JPWO2007013438A1 - 抗肥満剤および抗肥満食品 - Google Patents
抗肥満剤および抗肥満食品 Download PDFInfo
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- JPWO2007013438A1 JPWO2007013438A1 JP2007528471A JP2007528471A JPWO2007013438A1 JP WO2007013438 A1 JPWO2007013438 A1 JP WO2007013438A1 JP 2007528471 A JP2007528471 A JP 2007528471A JP 2007528471 A JP2007528471 A JP 2007528471A JP WO2007013438 A1 JPWO2007013438 A1 JP WO2007013438A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
Description
しかしながら、無理な食事制限で肥満を抑えようとすると、他の必要な栄養素の摂取量が不足したり、バランスを崩す結果、体に悪影響を与えることもあった。これは、いわゆるダイエット食品と呼ばれる、栄養素が少ないにもかかわらず満腹感を与える食品を摂取した場合にも同じことがいえる。
L.ロイテリ JCM1112Tの抗肥満効果試験:
下記の材料および方法により、L.ロイテリJCM1112Tの抗肥満効果を調べた。
(1)使用実験動物
SPFグレードのウイスター(Wistar)系ラット(8週齢−雄;日本SLC株式会社)の体重約180〜200gのものを使用し、実験室搬入日より7日間の馴致期間を設け、実験を開始した。飼育環境は、温度22±1℃、湿度55±5%、照明時間12時間(8時〜20時)に設定し、ラットは1匹/ゲージとし、滅菌蒸留水を給水瓶にて、またラット用放射線滅菌固形試料*(CE−2、日本クレア)を給餌器にて、それぞれ自由摂取とした。用いた飼育器具はすべて高圧蒸気滅菌器により滅菌したものを使用した。
* 飼育繁殖用(粗脂肪 4.6%)
被験菌として、L.ロイテリ JCM1112T(理化学研究所の標準菌であり、同所より分譲を受けた)およびL.ラムノーサス ATCC53103(GG株)を使用した。これら被験菌は、MRS液体培地(Oxid)に接種して37℃で一晩培養し、プレ培養液とした。このプレ培養液を1%量となるように新たなMRS液体培地に添加し、37℃で18時間培養したものを被験菌液とした。
前記実験動物を各被験菌投与群および対照群(1群は各5匹)に分け、上記被験菌液を被験菌投与群に経口投与により投与した。被験菌液の投与は、ゾンデを用い、強制的に行った。投与菌液は用時調製し、投与菌量はラット1匹当り109CFUとした。また、コントロール群には同容量のPBSを投与した。
実験動物の体重は毎日、体重計にて測定した。一般症状は毎日観察した。症状は個体別に記録し、顕著な変化がみられた症状項目を発現個体数で表した。
図2に、上記ラクトバチルス属細菌をそれぞれ投与したラット群および無投与のラット群について、経時的な体重の増加の様子を示す。この図から明らかなように、無投与群に比べ、L.ロイテリ JCM1112T投与群は、有意に体重の増加が抑制されており、この程度は、L.ラムノーサス ATCC53103投与群より大きかった。
L.ロイテリJCM1112Tのゲノム解析:
L.ロイテリJCM1112Tから、次の薬品を用い、以下の方法によりDNAを取得し、ゲノム解析を行った。
(薬 品)
・Nuclei Lysis solution(Wizard genome DNA purification kit;
Promega社製)
・生理食塩水
・50mM EDTA(pH7.0)
・50mg/ml リゾチーム溶液(使用当日に、所定量のリゾチームを、TE緩衝
液、0.25M Tris−HCl(pH8.0)又は10mM Tris−HCl(pH8.0)/
10mM EDTA/0.5%SDSで溶解し、調製する)
・2mg/ml EDTA
・フェノール・クロロホルム・イソアミルアルコール(25:24:1)混液
(以下、「PCI」と略す)
・クロロホルム・イソアミルアルコール(24:1)混液(以下、「CIA」と略す)
・99% エタノール
・70% エタノール
・TE 緩衝液
1.L.ロイテリJCM1112Tを、静置条件下、37℃で24時間培養し、得ら
れた培養液(50ml)を3,500r.p.mで、15分間遠心分離後、生理食塩
水で懸濁する。
2.1.で得られた懸濁液を3,500r.p.mで、15分間遠心分離し、上清を捨て
50mM EDTA 5mlで懸濁する。
3.2.で得られた懸濁液に、50mg/ml リゾチーム溶液を200μlを加え、
37℃で60分間インキュベートする。(この際、エアーインキュベーターを使用
する場合には、インキュベート時間を2−3時間とする。)
4.インキュベート後、3,500r.p.mで15分間遠心分離し、上清を捨てる。
5.4.で得た沈殿物に、Nuclei Lysis Solution 5mlを加え、80℃で10分間
インキュベートする。
6.2mg/ml RNase A 1μlを加え、37℃で45分間インキュベートする。
7.PCI 10mlを加え、混合し、3,500r.p.m.で15分間遠心分離する。
8.上清を新しいチューブに移し、さらにPCI 10mlを加え、混合する。
9.同様の操作を合計3回繰り返す。(タンパク質層が確認できなくなるまで行う)
10.CIA 10mlを加え、おだやかに混合後、3,500r.p.mで15分間遠心
分離する。(フェノールの除去)
11.上清を新しいチューブに移し、エタノール沈殿を行う。
12.11.で得た沈殿物を、TE緩衝液 1mlに溶解し、乳酸菌DNAを得る。
上記のようにして得たDNAについて、構造遺伝子予測とアノテーションを行った。この構造遺伝子予測等は、GENOMEGAMBLER(Sakiyama, T., Takami, H., Ogasawara, N., Kuhara, S., Kozuki, T., Doga, K., Ohyama, A., Horikoshi, K., An automated system for genome analysis to support microbial whole-genome shotgun sequencing., Biosci. Biotechnol. Biochem., 64:670-673. 2000.)、GLIMMER 2.0(Salzberg, SL., Delcher, AL., Kasif, S., and White, O., Microbial gene identification using interpolated Markov models., Nucleic. Acid. Res., 26:544-548. 1998.)およびBLAST program blastp (Altschul, SF., Gish, W,. Miller, W., Myers, EW., and Lipman, DJ., Basic local alignment search tool., J. Mol. Biol., 215:403-410. 1990.)の結果を併用して行った。
グリセロール分解遺伝子の増幅:
実施例2で精製されたDNAをテンプレート、次のヌクレオチド配列をプライマーとし、下記反応液を使用してPCRを行った。なお、PCR条件も下に示す。
pduCDE(F):
CACCATGAAACGTCAAAAACGATTT
pduCDE(R):
AAAAGCTTAGTTATCGCCCTTTAGC
テンプレートDNA: 1μl
KOD−plus : 1μl
10×KOD−plus緩衝液: 5μl
dNTP(各2mM) : 5μl
プライマー(20mm): 各1μl
MgSO4(25mm): 2μl
脱イオン水(D.W.): 34μl
(1)94℃で3分間保持する。
(2)94℃で15秒間保持する。
(3)56℃(Tm)で30秒間保持する。
(4)68℃で3分30秒間保持する。
(5)(2)ないし(4)を30サイクル行う。
(6)4℃で保存する。
L.ロイテリ由来リパーゼ遺伝子および接着性遺伝子の増幅:
プライマーを下記のものに代える以外は実施例3と同様にしてPCRを行ない、リパーゼ遺伝子および接着性遺伝子を増幅した。
リパーゼ(1)
5’-ATGGTGAAATTGATGACAAT
5’-TTATTTAAATTGATCGCCAA
リパーゼ(2)
5’-TTGATTTATGTTTTAAAAGA
5’-CTATGACCGAGTTAAATACT
リパーゼ(3)
5’-ATGGAAATTAAAAGTGTTAA
5’-CTAAATTAAATTCAGTTCAG
接着性遺伝子
5’-ATGTTCGGTCACGATGGCCG
5’-TCAAATTTCAGAAGGATCAT
Claims (60)
- ラクトバチルス・ロイテリに属する微生物が、ラクトバチルス・ロイテリ JCM1112Tである請求項第1項ないし第11項の何れかの項記載の抗肥満剤。
- ラクトバチルス・ロイテリに属する微生物が、ラクトバチルス・ロイテリ JCM1112Tである請求項第13項ないし第23項の何れかの項記載の抗肥満飲食品。
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EP (2) | EP2604689B1 (ja) |
JP (2) | JP5144263B2 (ja) |
KR (3) | KR101411144B1 (ja) |
CN (3) | CN101370729B (ja) |
ES (1) | ES2532481T3 (ja) |
PL (1) | PL2604689T3 (ja) |
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2006
- 2006-07-25 PT PT12189236T patent/PT2604689E/pt unknown
- 2006-07-25 WO PCT/JP2006/314640 patent/WO2007013438A1/ja active Application Filing
- 2006-07-25 KR KR1020087003891A patent/KR101411144B1/ko not_active IP Right Cessation
- 2006-07-25 CN CN2006800527329A patent/CN101370729B/zh not_active Expired - Fee Related
- 2006-07-25 CN CN201110150966XA patent/CN102225078B/zh active Active
- 2006-07-25 KR KR1020117015610A patent/KR20110095929A/ko not_active Application Discontinuation
- 2006-07-25 KR KR1020147004764A patent/KR20140043145A/ko not_active Application Discontinuation
- 2006-07-25 EP EP12189236.8A patent/EP2604689B1/en active Active
- 2006-07-25 CN CN2006800273816A patent/CN101233231B/zh not_active Expired - Fee Related
- 2006-07-25 US US11/997,034 patent/US20100216212A1/en not_active Abandoned
- 2006-07-25 PL PL12189236T patent/PL2604689T3/pl unknown
- 2006-07-25 EP EP06781552.2A patent/EP1918373B1/en not_active Not-in-force
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2011
- 2011-05-23 JP JP2011114716A patent/JP2011206057A/ja active Pending
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Also Published As
Publication number | Publication date |
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KR20140043145A (ko) | 2014-04-08 |
EP1918373B1 (en) | 2014-08-20 |
US8637001B2 (en) | 2014-01-28 |
KR20080034474A (ko) | 2008-04-21 |
PT2604689E (pt) | 2015-03-09 |
CN101370729A (zh) | 2009-02-18 |
US20110311501A1 (en) | 2011-12-22 |
JP5144263B2 (ja) | 2013-02-13 |
KR101411144B1 (ko) | 2014-06-26 |
ES2532481T3 (es) | 2015-03-27 |
US20120027736A1 (en) | 2012-02-02 |
CN101233231A (zh) | 2008-07-30 |
WO2007013438A1 (ja) | 2007-02-01 |
US8637000B2 (en) | 2014-01-28 |
CN102225078A (zh) | 2011-10-26 |
PL2604689T3 (pl) | 2015-08-31 |
CN102225078B (zh) | 2012-12-26 |
EP1918373A4 (en) | 2009-02-25 |
CN101370729B (zh) | 2012-08-29 |
US20100216212A1 (en) | 2010-08-26 |
EP1918373A1 (en) | 2008-05-07 |
JP2011206057A (ja) | 2011-10-20 |
CN101233231B (zh) | 2011-06-29 |
KR20110095929A (ko) | 2011-08-25 |
EP2604689B1 (en) | 2015-01-21 |
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