JP6849890B2 - デオキシリボ核酸保存用組成物およびその製造方法、デオキシリボ核酸を保存する方法、乳酸菌産生物およびその使用方法 - Google Patents
デオキシリボ核酸保存用組成物およびその製造方法、デオキシリボ核酸を保存する方法、乳酸菌産生物およびその使用方法 Download PDFInfo
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Description
本発明の実施の形態において、多糖体含有乳酸菌産生物には、多糖体を含有する乳酸菌発酵物(以下「多糖体含有乳酸菌発酵物」という。)、多糖体を含有する乳酸菌培養物(以下「多糖体含有乳酸菌培養物」という。)、多糖体を含有する乳酸菌代謝物等が含まれる。
本発明の実施の形態に係るDNA保存用組成物は、医薬品,サプリメントおよび食品添加剤等の製剤、飲食品(動植物そのものを除く。)ならびに飲食品組成物(加工された飲食品を含む。)等の形態を採り得る。
本発明の実施の形態に係るDNA保存用組成物は、多糖体の摂取量が200μg以上/日となるように、少なくとも7日間、経口摂取することが好ましい。このDNA保存用組成物をこのように摂取することによって、DNAの損傷を有効に抑制することができると共に、DNAの損傷修復を有効に促進させることができる。
(人体への必要投与用量(換算値))=(動物への必要投与用量)×(女性体重下限値:40kg)÷(安全係数:100)
以下、実験例を示して本発明をより詳細に説明する。なお、本発明は、以下の実験例に限定されることはない。
本実験例では、紫外線の照射下における皮膚DNA損傷および皮膚DNA損傷修復に対する多糖体の影響を検証した。具体的には、ヘアレスマウスに紫外線を照射することによって皮膚のDNAを損傷させ、この状態のヘアレスマウスに対する多糖体の影響を検証した。
10質量%の脱脂粉乳を含む培地にラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus)OLL1247菌およびストレプトコッカス・サーモフィラス(Streptococcus thermophilus)OLS3078菌を接種した後、その培地を43℃に保ちながら3時間発酵させた。このようにして得られたヨーグルトには、多糖体が110μg/g含まれていた。
ヨーグルトの一部を分取し、その上清に3倍量のエタノールを添加して冷凍保管した。その後、その上清を遠心分離処理したところ、沈殿物が得られた。そして、この沈殿物を凍結乾燥して多糖体抽出物を得た。なお、11.3gのヨーグルト中に70mgの多糖体抽出物が含まれていた。
72匹のヘアレスマウス(Hos:HR−1,雌,8週齢,日本エスエルシー株式会社)を4日間馴化させた後、それらのヘアレスマウスを8匹ずつの群(紫外線照射前水群、紫外線照射前ヨーグルト群、紫外線照射前多糖抽出物群、紫外線照射後24時間水群、紫外線照射後24時間ヨーグルト群、紫外線照射後24時間多糖体抽出物群、紫外線照射後48時間水群、紫外線照射後48時間ヨーグルト群、紫外線照射後48時間多糖体抽出物群)に分けて、各群のヘアレスマウスに水、ヨーグルトおよび多糖体抽出物(以下これらをまとめて「試料」と称することがある。)をそれぞれ皮膚採取の前日まで経口投与した。なお、ヨーグルトは11.3g/kg体重/日で投与し、多糖体抽出物群は70mg/kg体重/日で投与した。そして、各試料投与開始から7日経過後に紫外線照射前水群、紫外線照射前ヨーグルト群、紫外線照射前多糖抽出物群のヘアレスマウスの背部皮膚をイソフルラン麻酔下採取した。また、各試料投与開始から7日経過後に残りの群のヘアレスマウスの背部に、0.4mW/cm2の照度で紫外線を50秒間照射した(なお、紫外線照射装置として三共電気株式会社製のGL20SE(波長領域:280nmから400nm,ピーク波長:306nm)を用いた。このときの紫外線量は20mJ/cm2(=0.4mW/cm2×50秒)である。)。そして、この紫外線照射から24時間経過時および72時間経過時に各群のヘアレスマウスの背部皮膚をイソフルラン麻酔下採取した。なお、以下、説明の便宜上、水を投与したヘアレスマウス群(比較例に相当)を「コントロール群」と称し、ヨーグルトを11.3g/kg体重/日で経口投与したヘアレスマウス群(実施例に相当)を「ヨーグルト群」と称し、多糖体抽出物を70mg/kg体重/日で経口投与したヘアレスマウス群(実施例に相当)を「多糖体抽出物群」と称する。
(4−1)紫外線によるDNA損傷の抑制効果の検証
上述の通りに採取した各群のヘアレスマウスの背部皮膚からquagen QIAamp DNA Mini Kit(キアゲン社製)を用いてDNAを採取して精製した。紫外線によるDNA損傷マーカーであるシクロブタンピリミジンダイマーの存在量をELISAキット(High Sensitivity CPD ELISA kit Ver.2, コスモバイオ社製)を用いて測定した。なお、標準品として、孔牛胸腺のDNAを0、2.5、5.0、7.5、10J/m2の紫外線量(紫外線波長は主に254nmであった。)で照射したDNAサンプル(UVC-irradiated DNA samples,コスモバイオ社製)を用いた。
上述の通りに採取した各群のヘアレスマウスの背部皮膚からqiagen QIAamp DNA Mini Kit(キアゲン社製)を用いてDNAを採取して精製した。そして、DNase I、alkaline phosphatase、phosphodiesterase (8−OHdG Assay Preparation Reagent Set,和光純薬工業社製)を用いてこのDNAを分解した。その後、DNA損傷マーカーである8−オキソ−2’−デオキシグアノシン(8−OHdG)の存在量を定量した。
・Gas2:80psi
・ion spray voltage(IS):5500V
・turbo heater temperature(TEM):500℃
・entrance potential(EP):10V
・collision activation dissociation(CAD):8psi
・curtain gas(CUR):30psi
・declustering potential(DP):55V
・collision energy(CE):18V
・cell exit potential(CXP):7V
上述の通りに採取した各群のヘアレスマウスの背部皮膚からRNeasy Mini Kit(キアゲン社製)を用いてTotal RNAを抽出した。その後、抽出したTotal RNAからRivertAid First Strand cDNA Synthesis Kit(サーモフィッシャー社製)を用いてcDNAを合成した。そして、ABI 7500 Fast Realtime PCR system(アプライドバイオシステムズ社製)を用いて、そのcDNAおよび内在性遺伝子としてのGAPDHからカタラーゼのmRNAの相対量を定量した。
上述の通りに採取した各群のヘアレスマウスの背部皮膚からRNeasy Mini Kit(キアゲン社製)を用いてTotal RNAを抽出した。その後、抽出したTotal RNAからRivertAid First Strand cDNA Synthesis Kit(サーモフィッシャー社製)を用いてcDNAを合成した。そいsて、ABI 7500 Fast Realtime PCR system(アプライドバイオシステムズ社製)を用いて、そのcDNAおよび内在性遺伝子としてのGAPDHからxeroderma pigmentosum complementation group A(XPA)のmRNAの相対量を定量した。
NITE BP−01814
Claims (7)
- ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスOLL1247菌(受託番号:NITE BP−01814)により乳を発酵させることによって得られる多糖体を含有する乳酸菌産生物を有効成分とするデオキシリボ核酸保存用組成物。
- 前記乳酸菌産生物は、ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスOLL1247菌(受託番号:NITE BP−01814)とストレプトコッカス・サーモフィラス(Streptococcus thermophilus)との組合せによって生成される
請求項1に記載のデオキシリボ核酸保存用組成物。 - 前記ストレプトコッカス・サーモフィラスは、ストレプトコッカス・サーモフィラスOLS3078菌(受託番号:NITE BP−01697)である
請求項2に記載のデオキシリボ核酸保存用組成物。 - ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスOLL1247菌(受託番号:NITE BP−01814)により乳を発酵させることによって得られる多糖体を含有する乳酸菌産生物をデオキシリボ核酸保存用組成物として使用する方法(ただし、人を治療する行為を除く。)。
- 請求項1から3のいずれかに記載のデオキシリボ核酸保存用組成物を経口投与させることによりデオキシリボ核酸を保存させる方法(ただし、人を治療する行為を除く。)。
- 前記デオキシリボ核酸保存用組成物を、前記多糖体の摂取量が200μg以上/日となるように、少なくとも7日間、経口投与することによりデオキシリボ核酸を保存する、請求項5に記載の方法。
- ラクトバチルス・デルブルッキー・サブスピーシーズ・ブルガリクスOLL1247菌(受託番号:NITE BP−01814)で乳原料を発酵することによりデオキシリボ核酸保存用発酵乳を製造する方法。
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JP2016141114A JP6849890B2 (ja) | 2016-07-19 | 2016-07-19 | デオキシリボ核酸保存用組成物およびその製造方法、デオキシリボ核酸を保存する方法、乳酸菌産生物およびその使用方法 |
PCT/JP2017/025335 WO2018016393A1 (ja) | 2016-07-19 | 2017-07-12 | デオキシリボ核酸保存用組成物およびその製造方法、デオキシリボ核酸を保存する方法、乳酸菌産生物およびその使用方法 |
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