ES2611028T3 - Lactobacillus brevis productor de reuterina - Google Patents

Lactobacillus brevis productor de reuterina Download PDF

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ES2611028T3
ES2611028T3 ES11799493.9T ES11799493T ES2611028T3 ES 2611028 T3 ES2611028 T3 ES 2611028T3 ES 11799493 T ES11799493 T ES 11799493T ES 2611028 T3 ES2611028 T3 ES 2611028T3
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pylori
reuterine
lactobacillus brevis
strain
producer
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Peggy Garault
Gaëlle QUERE
Raphaelle Bourdet-Sicard
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Gervais Danone SA
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Gervais Danone SA
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • 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
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/121—Brevis
    • 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
    • C12R2001/24—Lactobacillus brevis

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Dairy Products (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

Una cepa acumuladora de reuterina de Lactobacillus brevis que es la cepa CNCM I-4431 de Lactobacillus brevis.

Description

imagen1
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Ejemplo 3: Efecto de L. Brevis CNCM I-4431 sobre la infección por H. Pylori en un modelo con C. Elegans
Cultivos de L. brevis y H. pylori
Se cultivó la cepa L. brevis CNCM I-4431 en Caldo MRS como se describió en el Ejemplo 1.
Se cultivó la cepa NCTC11637 (ATCC 43504T) de H. pylori en medio BHI (Oxoid) que contenía sangre de caballo al 5% (v/v) (Oxoid).
De las células se separaron los sobrenadantes por centrifugación y se neutralizaron con NaOH (1M). A continuación, los sobrenadantes se filtraron (0,22 µm) y se añadieron al sedimento (“pellet”) celular.
En el caso de los cultivos de H. pylori, se separó por centrifugación el medio BHI que contenía sangre de caballo (Oxoid) y las células se lavaron con solución salina.
Las placas de agar NG usadas para los ensayos de infección de C. elegans se prepararon por adición de 100 µL de una mezcla que contenía cada cultivo LAB a D.O.= 2,22 (incluyendo las células y el sobrenadante) con las correspondientes células de la cepa de H. pylori (D.O.= 0,022). En la condición de referencia, en las que los nematodos no se alimentan con LAB, las células de H. pylori se volvieron a poner en suspensión en solución salina y se añadieron a las placas.
Ensayos de infección de C. elegans.
Los ensayos de infección se realizaron con la cepa de tipo silvestre de C. elegans (N2). Se empleó la cepa NCTC11637 de H. pylori para la infección de nematodos. Los experimentos se iniciaron con adultos jóvenes sincronizados (nematodos de tres días) que se transfirieron a las diferentes condiciones de cultivo:
-medio NG + E. coli OP50 (control),
-medio NG + E. coli OP50 + 100 µL de medios de cultivo LAB (control de los medios de cultivo),
-medio NG + E. coli OP50 + 100 µL (H. pylori NCTC 11637T + LAB).
Los gusanos se incubaron a 25 ºC durante 8 días, transfiriéndolos a placas nuevas cada dos días. Se anotó la supervivencia de la población de nematodos en cada condición de cultivo. Después del período de incubación se tomaron muestras de 15 gusanos por condición, lavándolas con solución tampón M9 que contenía Triton X-100 al 0,1 %. Los gusanos se rompieron usando 0,4 g de perlas de carburo de silicio y agitando en vórtex.
El ADN de los gusanos infectados se aisló con un equipo comercial “High Pure PCR Template Preparation Kit” (Roche), seguido de una precipitación posterior y un proceso de lavado con etanol absoluto y acetato de potasio 5M. Se usaron diluciones de muestras de ADN para cuantificar, por RT-PCR, la presencia de H. pylori en las muestras.
RT-PCR
Los cebadores usados en este estudio están dirigidos al gen vacA (Nayak y Rose, J Appl Microbiol, 103, 1.931-41, 2007). Se denominan HpylF (5’ CAA TAG CAA TCA AGT GGC TTT G 3’, SEQ ID NO: 5) y HpylR (5’ GCG CGC TTC CAC ATT AGC 3’; SEQ ID NO: 6). La especificidad se comprobó previamente in silico mediante la herramienta en línea BLAST e, in vitro, mediante Q-PCR con cepas de la especie H. pylori, diferentes cepas probióticas y E. coli OP50 usadas para alimentar a C. elegans (véase el informe previo sobre optimización). Se utilizó la Mezcla Maestra de PCR SYBR® Green (Applied Biosystem) y el termociclador en Tiempo Real StepOne (Applied Biosystem). La siguiente Tabla 2 resume las condiciones optimizadas para la reacción Q-PCR sobre H. pylori.
7
Tabla 2.
Mezcla de reacción
Mezcla maestra 2x
1x
cebador directo (“For”): 325 nM
cebador inverso (“Rev”): 325 nM
Ciclado térmico
Etapa de mantenimiento
50 ºC 2 min
95 ºC
10 min
Etapa de ciclado (x40)
95 ºC 15 s
60 ºC
1 min
El ADN para la curva patrón se preparó mediante la cuantificación del ADN de H. pylori por espectrofotometría (A260). El número de los correspondientes genomas se calculó como siguiente: 5 -Número de Genoma = ADN (g) x NA/Mm -Tamaño del genoma de H. Pylori =1,6 Mpb -NA= 6,023 x 1023 -Número de Genoma = ADN (g) x 6,023 x 1023/1,6 x 106 x 2 x 309 La curva patrón se realizó usando cuatro diluciones 1/10 de ADN.
10 Los resultados obtenidos con la cepa I-4431 cultivada en MRS o cultivada en MRS + glicerol se muestran en la Figura 2. Estos resultados muestran que la cepa CNCM I-4431 reduce la carga de H. pylori NCTC 11637T en C. elegans. La reducción de la infección es más importante cuando la cepa I-4431 se cultiva en MRS + glicerol, lo que demuestra que al menos parte del efecto antiinfeccioso de esta cepa es resultado de la producción de reuterina.
15
8

Claims (1)

  1. imagen1
ES11799493.9T 2011-11-30 2011-11-30 Lactobacillus brevis productor de reuterina Active ES2611028T3 (es)

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Application Number Priority Date Filing Date Title
PCT/IB2011/055391 WO2013079992A1 (en) 2011-11-30 2011-11-30 Reuterin-producing lactobacillus brevis

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CN107739747B (zh) * 2017-09-27 2020-06-02 浙江工商大学 一种利用罗伊氏乳杆菌与醋杆菌共发酵高密度生产罗伊特林素的方法
KR102101692B1 (ko) * 2018-03-05 2020-04-20 주식회사 엠디헬스케어 락토바실러스 속 세균 유래 나노소포 및 이의 용도
CN108741090A (zh) * 2018-08-28 2018-11-06 安徽谷益生物科技有限公司 一种抑制幽门螺旋杆菌的复合益生菌功能性食品
CN114032230B (zh) * 2021-12-29 2023-07-04 安徽省农业科学院农业工程研究所 一种细菌甘油脱水酶突变体Gt9及其表达菌株的构建和应用

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ATE332649T1 (de) * 1987-05-01 2006-08-15 Biogaia Biolog Ab Reuterin enthaltende nährungszusammensetzung
JP4021951B2 (ja) * 1996-03-01 2007-12-12 わかもと製薬株式会社 乳酸菌を有効成分とする抗胃炎剤、抗潰瘍剤および醗酵食品
IT1306716B1 (it) * 1999-06-21 2001-10-02 Mendes S U R L Associazione di batteri lattici e suo uso per la prevenzione e/o iltrattamento terapeutico di infezioni e di stati infiammatori.
US7105336B2 (en) * 2002-10-07 2006-09-12 Biogaia Ab Selection and use of lactic acid bacteria for reducing inflammation caused by Helicobacter
JP2005304362A (ja) * 2004-04-20 2005-11-04 Nippon Shokubai Co Ltd １，３−プロパンジオール及び／又は３−ヒドロキシプロピオン酸を製造する方法
EP1731604A4 (en) * 2004-03-26 2007-04-04 Nippon Catalytic Chem Ind PROCESS FOR THE PREPARATION OF 1,3-PROPANEL AND / OR 3-HYDROXYPROPIONIC ACID
JP2005278414A (ja) * 2004-03-26 2005-10-13 Nippon Shokubai Co Ltd １，３−プロパンジオール及び３−ヒドロキシプロピオン酸を製造する方法
JP2007082476A (ja) * 2005-09-22 2007-04-05 Nippon Shokubai Co Ltd グリセリンから３−ヒドロキシプロピオン酸を製造する方法
US20080254011A1 (en) * 2007-04-11 2008-10-16 Peter Rothschild Use of selected lactic acid bacteria for reducing atherosclerosis
JP2010183858A (ja) * 2009-02-10 2010-08-26 Nippon Shokubai Co Ltd 遺伝子組み換え微生物及びこれを用いた３−ヒドロキシプロピオン酸の製造方法

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JP2015500016A (ja) 2015-01-05
US9387229B2 (en) 2016-07-12
EP2785878A1 (en) 2014-10-08
AR089034A1 (es) 2014-07-23
PL2785878T3 (pl) 2017-04-28
JP5868519B2 (ja) 2016-02-24
CN104053766A (zh) 2014-09-17
MX2014006556A (es) 2014-10-24
WO2013079992A1 (en) 2013-06-06
RU2577994C1 (ru) 2016-03-20
MX349944B (es) 2017-08-21
BR112014012887A2 (pt) 2020-10-20
CN104053766B (zh) 2016-05-04
US20140341855A1 (en) 2014-11-20
EP2785878B1 (en) 2016-08-31

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