for 50 minutes after preparation. Immunization was carried out intraperitoneally with 50 μg of β-LG per animal together with aluminum hydroxide gel (40-50 mg / ml) in the first and third weeks from the start of administration.
（５）抗体の測定
４週目に採血を行い、血清中のβ−ＬＧ特異的ＩｇＥ抗体量をＥＬＩＳＡにより測定した。
(5) Measurement of antibody Blood was collected at 4 weeks, and the amount of β-LG-specific IgE antibody in the serum was measured by ELISA.
（６）結果
各投与区のβ−ＬＧ特異的ＩｇＥ抗体量を図１８に示す。ＴＥＳＴ１を対照としたｔ検定により、ＴＥＳＴ２の乾燥卵白のみ、あるいはＴＥＳＴ３のＥ１２のみの投与ではβ−ＬＧ特異的ＩｇＥ抗体産生の低下は認められなかった。しかし、単独では効果の認められなかった乾燥卵白とＥ１２を組み合わせたＴＥＳＴ４では有意にβ−ＬＧ特異的ＩｇＥ抗体の産生の低下が認められた（Ｐ≦０．０５％）。
(6) Results The amount of β-LG-specific IgE antibody in each administration group is shown in FIG. According to the t-test using TEST1 as a control, a decrease in β-LG-specific IgE antibody production was not observed when only TEST2 dried egg white or TEST3 E12 alone was administered. However, a significant decrease in the production of β-LG-specific IgE antibody was observed in TEST4, which was a combination of dry egg white and E12, which was not effective by itself (P ≦ 0.05%).
（７）考察
卵白成分のオボムコイドが乳、ダニ、花粉などに対する過剰な免疫を抑制する作用を有することを本発明者らにより明らかにしているが、今回の試験では、通常の食品である卵白を投与した場合、あるいは卵白に免疫調節機能を有する乳酸菌を加えた場合の免疫応答に対する影響を検討した。その結果、卵白の投与は、乳の主要アレルゲンであるβ−ＬＧに対する特異的ＩｇＥ抗体の産生を抑制する傾向にあるものの有意ではなかった。また、実施例６ではＥ１２の１０ｍｇ／ｍｌ投与によりＯＶＡに対する特異的ＩｇＥ抗体抑制作用を示したが、実施例７では、ＯＶＡと同じ濃度のβ−ＬＧに対し、特異的ＩｇＥ抗体の産生を抑制する傾向にあるものの有意ではなかった。しかし、有意な抑制効果を示さない乾燥卵白とＥ１２を組み合わせることにより相乗的な免疫調節機能を示し、β−ＬＧ特異的ＩｇＥ抗体の産生を抑制すること（ＴＥＳＴ４）が明らかとなった。このことから、免疫調製剤は、単一性分を利用するよりも、複数の有効成分の組み合わせにより、より効果的な商品設計が可能になると考えられた。さらに、乳酸菌の製造コストが最終製品のコストアップにつながるが、乳酸菌を他の食品成分と組み合わせることにより相乗的な効果が得られることが明らかとなり、免疫調製剤のコストダウンにも貢献できるものと考えられた。
(7) Discussion Although the present inventors have clarified that ovomucoid, an egg white component, has an action of suppressing excessive immunity against milk, mites, pollen, etc., in this test, egg white, which is a normal food, The effect on immune response when administered or when lactic acid bacteria having an immunomodulating function were added to egg white was examined. As a result, the administration of egg white was not significant, although it tended to suppress the production of specific IgE antibodies against β-LG, which is the major milk allergen. Moreover, in Example 6, the specific IgE antibody inhibitory action with respect to OVA was shown by administration of E12 at 10 mg / ml, but in Example 7, the production of specific IgE antibody was suppressed against β-LG having the same concentration as OVA. Although there was a tendency to do, it was not significant. However, it was revealed that the combination of dried egg white that does not show a significant inhibitory effect and E12 exhibits a synergistic immunoregulatory function and suppresses the production of β-LG-specific IgE antibody (TEST4). From this, it was considered that an immunopreparation agent can be designed more effectively by combining a plurality of active ingredients than using a single component. Furthermore, although the production cost of lactic acid bacteria leads to an increase in the cost of the final product, it has become clear that combining lactic acid bacteria with other food ingredients can provide a synergistic effect, which can contribute to the cost reduction of immune preparations. it was thought.
［オボムコイド及び乳酸菌の組み合わせによるマウスの免疫応答に対する効果］
（１）乳酸菌の培養
Ｅ１２（Enterococcus faecium（ＦＥＲＭ Ｐ−２０６３７））は、ＳＣＤ−ｂｒｏｔｈ培地（Difco Laboratories）で３５℃・１８時間培養を行った。
[Effects of the combination of ovomucoid and lactic acid bacteria on immune responses in mice]
(1) Culture of lactic acid bacteria E12 (Enterococcus faecium (FERM P-20637)) was cultured in an SCD-broth medium (Difco Laboratories) at 35 ° C. for 18 hours.
（２）ニワトリオボムコイド（以下「ＯＭ」という）の調製
新鮮卵白よりアセトン沈殿法（Lineweaver & Murry）により粗ＯＭを調製した。さらに０．１Ｍ酢酸緩衝液に対し透析後、８，０００ｒｐｍ×２０分遠心し、上精を回収した。さらに、ＴＳＫ ｇｅｌ ＤＥＡＥ ６５０Ｓ（ＴＯＳＯＨ）を用いたイオン交換クロマトグラフィーにより精製した。移動相には５０ｍＭイミダゾール−塩酸緩衝液（ｐＨ６．４）を用い、ＮａＣｌの０から０．３ＭのリニアグラジェントによりＯＭを分画し、透析による脱塩後、凍結乾燥を行った。
(2) Preparation of chicken ovomucoid (hereinafter referred to as “OM”) Crude OM was prepared from fresh egg white by the acetone precipitation method (Lineweaver & Murry). Furthermore, after dialysis against 0.1 M acetate buffer, the supernatant was collected by centrifugation at 8,000 rpm × 20 minutes. Further, the product was purified by ion exchange chromatography using TSK gel DEAE 650S (TOSOH). The mobile phase was 50 mM imidazole-hydrochloric acid buffer (pH 6.4), OM was fractionated with a linear gradient of NaCl from 0 to 0.3 M, desalted by dialysis, and lyophilized.
（３）マウスへの投与及び免疫
投与試験は、１試験区あたりマウス８匹を用いた。経口投与は、下記４試験区（表２）で実施した。
乳酸菌及びＯＭは７日間連続で投与した。ＯＭ及び乳酸菌は生理食塩水で調製し、乳酸菌は調製後１００℃・５０分加熱したものを用いた。また、免疫は投与開始から１週目及び３週目に、１匹あたりβ−ＬＧ５０μｇを水酸化アルミニウムゲル（４０〜５０ｍｇ／ｍｌ）とともに腹腔内に注射した。
(3) Administration to mice and immunization In the administration test, 8 mice were used per test group. Oral administration was performed in the following 4 test sections (Table 2).
Lactic acid bacteria and OM were administered for 7 consecutive days. OM and lactic acid bacteria were prepared with physiological saline, and lactic acid bacteria were heated at 100 ° C. for 50 minutes after preparation. Immunization was carried out intraperitoneally with 50 μg of β-LG per animal together with aluminum hydroxide gel (40-50 mg / ml) in the first and third weeks from the start of administration.
（４）抗体の測定
４週目に採血を行い、血清中の総ＩｇＥ抗体量をＥＬＩＳＡにより測定した。
(4) Measurement of antibody Blood was collected on the 4th week, and the total amount of IgE antibody in the serum was measured by ELISA.
（５）結果
各投与区の総ＩｇＥ抗体量を図１９に示す。ＴＥＳＴ１を対照としたｔ検定により、ＴＥＳＴ２のＯＭのみ、あるいはＴＥＳＴ３のＥ１２のみの投与では総ＩｇＥ抗体量の低下は認められなかった。しかし、単独では効果の認められなかったＯＭとＥ１２を組み合わせたＴＥＳＴ４では有意に総ＩｇＥ抗体量の低下が認められた（Ｐ≦０．０５％）。
(5) Results FIG. 19 shows the total IgE antibody amount in each administration group. According to the t-test using TEST1 as a control, the administration of TEST2 OM alone or TEST3 E12 alone showed no decrease in the total IgE antibody amount. However, a significant decrease in the amount of total IgE antibody was observed in TEST4 which was a combination of OM and E12, which was not effective by itself (P ≦ 0.05%).
（６）考察
単独で免疫抑制効果を示さない少量のＥ１２と、卵白アレルゲンのＯＭと組み合わせることにより、免疫抑制効果を示すことが明らかとなった。これらの結果は実施例７と同様の傾向を示し、実施例７でβ−ＬＧ特異的ＩｇＥ抗体を抑制していた卵の成分は主にＯＭと考えられ、ＯＭとＥ１２を組み合わせることにより、効果的な免疫調節機能を発現できるものと考えられた。
(6) Discussion It became clear that an immunosuppressive effect was shown by combining a small amount of E12 that does not show an immunosuppressive effect alone with OM of an egg white allergen. These results show the same tendency as in Example 7. The egg component that suppressed the β-LG-specific IgE antibody in Example 7 was considered to be mainly OM, and the effect was obtained by combining OM and E12. It was thought that it could express a typical immunoregulatory function.
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