JP2017014231A - NK cell activator - Google Patents

NK cell activator Download PDF

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JP2017014231A
JP2017014231A JP2016146787A JP2016146787A JP2017014231A JP 2017014231 A JP2017014231 A JP 2017014231A JP 2016146787 A JP2016146787 A JP 2016146787A JP 2016146787 A JP2016146787 A JP 2016146787A JP 2017014231 A JP2017014231 A JP 2017014231A
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polysaccharide
cells
cell
polysaccharides
acidic
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聖也 牧野
Kiyonari Makino
聖也 牧野
秀二 池上
Hideji Ikegami
秀二 池上
紀宏 指原
Norihiro Yubihara
紀宏 指原
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Meiji Co Ltd
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Abstract

PROBLEM TO BE SOLVED: To provide a NK cell activator that enables prevention of infections such as influenza, prevention of cancer, prevention of progression, and so on.SOLUTION: In the food and drink and/or medicine for immunostimulation of the present invention, phosphorylated polysaccharides obtained from the Lactobacillus bulgaricus OLLL1073R-1 strain (FERM P-17227) have excellent abilities to activate NK cells and the phosphorylated polysaccharides are excellent in safety and has a high NK-cell activation effect.SELECTED DRAWING: None

Description

本発明は、ナチュラルキラー(以下「NK」ともいう)細胞活性化作用を有する組成物に関するものである。   The present invention relates to a composition having a natural killer (hereinafter also referred to as “NK”) cell activation effect.

NK細胞は、名前のとおりウイルス感染細胞やがん細胞をいち早く発見して殺す働きがあり、インフルエンザなどの感染症予防、癌予防の決め手として注目されている。しかしながらNK細胞の活性(破壊能力)は加齢とともに低下し、高齢になるほどガン発生率が高くなるのは、NK細胞の活性化の衰えに関連しているとされている。また、NK細胞は自律神経に影響を受けるため、ストレスを受けて交感神経が優位になるとNK細胞の活性(破壊能力)は激減する。このように、健康を維持するためには、NK細胞の活性化が不可欠である。そして、NK細胞活性化を促進する物質が探索されているが、薬剤として利用されるものがほとんどである。食品として利用可能な素材としては、多糖(レンチナン、キチン、キトサン、マイタケD-フラクション、ニゲロオリゴ糖等)および菌体(Lactobacillus casei shirota、Bifidobacterium lactis HN019等)が知られている(非特許文献1〜6、特許文献1)が、必ずしも十分な活性を得ることが出来なかった。しかしながら、食品として利用されている素材由来であれば、日常生活において安全かつ簡便に摂取し、免疫力の増強が期待される。   As the name suggests, NK cells have the function of quickly discovering and killing virus-infected cells and cancer cells, and are attracting attention as a deciding factor in preventing infectious diseases such as influenza and cancer. However, the activity (destructive ability) of NK cells decreases with aging, and the fact that the incidence of cancer increases with age is considered to be related to the decline in activation of NK cells. In addition, since NK cells are affected by autonomic nerves, the activity (destructive ability) of NK cells drastically decreases when stress is applied and sympathetic nerves become dominant. Thus, in order to maintain health, activation of NK cells is indispensable. And although the substance which promotes NK cell activation is searched, most are utilized as a chemical | medical agent. As materials that can be used as food, polysaccharides (lentinan, chitin, chitosan, maitake D-fraction, nigerooligosaccharide, etc.) and bacterial cells (Lactobacillus casei shirota, Bifidobacterium lactis HN019, etc.) are known (Non-Patent Documents 1 to 4). 6, Patent Document 1) cannot always obtain sufficient activity. However, if it is derived from a material used as a food, it is expected to be safely and easily ingested in daily life and enhance immunity.

特開2001-31573号公報JP 2001-31573 A

秋山由紀雄, 羽室淳爾, 蛋白質 核酸 酵素, 26(3), 208-224(1981)Yukio Akiyama, Jun Hamuro, Protein Nucleic Acid Enzyme, 26 (3), 208-224 (1981) Nishimura K, Nishimura S, Nishi N, Numata F, Tone Y, Tokura S, Azuma I, Vaccine,3(5),379-84(1985)Nishimura K, Nishimura S, Nishi N, Numata F, Tone Y, Tokura S, Azuma I, Vaccine, 3 (5), 379-84 (1985) Kodama N, Komuta K, Sakai N, Nanba H, Biol Pharm Bull,25(12),1647-50(2002)Kodama N, Komuta K, Sakai N, Nanba H, Biol Pharm Bull, 25 (12), 1647-50 (2002) Murosak S, Muroyama K, Yamamoto Y, Liu T, Yoshikai Y,Int Immunopharmacol,2(1),151-9(2002)Murosak S, Muroyama K, Yamamoto Y, Liu T, Yoshikai Y, Int Immunopharmacol, 2 (1), 151-9 (2002) Hori T, Kiyoshima J, Yasui H, Biosci Biotechnol Biochem,67(2),420-2(2003)Hori T, Kiyoshima J, Yasui H, Biosci Biotechnol Biochem, 67 (2), 420-2 (2003) Gill HS, Rutherfurd KJ, Cross ML, Gopal PK., Am J Clin Nutr,74(6),833-9(2001)Gill HS, Rutherfurd KJ, Cross ML, Gopal PK., Am J Clin Nutr, 74 (6), 833-9 (2001)

そこで、NK細胞を活性化し、免疫力を高める食品由来の組成物が望まれている。   Therefore, a food-derived composition that activates NK cells and enhances immunity is desired.

本発明者らは、NK細胞を活性化する食品由来の組成物を検討したところ、酸性多糖類にNK細胞活性化作用があることを見出して本発明を完成させた。
本発明は、具体的には、
[1] 酸性多糖類を有効成分として含有することを特徴とするNK細胞活性化剤、
[2] 酸性多糖類がリン酸化多糖類である上記[1]記載のNK細胞活性化剤、
[3] 多糖類が乳酸菌由来である上記[1]または[2]記載のNK細胞活性化剤、
[4] 上記[1]〜[3]のいずれか1つに記載のNK細胞活性化剤を含み、NK細胞活性化作用を有するものであることを特徴とし、NK細胞活性化のために用いられる旨の表示を付した発酵乳、
を提供するものである。
When the present inventors examined the composition derived from the foodstuff which activates NK cell, it discovered that acidic polysaccharide had NK cell activation effect | action, and completed this invention.
The present invention specifically includes:
[1] An NK cell activator comprising an acidic polysaccharide as an active ingredient,
[2] The NK cell activator according to [1] above, wherein the acidic polysaccharide is a phosphorylated polysaccharide,
[3] The NK cell activator according to [1] or [2] above, wherein the polysaccharide is derived from lactic acid bacteria,
[4] The NK cell activator according to any one of [1] to [3] above, which has an NK cell activation action, and is used for NK cell activation Fermented milk with an indication that
Is to provide.

酸性多糖類からなるNK細胞活性化剤により、インフルエンザなどの感染予防、癌の予防、進行の防止などが可能となった。根本治療法のないこれらの疾患の治療または予防薬の一つを新たに提供することができる。   An NK cell activator comprising an acidic polysaccharide has made it possible to prevent infection such as influenza, prevent cancer, and prevent progression. One of the therapeutic or prophylactic drugs for these diseases without a fundamental treatment can be newly provided.

陰イオン交換樹脂 DEAE sepharoseによる中性多糖と酸性多糖の分離を示す図である。It is a figure which shows isolation | separation of neutral polysaccharide and acidic polysaccharide by anion exchange resin DEAE sepharose. 酸性多糖のSephacryl S-400HRを用いたゲルろ過による分離を示す図である。It is a figure which shows isolation | separation by gel filtration using Sephacryl S-400HR of acidic polysaccharide. 酸性多糖を3週間経口投与したマウスにおける脾臓細胞のNK活性を示す図である。(検定方法:Student's t-test)It is a figure which shows the NK activity of the spleen cell in the mouse | mouth which orally administered acidic polysaccharide for 3 weeks. (Test method: Student's t-test) 多糖産生乳酸菌を利用した発酵乳を4週間経口投与したマウスにおける脾臓細胞のNK活性を示す図である。(検定方法:Student's t-test)It is a figure which shows the NK activity of the spleen cell in the mouse | mouth which orally administered fermented milk using a polysaccharide-producing lactic acid bacterium for 4 weeks. (Test method: Student's t-test)

本発明のNK細胞活性化剤は、酸性多糖類、好ましくはリン酸化多糖類を有効成分とする。リン酸化多糖類には、多糖類を産生する乳酸菌の培養物、該培養物から精製した多糖類、多糖類を化学的又は酵素的にリン酸化して得られた多糖類などが挙げられる。リン酸化多糖類中のリン含有量は0.01%以上であることが好ましく、0.1%以上であることがさらに好ましい。また、本願発明でいう多糖類には、当該多糖を化学的又は酵素的に分解して得られたオリゴ糖を含む。
本発明に用いられる乳酸菌としては、多糖類を生産する乳酸菌であれば、種類を問わない。酸性多糖類は単独で、または種類の異なる2種以上を組み合わせて用いてもよい。
乳酸菌としては、ラクトバチルス・ブルガリクス(Lactobacillus bulgaricus)、またはラクトコッカス・ラクティス・クレモリス(Lactococcus lactis ssp. cremoris) 等が、単独でまたは組み合わせて用いられる。これらの中でもLactococcus lactis ssp. cremoris KVS20株(Kitazawa H, Yamaguchi T, Miura M, Saito T, Itoh T, J Dairy Sci,76,1514-9(1993)), Lactobacillus bulgaricusOLL1073R-1株(FERM P-17227)が好適である。
The NK cell activator of the present invention contains an acidic polysaccharide, preferably a phosphorylated polysaccharide as an active ingredient. Examples of phosphorylated polysaccharides include cultures of lactic acid bacteria that produce polysaccharides, polysaccharides purified from the cultures, polysaccharides obtained by phosphorylating polysaccharides chemically or enzymatically, and the like. The phosphorus content in the phosphorylated polysaccharide is preferably 0.01% or more, and more preferably 0.1% or more. In addition, the polysaccharide referred to in the present invention includes an oligosaccharide obtained by chemically or enzymatically degrading the polysaccharide.
The lactic acid bacteria used in the present invention are not limited as long as they are lactic acid bacteria producing polysaccharides. Acidic polysaccharides may be used alone or in combination of two or more different types.
As lactic acid bacteria, Lactobacillus bulgaricus, Lactococcus lactis ssp. Cremoris, etc. are used alone or in combination. Among these, Lactococcus lactis ssp. Cremoris KVS20 strain (Kitazawa H, Yamaguchi T, Miura M, Saito T, Itoh T, J Dairy Sci, 76, 1514-9 (1993)), Lactobacillus bulgaricus OLL1073R-1 strain (FERM P-17227 ) Is preferred.

多糖類を培養物そのままを用いても構わないが、特開2000-247895号公報記載の方法で中性多糖類を除く、または、必要に応じて下記の様にして精製したものを用いても構わない。尚、下記の工程の一部を省略、追加しても構わない。
1、遠心分離による培養物からの菌体の除去
2、エタノール沈殿によって高分子量の多糖類、タンパク質を沈殿として回収
3、タンパク質の除去
a)final 5-10%程度のトリクロロ酢酸でタンパク沈殿、遠心
b)boilすることによりタンパク質を熱変性、遠心
c)b)のあとにプロテイナーゼによるタンパク分解、あるいはDNAaseによる核酸の分解処理等も行い、透析
4、陰イオン交換樹脂による酸性多糖体類の吸着と溶出による回収
5、ゲルろ過により分子量100万以上の物質として酸性多糖体を精製する。
この時、酸性かつ分子量が近いタンパク質が混合していた場合は、ゲルろ過の前、あるいは後に何らかのタンパク除去操作を加えることも可能である。
The culture may be used as it is, but the neutral polysaccharide may be removed by the method described in JP-A-2000-247895, or a product purified as described below may be used if necessary. I do not care. Note that some of the following steps may be omitted or added.
1. Removal of bacterial cells from the culture by centrifugation 2. Recovery of high molecular weight polysaccharides and proteins as precipitates by ethanol precipitation 3. Removal of proteins
a) Protein precipitation with about 5-10% final trichloroacetic acid, centrifugation
b) Heat denaturation and centrifugation of the protein by boiling
c) After b), protein degradation by proteinase or nucleic acid degradation by DNAase is also performed, dialysis 4, recovery by adsorption and elution of acidic polysaccharides by anion exchange resin, and molecular weight of 1 million or more by gel filtration. Acidic polysaccharides are purified as a substance.
At this time, if proteins with acidic and close molecular weight are mixed, it is possible to add some protein removal operation before or after gel filtration.

酸性多糖類を製剤化して医薬品とする場合には、治療目的や投与経路等に応じて剤型を選択することができ、例えば、錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、注射剤、坐剤、シロップ剤、浸剤、煎剤、チンキ剤等が挙げられる。また、製剤化のために、必要に応じて充填剤、増量剤、結合剤、保湿剤、崩壊剤、界面活性剤、滑沢剤等の希釈剤あるいは賦形剤を用いることができる。また、この医薬製剤中に着色剤、保存剤、香料、風味剤、甘味剤等や他の医薬品を医薬製剤中に含有させてもよい。   When an acidic polysaccharide is formulated into a pharmaceutical product, the dosage form can be selected according to the therapeutic purpose, administration route, etc., for example, tablets, pills, powders, solutions, suspensions, emulsions, granules Agents, capsules, injections, suppositories, syrups, soaking agents, decoction, tinctures and the like. For formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants, lubricants and the like can be used as necessary. In addition, a colorant, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals may be included in the pharmaceutical preparation.

酸性多糖類を含む食品の形態としては、ヨーグルトの様な発酵乳、飲料等を挙げることができ、健康食品、特定保健用食品、栄養補助食品もしくはNK細胞活性化作用を表示した食品等として使用できる。酸性多糖類と食品衛生上許容できる配合物、例えば、安定化剤、保存料、着色料、香料、ビタミン等の配合物を上記リン酸化多糖類に適宜添加し、混合し、定法により、錠剤、粒状、顆粒状、粉末状、カプセル状、液状、ゼリー状、クリーム状、飲料等の食品とすることが出来る。
このような形態の、NK細胞活性化補助食品を摂取することにより、NK細胞活性化が図られるため、本発明で得られる食品は、インフルエンザなどの感染予防、癌の予防、進行の防止などに寄与することが可能である。
Examples of foods containing acidic polysaccharides include fermented milk and beverages such as yogurt, and can be used as health foods, foods for specified health use, dietary supplements, foods displaying NK cell activation, etc. it can. Acidic polysaccharides and food hygiene acceptable formulations, for example, stabilizers, preservatives, coloring agents, fragrances, vitamins and the like are added to the phosphorylated polysaccharides as appropriate, mixed, and tableted in a conventional manner. Granules, granules, powders, capsules, liquids, jellies, creams, beverages and other foods can be used.
Since NK cell activation is achieved by ingesting NK cell activation supplements in this form, the food obtained in the present invention can be used to prevent infection such as influenza, cancer, and progression. It is possible to contribute.

以下、本発明を説明するが、本発明がこの実施例に限定されないことはいうまでもない。   Hereinafter, the present invention will be described, but it goes without saying that the present invention is not limited to these examples.

〔実施例1〕
乳酸菌由来多糖の製造
1.乳酸菌の培養
10%乳清をプロテアーゼで55℃、8時間処理した後酵母エキスを0.5%添加、pH7.0に調製したものにLactobacillus bulgaricusOLL1073R-1株(FERM P-17227)を接種し、37℃、24時間の静置培養を行った。
2、多糖の精製
10-20Lの培養液より菌体を遠心分離(22800 x g,4℃,20min)した後、エタノール沈殿(60-70%(v/v))を2回行った。沈殿物はMilliQ水に溶解し、トリクロロ酢酸(final 10%)によるタンパク変性、遠心分離(22800 x g,4℃,20min)による不溶物の除去を行った。
MilliQ水に対する透析、凍結乾燥を行った後、0.02M Tris-HCl(pH8.6)に溶解し、陰イオン交換樹脂であるDEAE-Sepharose(Amersham Biosciences)を用いて中性多糖と酸性多糖の分離を行った(図1)。中性多糖はPass画分として回収し、酸性多糖はNaClを用いて0-0.5Mイオン強度勾配による溶出で回収した。
回収された酸性多糖はSephacryl S-400HR(Amersham Biosciences)を用いてゲルろ過を行い、分子量による分離を行った。本多糖は分子量1×106Da以上の画分と分子量がより小さい画分に分離され、分子量1×106Da以上の画分について分取した(図2)。
分取した多糖はMilliQ水に対して透析後、凍結乾燥し、酸性多糖精製品を得た。
なお、多糖はグルコースを標準物質としてフェノール・硫酸法を用いて検出・定量した。
また、文献記載(Uemura J, Milchwissenschaft,53(8),443-6(1998))の方法で当該多糖を分析したところ、リン酸化多糖を含有していることが確認された。
[Example 1]
Production of polysaccharides derived from lactic acid bacteria Culture of lactic acid bacteria
10% whey was treated with protease at 55 ° C for 8 hours, 0.5% yeast extract was added, and the pH adjusted to 7.0 was inoculated with Lactobacillus bulgaricus OLL1073R-1 strain (FERM P-17227) at 37 ° C for 24 hours Was statically cultured.
2. Purification of polysaccharides
Bacterial cells were centrifuged (22800 × g, 4 ° C., 20 min) from 10-20 L of the culture solution, and then ethanol precipitation (60-70% (v / v)) was performed twice. The precipitate was dissolved in MilliQ water, and protein denaturation with trichloroacetic acid (final 10%) and insoluble matter were removed by centrifugation (22800 xg, 4 ° C, 20 min).
After dialysis against MilliQ water and freeze-drying, dissolve in 0.02M Tris-HCl (pH 8.6) and separate neutral and acidic polysaccharides using DEAE-Sepharose (Amersham Biosciences), an anion exchange resin (FIG. 1). Neutral polysaccharides were collected as the Pass fraction, and acidic polysaccharides were collected by elution with 0-0.5M ionic strength gradient using NaCl.
The collected acidic polysaccharide was subjected to gel filtration using Sephacryl S-400HR (Amersham Biosciences) and separated by molecular weight. Honda sugar molecular weight 1 × 10 6 Da or more fractions and the molecular weight are separated into smaller fractions were collected for partial molecular weight 1 × 10 6 Da or more fractions (Figure 2).
The collected polysaccharide was dialyzed against MilliQ water and freeze-dried to obtain a purified acidic polysaccharide product.
The polysaccharide was detected and quantified using the phenol / sulfuric acid method with glucose as a standard substance.
Further, when the polysaccharide was analyzed by the method described in the literature (Uemura J, Milchwissenschaft, 53 (8), 443-6 (1998)), it was confirmed that it contained a phosphorylated polysaccharide.

〔実施例2〕
多糖のNK活性
1.多糖のマウスへの経口投与試験
マウスBALB/c(雄、8週齢、SLC社)30匹を1群10匹で3群に分け、以下の投与物を3週間、ゾンデにより強制経口投与を行った。
1) 蒸留水投与群:0.5ml/body/day
2) 実施例1で得た酸性多糖投与群:5mg/kg/day
3) 実施例1で得た酸性多糖投与群:30mg/kg/day
多糖は各濃度で蒸留水に溶解し、0.5ml/body/dayで投与を行った。
2.NK活性測定
3週間の投与終了後、個々のマウスよりそれぞれ脾臓を取り出し、細胞をRPMI1640(10%FCS)に懸濁することにより脾臓細胞懸濁液(1×107cells/ml)を調製した。
この脾臓細胞をエフェクター細胞、YAC-1細胞をターゲット細胞としてNK活性をfluorescence activated cell sorter(以下FACSとする)を用いて測定した。ターゲット細胞であるYAC-1は3,3’-dioctadecyloxacarbocyanine perchlorate(以下Dioとする)により蛍光染色した後、脾臓細胞とともに96穴マイクロプレート(丸底)を用いて37℃、5%CO2条件下で4時間培養を行った。培養終了前にPropidium Iodide(以下PIとする)を培養液に加え、死細胞を染色した。培養終了後、Johannの論文を参考にFACSを用いてDio、PIともに染色された細胞を死滅したYAC-1細胞として検出した(Johann S, Blumel G, Lipp M, Forster R,J Immunol Methods,185(2),209-16(1995))。なお、エフェクター細胞とターゲット細胞の比率は40:1とした。
その結果、酸性多糖30mg/kg/day投与群の脾臓細胞NK活性はコントロールである水投与群に比べ有意に高かった(P<0.01)。また、酸性多糖5mg/kg/day投与群においてもコントロールに比べ高い傾向(P<0.1)が見られた(図3)。
[Example 2]
NK activity of polysaccharides Oral administration test of polysaccharides to mice 30 mice BALB / c (male, 8 weeks old, SLC) were divided into 3 groups with 10 mice per group, and the following doses were administered by oral gavage with a sonde for 3 weeks It was.
1) Distilled water administration group: 0.5ml / body / day
2) Acid polysaccharide administration group obtained in Example 1: 5 mg / kg / day
3) Acid polysaccharide administration group obtained in Example 1: 30 mg / kg / day
The polysaccharide was dissolved in distilled water at each concentration and administered at 0.5 ml / body / day.
2. NK activity measurement After 3 weeks of administration, the spleen was removed from each mouse and the cells were suspended in RPMI1640 (10% FCS) to prepare a spleen cell suspension (1 × 10 7 cells / ml). .
The NK activity was measured using a fluorescence activated cell sorter (hereinafter referred to as FACS) using the spleen cells as effector cells and YAC-1 cells as target cells. The target cell, YAC-1, was fluorescently stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (hereinafter referred to as Dio), and then used together with spleen cells in a 96-well microplate (round bottom) at 37 ° C and 5% CO 2 Incubated for 4 hours. Before completion of the culture, Propidium Iodide (hereinafter referred to as PI) was added to the culture solution to stain dead cells. After completion of the culture, cells stained with Dio and PI were detected as dead YAC-1 cells using FACS with reference to Johann's paper (Johann S, Blumel G, Lipp M, Forster R, J Immunol Methods, 185 (2), 209-16 (1995)). The ratio of effector cells to target cells was 40: 1.
As a result, the spleen cell NK activity in the acidic polysaccharide 30 mg / kg / day administration group was significantly higher than that in the water administration group as a control (P <0.01). In addition, the acidic polysaccharide 5 mg / kg / day administration group showed a higher tendency (P <0.1) than the control (FIG. 3).

〔実施例3〕
多糖産生乳酸菌を利用した発酵乳の作製
NK活性上昇作用を有する酸性多糖体を産生するL. bulgaricus OLL1073R-1を用いて発酵乳を作製した。生乳、脱脂粉乳、砂糖を使用し、SNF9.7%、FAT3.05%、砂糖3.0%に調製した溶液(以下発酵乳Mix)にL. bulgaricus OLL1073R-1、S. thermophilus OLS3059(FERM P-15487)をスターター菌として加え、43℃で発酵を行った。酸度0.7で発酵を終了し、4℃で1日保存することで最終酸度0.78となった。また、対照発酵乳としてL. bulgaricus OLL1256及びS. thermophilus OLS3295をスターター菌として用いた発酵乳についても作製した。
Example 3
Preparation of fermented milk using polysaccharide-producing lactic acid bacteria Fermented milk was prepared using L. bulgaricus OLL1073R-1 which produces an acidic polysaccharide having an NK activity increasing action. Using raw milk, skim milk powder and sugar, a solution prepared to SNF9.7%, FAT3.05%, sugar 3.0% (hereinafter fermented milk Mix) was added to L. bulgaricus OLL1073R-1, S. thermophilus OLS3059 (FERM P-15487 ) As a starter fungus and fermented at 43 ° C. Fermentation was completed at an acidity of 0.7, and a final acidity of 0.78 was obtained by storing at 4 ° C for 1 day. In addition, fermented milk using L. bulgaricus OLL1256 and S. thermophilus OLS3295 as starter fungi as control fermented milk was also produced.

〔実施例4〕
多糖産生乳酸菌を利用した発酵乳のNK活性
1.多糖産生乳酸菌を利用した発酵乳のマウスへの経口投与試験
マウスBALB/c(雄、11週齢、SLC社)32匹を1群8匹で4群に分け、以下の投与物を4週間、ゾンデにより強制経口投与を行った。
1) 蒸留水投与群:1.0ml/body/day
2) L. bulgaricus OLL1073R-1発酵乳(実施例3)投与群:200mg/body/day
3) 対照発酵乳(実施例3)投与群:200mg/body/day
4) 発酵乳Mix(実施例3)投与群:200mg/body/day
発酵乳、発酵乳Mixは凍結乾燥物を用い、投与時に200mg/mlで蒸留水に懸濁した。
2.NK活性測定
4週間の投与終了後、個々のマウスよりそれぞれ脾臓を取り出し、細胞をRPMI1640(10%FCS)に懸濁することにより脾臓細胞懸濁液(1×107cells/ml)を調製した。
この脾臓細胞をエフェクター細胞、YAC-1細胞をターゲット細胞としてNK活性をfluorescence activated cell sorter(以下FACSとする)を用いて測定した。ターゲット細胞であるYAC-1は3,3’-dioctadecyloxacarbocyanine perchlorate(以下Dioとする)により蛍光染色した後、脾臓細胞とともに96穴マイクロプレート(丸底)を用いて37℃、5%CO2条件下で4時間培養を行った。培養終了前にPropidium Iodide(以下PIとする)を培養液に加え、死細胞を染色した。培養終了後、Johannの論文を参考にFACSを用いてDio、PIともに染色された細胞を死滅したYAC-1細胞として検出した(Johann S, Blumel G, Lipp M, Forster R,J Immunol Methods,185(2),209-16(1995))。なお、エフェクター細胞とターゲット細胞の比率は40:1とした。
その結果、発酵乳投与では多糖体産生菌であるL. bulgaricus OLL1073R-1を使用した発酵乳投与群においてNK活性がコントロールである蒸留水投与群に比べ有意に高く(p<0.05)、発酵乳Mixと対照発酵乳に対しては高い傾向(p<0.1)が見られた(図4)。
Example 4
NK activity of fermented milk using polysaccharide-producing lactic acid bacteria Oral administration test to mice of fermented milk using polysaccharide-producing lactic acid bacteria 32 mice BALB / c (male, 11 weeks old, SLC) divided into 4 groups with 8 mice per group, the following administrations for 4 weeks, Forced oral administration was performed with a sonde.
1) Distilled water administration group: 1.0ml / body / day
2) L. bulgaricus OLL1073R-1 fermented milk (Example 3) administration group: 200 mg / body / day
3) Control fermented milk (Example 3) administration group: 200 mg / body / day
4) Fermented milk mix (Example 3) administration group: 200 mg / body / day
Fermented milk and fermented milk Mix were lyophilized and suspended in distilled water at 200 mg / ml at the time of administration.
2. NK activity measurement After completion of administration for 4 weeks, spleens were removed from individual mice, and cells were suspended in RPMI1640 (10% FCS) to prepare a spleen cell suspension (1 × 10 7 cells / ml). .
The NK activity was measured using a fluorescence activated cell sorter (hereinafter referred to as FACS) using the spleen cells as effector cells and YAC-1 cells as target cells. The target cell, YAC-1, was fluorescently stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (hereinafter referred to as Dio), and then used together with spleen cells in a 96-well microplate (round bottom) at 37 ° C and 5% CO 2 Incubated for 4 hours. Before completion of the culture, Propidium Iodide (hereinafter referred to as PI) was added to the culture solution to stain dead cells. After completion of the culture, cells stained with Dio and PI were detected as dead YAC-1 cells using FACS with reference to Johann's paper (Johann S, Blumel G, Lipp M, Forster R, J Immunol Methods, 185 (2), 209-16 (1995)). The ratio of effector cells to target cells was 40: 1.
As a result, fermented milk administration showed significantly higher NK activity in the fermented milk administration group using L. bulgaricus OLL1073R-1 which is a polysaccharide-producing bacterium (p <0.05). A high tendency (p <0.1) was observed for Mix and control fermented milk (FIG. 4).

本発明によれば、NK細胞活性化効果が高いので、免疫賦活剤として有用であるだけでなく、免疫賦活用飲食品としても利用することができる。   According to the present invention, since the NK cell activation effect is high, it is not only useful as an immunostimulator, but can also be used as an immunostimulatory food or drink.

Claims (4)

酸性多糖類を有効成分として含有することを特徴とするNK細胞活性化剤。   An NK cell activator comprising an acidic polysaccharide as an active ingredient. 酸性多糖類がリン酸化多糖類である請求項1記載のNK細胞活性化剤。   The NK cell activator according to claim 1, wherein the acidic polysaccharide is a phosphorylated polysaccharide. 多糖類が乳酸菌由来である請求項1または2記載のNK細胞活性化剤。   The NK cell activator according to claim 1 or 2, wherein the polysaccharide is derived from lactic acid bacteria. 請求項1〜3のいずれか1つに記載のNK細胞活性化剤を含み、NK細胞活性化作用を有するものであることを特徴とし、NK細胞活性化のために用いられる旨の表示を付した発酵乳。   It contains the NK cell activator according to any one of claims 1 to 3 and has an NK cell activation action, and is labeled for use for NK cell activation. Fermented milk.
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