JP5891337B2 - ラクトバチルス属菌含有組成物 - Google Patents
ラクトバチルス属菌含有組成物 Download PDFInfo
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- JP5891337B2 JP5891337B2 JP2015512501A JP2015512501A JP5891337B2 JP 5891337 B2 JP5891337 B2 JP 5891337B2 JP 2015512501 A JP2015512501 A JP 2015512501A JP 2015512501 A JP2015512501 A JP 2015512501A JP 5891337 B2 JP5891337 B2 JP 5891337B2
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- intestinal tract
- bacteria
- lactic acid
- lactobacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A61K2035/11—Medicinal preparations comprising living procariotic cells
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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
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- Microbiology (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Food Science & Technology (AREA)
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- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Public Health (AREA)
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- Biochemistry (AREA)
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- Hematology (AREA)
- Diabetes (AREA)
- Child & Adolescent Psychology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明者らが保有する乳酸菌のうち、主に植物性乳酸菌である約480株(JCM株を含む)について、人工腸液における増殖能を評価した。
<人工腸液(pH6.45)>
MRS培地 9mL
10w/v%胆汁(和光純薬工業株式会社)溶液 1mL
1w/v%パンクレアチン(from Porcine:SIGMA) 100μL
なお、胆汁溶液及びパンクレアチン溶液は0.22μmのフィルター(PVDF membrane、Millipore社製)で処理することにより無菌にしたものを用いた。
高脂肪食を自由摂取させたマウスに以下のように調製したTUA4337L株を投与して、***される菌数を定量した。具体的には、以下のように調製した投与サンプルを用いて、乳酸菌約10億cells(1.0×109cells)分(菌体懸濁液250μLに相当)をC57BL/6Jマウス(10週齢、雄)に午前10時ごろに1回投与した(n=5)。
<投与サンプル(生菌含有サンプル)の調製>
〔1〕TUA4337L株のグリセロールストックから、MRS培地(30mL)へ1v/v%植菌
〔2〕培養(35℃、20hr)
〔3〕培養液を遠心(8000rpm、5min)して上清除去後、PBS(−)30mLで懸濁
〔4〕〔3〕の懸濁液を遠心(8000rpm、5min)して上清除去後、PBS(−)5mLで再懸濁
〔5〕顕微鏡で菌数カウント
〔6〕15mLの遠心管に200億cells分の溶液を分取し、遠心(8000rpm、5min)して上清除去後、液状飼料(高脂肪食 60kcal%FAT:Research Diet)5mLに懸濁して菌体懸濁液を調製(液状飼料はPBS(−)で調製)
<リアルタイムPCRによる菌数測定法>
〔1〕糞便100mg(湿重量)に対してPBS(−)1mLを添加後、スパチュラーを用いて解砕
〔2〕糞便100mg分をエッペンチューブ(登録商標)に採取後、遠心(15000rpm、5min)して上清除去後、PBS(−)1mLで懸濁(遠心から懸濁までの操作を2回繰り返す)
〔3〕〔2〕の懸濁液から上清除去後、キット(QIAamp DNA Stool Mini Kit:キアゲン)を用いてDNAを抽出
(細胞破砕は、Glass beads(150〜212μm:SIGMA)300mg、phenol/chloroform/isoamylalcohol(25:24:1)300μL、及びbuffer ASL(キット中の試薬)900μLを添加し、MULTI−BEADS SHOCKER MB−200(YASUI KIKAI)で3000rpm、1min遠心後、氷上1min静置の操作を3回繰り返すことで行う)
〔4〕下記に示す条件のリアルタイムPCRにより腸管内容物中の乳酸菌を定量
((リアルタイムPCR条件))
(1) SYBR Premix Ex Taq ll(Takara Bio)10μL、each primer(10μM)0.8μL、ROX reference DyeII 0.4μL、滅菌水6μL、DNA溶液2μLを混合してPCR反応液を調製する。プライマーはラクトバチルス・ペントーサス及びラクトバチルス・プランタラムの16S rDNAを特異的に検出する以下のプライマーを用いる(ラクトバチルス・ペントーサスとラクトバチルス・プランタラムの16S rDNAの配列は100%同一である)。
primer1:5’-GCAAGTCGAACGAACTCTGGTATT-3’(配列番号2)
primer2:5’-CGGACCATGCGGTCCAA-3’(配列番号3)
(2) PCRは7500 Real Time PCR System(Applied Biosystems)を用いて、95℃ 30秒の後、95℃ 5秒、60℃ 34秒を1サイクルとして、合計60サイクル反応を行い、得られた蛍光強度と腸管内容物合計量と希釈倍率から腸管内容物1g当りのコピー数を求める。
(3) 別途1cell当たりの16S rDNAのコピー数を求め、菌数に換算する。なお、乳酸菌を投与していないマウスにおいては、前記リアルタイムPCRによりラクトバチルス・ペントーサス及びラクトバチルス・プランタラムのいずれも検出されないことを確認する。
C57BL/6Jマウス(8週齢、雄)を、通常食群、高脂肪食群、高脂肪食+生菌群、高脂肪食+死菌群の4群に群分けし(各n=10)、それぞれに以下の表5に示す食餌を32日間続け、体重を毎日測定して平均値を算出した。平均値の推移を図2に示す。なお、群間比較は、有意水準0.05によるt検定を用いて行った。
〔1〕TUA4337L株のグリセロールストックから、MRS培地(30mL)へ1v/v%植菌
〔2〕培養(35℃、20hr)
〔3〕培養液を遠心(8000rpm、5min)して上清除去後、PBS(−)30mLで懸濁
〔4〕〔3〕の懸濁液を遠心(8000rpm、5min)して上清除去後、PBS(−)5mLで再懸濁
〔5〕顕微鏡で菌数カウント
〔6〕15mLの遠心管に200億cells分の溶液を分取し、遠心(8000rpm、5min)して上清除去後、人工胃液(125mM NaCl、7mM KCl、pH1.0)5mLを添加して攪拌後60min放置
〔7〕〔6〕の溶液を遠心(8000rpm、5min)して上清除去後、液状飼料(60kcal%FAT)5mLで懸濁して菌体懸濁液を調製
実施例3における通常食群、高脂肪食群、高脂肪食+生菌群の群構成で(各n=12)、それぞれに実施例3と同じ内容の食餌を2週間続けた。その後、一晩絶食させた後、オリーブオイル(nacalai tesque)を投与し(5mL/kg)、さらに3時間後に解剖して大静脈から血清を採取した。血清中のトリグリセライド(TG)をトリグリセライド E−テストワコー(和光純薬工業株式会社)を用いて測定した。結果を図3に示す。なお、群間比較は、有意水準0.05によるt検定による有意差判定を行った。
以下に示す方法により、TUA4337L株を含有する医薬品(錠剤)を製造する。
TUA4337L株の乾燥粉砕物66.7gを、乳糖232.0gおよびステアリン酸マグネシウム1.3gとともに混合し、単発式打錠機にて打錠することにより、直径10mm、重量300mgの錠剤を製造する。
牛乳、脱脂粉乳、水を混合したミックスを調製し、加熱殺菌した後、40℃前後まで冷却し、スターターとしてTUA4337L株を接種したのち、発酵室にて静置発酵させる。ここで、静置発酵の温度は、適宜選択できる。また、発酵開始時における溶存酸素濃度を抑えるため、窒素などの不活性ガスによる置換処理を行ってもよい。このようにして得られたTUA4337L発酵乳を、市販の牛乳に添加し3日間静置しヨーグルトを調製する。
TUA4337L株を用いて表6に示した原料を混合して乳酸菌飲料を調製する。
モモ果汁に、TUA4337Lを2重量%量を接種し、30℃で38時間培養してモモ発酵果汁を製造する。また、同様にニンジン汁を発酵させてニンジン発酵汁を製造する。
配列表の配列番号2は、ラクトバチルス・ペントーサス/プランタラム特異的プライマーの塩基配列である。
配列表の配列番号3は、ラクトバチルス・ペントーサス/プランタラム特異的プライマーの塩基配列である。
Claims (14)
- 腸管内での増殖能を有するラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)を含有する組成物。
- 腸管からの脂肪吸収抑制作用を有する請求項1記載の組成物。
- 高脂肪食による腸管での脂肪吸収を抑制する請求項1又は2記載の組成物。
- 食した場合、ラクトバチルス・ペントーサス TUA4337L株が腸管内で増殖し、それにより増殖能のない乳酸菌を食した場合と比べて高い脂肪吸収抑制作用を発揮する、請求項1〜3いずれか記載の組成物。
- 食した場合、ラクトバチルス・ペントーサス TUA4337L株が腸管内で増殖し、それにより増殖能のない乳酸菌を食した場合と比べて脂肪吸収抑制作用が持続する、請求項1〜4いずれか記載の組成物。
- 体重増加の抑制用又は体重低減用に供される、請求項1〜5いずれか記載の組成物。
- 肥満の防止用又は肥満改善用に供される、請求項1〜6いずれか記載の組成物。
- 高脂肪食と共に、あるいは高脂肪食を食する前に供される、請求項1〜7いずれか記載の組成物。
- 飲料又は食品である、請求項1〜8いずれか記載の組成物。
- ヨーグルトである、請求項1〜9いずれか記載の組成物。
- 食事由来の脂肪の腸管からの吸収抑制剤である、請求項1〜8いずれか記載の組成物。
- 1日1回、朝食時に食する、請求項1〜11いずれか記載の組成物。
- ダイエット用である、請求項1〜12いずれか記載の組成物。
- 脂肪吸収を抑制させる使用のための、ラクトバチルス・ペントーサス TUA4337L株(受託番号NITE BP-1479)を含有する組成物。
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CA2908032A1 (en) | 2013-04-17 | 2014-10-23 | Suntory Holdings Limited | Composition containing bacterium belonging to genus lactobacillus |
JP6645756B2 (ja) * | 2015-06-16 | 2020-02-14 | サントリーホールディングス株式会社 | 体温低下抑制剤 |
TWI645854B (zh) | 2017-03-20 | 2019-01-01 | 大江生醫股份有限公司 | 胚芽乳酸桿菌tci378及其於減脂及改善腸胃功能之應用 |
CN109207389B (zh) * | 2017-07-07 | 2023-06-06 | 西北民族大学 | 一种消栓降脂益生复合菌中药口服液及其制备方法 |
CN107929329B (zh) * | 2017-07-07 | 2023-06-06 | 西北民族大学 | 一种消栓降脂益生复合菌中药颗粒及其制备方法 |
JP7046549B2 (ja) * | 2017-09-28 | 2022-04-04 | サントリーホールディングス株式会社 | 乳酸菌入り飲料 |
TWI739078B (zh) * | 2018-04-25 | 2021-09-11 | 日商曾根農場股份有限公司 | 脂肪累積抑制用組成物 |
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CN105121627B (zh) | 2017-12-15 |
ES2684074T3 (es) | 2018-10-01 |
US20170112168A1 (en) | 2017-04-27 |
EP3018199B1 (en) | 2018-05-30 |
WO2014171478A1 (ja) | 2014-10-23 |
HK1213011A1 (zh) | 2016-06-24 |
CA2908032A1 (en) | 2014-10-23 |
AU2014254813B2 (en) | 2019-08-01 |
CN105121627A (zh) | 2015-12-02 |
EP3018199A1 (en) | 2016-05-11 |
KR20150143803A (ko) | 2015-12-23 |
AU2014254813A1 (en) | 2015-10-22 |
NZ712603A (en) | 2020-02-28 |
US9603880B2 (en) | 2017-03-28 |
US10159269B2 (en) | 2018-12-25 |
US20160058807A1 (en) | 2016-03-03 |
MY183171A (en) | 2021-02-18 |
JPWO2014171478A1 (ja) | 2017-02-23 |
EP3018199A4 (en) | 2016-11-30 |
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