WO2001088150A1 - Bacteries d'acide lactique probiotiques incapables d'utiliser le lactose - Google Patents

Bacteries d'acide lactique probiotiques incapables d'utiliser le lactose Download PDF

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Publication number
WO2001088150A1
WO2001088150A1 PCT/EP2001/004941 EP0104941W WO0188150A1 WO 2001088150 A1 WO2001088150 A1 WO 2001088150A1 EP 0104941 W EP0104941 W EP 0104941W WO 0188150 A1 WO0188150 A1 WO 0188150A1
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WO
WIPO (PCT)
Prior art keywords
lactic acid
lactose
acid bacterium
milk
probiotic
Prior art date
Application number
PCT/EP2001/004941
Other languages
English (en)
Inventor
Jacques Edouard Germond
Beat Mollet
Original Assignee
Societe Des Produits Nestle S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Societe Des Produits Nestle S.A. filed Critical Societe Des Produits Nestle S.A.
Priority to AU2001262250A priority Critical patent/AU2001262250A1/en
Publication of WO2001088150A1 publication Critical patent/WO2001088150A1/fr

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Classifications

    • 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
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • 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
    • 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
    • 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
    • C12R2001/23—Lactobacillus acidophilus

Definitions

  • the present invention pertains to a Lactic acid bacterium having probiotic properties and being deficient in using lactose as a carbon source for the preparation while still retaining the capability of translating ⁇ -galactosidase.
  • the present invention relates to the use of such jmicro-organisms for the preparation of a food product containing lactose.
  • the present invention also relates to food compositions containing such micro-organisms.
  • Micro-organisms producing lactic acid upon growth in a lactose containing medium have been known for long and are generically summarized under the group "Lactic acid bacteria”.
  • This group comprises micro-organisms exhibiting quite different phentoypic traits, such as Lactococci, Bifidobacteria, Lactobacilli, Pediococci etc..
  • Lactic acid bacteria have been utilized for different purposes such as for the preservation of food taking benefit of a low pH and the action of fermentation products generated during the fermentative activity thereof. Also for the direct preparation of a variety of different foodstuff such as cheese, yogurt and other fermented dairy products, use is made of such bacteria.
  • Lactobacillus GG (ATCC 53103), is described.
  • This micro-organism shall be employed in a method of preventing or treating food induced hypersensitivity reactions. To this end, it is administered to a recipient together with a food material that has been subjected to a hydrolysis treatment.
  • the Lactobacillus strain selected is described to exhibit a protease enzyme system, thus being capable to further hydrolyze the food stuff by means of proteases secreted by the specific Lactobacillus strain.
  • EP 0 577 903 a strain of Lactic acid bacteria is described that has the capability of displacing Heliobacter pylori, a micro-organism known to cause ulcer, on intestinal or gastric cells. This micro-organism is proposed to be included in an ingestable carrier to be used for the therapeutic or prophylactic treatment of an ulcer associated with the action of Heliobacter pylori.
  • the lactose fermentative activity of the micro-organisms is not desirable.
  • the microorganisms should assist in the degradation of lactose in the gut, when lactose containing food material is incorporated.
  • Lactic acid bacteria as such probiotic strains, that are deficient in using lactose as a carbon source and that are still capable to translate the ⁇ -galactosidase gene.
  • the present inventors have now found that when using probiotic Lactic acid bacteria that are deficient in using lactose as a carbon source, such that no lactic acid is produced, the acidification and maintaining quality of products to which such strains are added are not essentially changed.
  • the probiotic Lactic acid bacteria are preferably selected from the group comprising Lactobacillus johnsonii, L. gasseri, L. acidophilus, L. rhamnosus, L. casei, and L. reuteri.
  • the Lactic acid bacterium preferably has a mutation in the lac-operon such that lactose may not be utilized as the carbon source. This mutation may relate to a regulatory region, or gene or to one or more of the structural genes selected from, e.g. the permease gene.
  • the Lactic acid bacteria are CNCM - I 2369 and CNCM - 1 2474, which micro-organisms have been deposited according to the Budapest Treaty with the Pasteur Institute, Paris, France on December 17 th 1999 and May 9 th , 2000, respectively.
  • strains since the strains are still able to translate the ⁇ -galactosidase gene and produce the corresponding polypeptide they may assist in the digestion of lactose in the gut, since upon lysis of the micro-organisms in the gut the cellular material inclusive the ⁇ -galactosidase will be set free and will exert its enzymatic activity.
  • the probiotic strain will, however, not grow in the lactose containing food product, such as milk, yoghurt etc, such that the product may be stored in the refrigerator for the same time period as the food product without being complemented with the probiotic strain.
  • a probiotic Lactic acid bacteria strain may be constructed, that has a mutation in the lac-operon such that lactose may not be utilized as the carbon source but still translates the ⁇ -galactosidase gene.
  • the mutation lies within a structural gene, that is within the lactose operon, e.g. the permease, but it may well lie within a regulatory region of the Lac operon or a control gene outside of the Lac operon.
  • probiotic strains deficient in utilizing lactose as the carbon source may be effected by means of recombinant technology.
  • a structural gene selected from the permease gene that has been mutated such that the gene product thereof is not or is essentially not functional any more is inserted into the location of the natural structural gene, by means of e.g. homologous recombination, with the effect that lactose may not be utilized by the micro-organism any more.
  • Methods to genetically engineer Lactic acid bacteria are within the general knowledge of the skilled artisan.
  • strains are preferably produced by common methods, such as mutation and selection or by simply selecting for a spontaneous mutation exerting a selective pressure on a culture of Lactic acid bacteria.
  • the probiotic strains are the micro- organisms deposited under the accession no. CNCM 1-2369 and CNCM 1-2474. These strains have been derived from the lactic acid bacterium CNCM 1-1225, available from the Pasteur Institute, that has the capability to displace Heliobacter pylori from intestinal cells.
  • the products to which the strains described herein are to be added are preferably milk derived products, since milk contains mainly lactose as the carbon source for lactic acid bacteria.
  • value added products such as milk, yoghurt, curd, cheese, fermented milks, ice-cream etc. may be produced by simply adding the probiotic Lactic acid bacteria, deficient in using lactose as the carbon source to the product.
  • Natural mutants of Lai were prepared in a two step process. In a first step the bacterial strain was grown in a medium containing 1 % yeast extract, 1 % tryptone, 1 % lactose and different concentrations of penicillin (2.5 g/ml).
  • the stability was assessed on the basis of the natural mutants bSing unable to acidify the supplemented milk supplemented with yeast extract (0.2%)as above upon incubation, which can be visualized by the coagulation of casein micelles occurring at lower pH values.
  • the bacteria were further investigated whether they were derived from the original starter culture Lai. In this respect total DNA was isolated from both the mutant and the starter strain and were digested with the restriction enzyme Muni under the conditions recommended by the supplier (Boehringer, Mannheim).
  • Bacteria were grown in milk supplemented with 0.2% yeast extract in the presence or absence of 1 % glucose and incubated overnight. Growth of bacteria was determined by measurement of the pH of the fermented products (table 1).
  • NCC Internal reference (NCC) Accession no at the pasteur institute (CNCM) pH of fermented milk (pH), pH of fermented milk supplemented with 1 % glucose (pH glu); and ⁇ -galactosidase activity (OD 420 ) of Lai and its mutants.
  • the two mutants are unable to grow in milk. Hence, they can not ferment lactose, and have a lactose-negative phenotype. In the presence of glucose, bacteria can grow and growth is comparable to that of the wild type Lai .
  • the ⁇ -galactosidase activity was determined by incubating equivalent amounts of the bacteria (by optical density at 600nm) in a phosphate buffer (100mm sodium phosphate pH 7.0, 1 mM MgCl 2 ) in the presence of ONPG (0.5 mg/ml ortho-nitrophenyl- ⁇ -D- galactopyranoside) after permeabilisation with 0.1% Triton X-100 (30 min at room temperature). Although that both mutants have a Lac-minus phenotype, they are genetically different. NCC 2680 is unable to metabolise ONPG after permeabilisation. This result indicates that the mutation had affected the ⁇ -galactosidase gene or a region/gene directly regulating its expression.
  • UHT processed milk at 3.7% fat was inoculated with the mutants of Lai (CNCM 1-2369 or CNCM 1-2474) (2% in a volume of frozen concentrated culture).
  • the frozen concentrate contained 5x10 9 cells/ml.
  • the inoculated milk was mixed, distributed in 150ml plastic jars or tetra-pack type packaging and directly stored at temperature of 8°C.
  • a milk product was obtained which did not acidify during a shelf life of 14 days.
  • mutants described in the present specification are particularly suitable as an addition to milk products, since they impart the probiotic properties to said products, while not negatively affecting the shelf life of the resulting product.
  • the order number given by the CNCM is identical with the registration number and the date of the deposit is the date of the registration.

Abstract

L'invention concerne une bactérie d'acide lactique présentant, d'une part, des propriétés probiotiques et, d'autre part, une incapacité à utiliser le lactose, tel qu'une source de carbone à des fins de préparation, mais conservant une capacité à transformer la ß-galactosidase. Plus particulièrement, l'invention concerne l'utilisation de tels micro-organismes pour la préparation d'un produit alimentaire contenant du lactose. En outre, l'invention concerne des compositions alimentaires contenant de tels micro-organismes.
PCT/EP2001/004941 2000-05-15 2001-05-02 Bacteries d'acide lactique probiotiques incapables d'utiliser le lactose WO2001088150A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001262250A AU2001262250A1 (en) 2000-05-15 2001-05-02 Probiotic lactic acid bacteria, unable to utilize lactose

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP00110379 2000-05-15
EP00110379.5 2000-05-15

Publications (1)

Publication Number Publication Date
WO2001088150A1 true WO2001088150A1 (fr) 2001-11-22

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AU (1) AU2001262250A1 (fr)
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003086093A1 (fr) * 2002-04-12 2003-10-23 Meiji Dairies Corporation Fromage ayant un effet de desinfection contre l'helicobacter pylori
EP1820851A1 (fr) * 2006-02-20 2007-08-22 Compagnie Gervais Danone Souches de Lactobacillus helveticus ne fermentant pas le lactose
WO2010139333A1 (fr) * 2009-06-04 2010-12-09 Chr. Hansen A/S Prématuration de lait
CN102367428A (zh) * 2011-10-24 2012-03-07 甘肃农业大学 低温发酵的干酪乳杆菌及用其制备牦牛乳硬质干酪的方法
US8399238B2 (en) 2004-03-24 2013-03-19 Nestec S.A. Shelf-stable product with living micro-organisms
WO2019206754A1 (fr) * 2018-04-24 2019-10-31 Chr. Hansen A/S Composition et procédé destinés à la production d'un produit laitier fermenté comprenant l'application d'une souche s thermophilus déficiente en lactose, d'une souche l bulgaricus déficiente en lactose et d'une souche probiotique
JP7089818B1 (ja) 2022-01-11 2022-06-23 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳
EP3821712B1 (fr) 2017-01-13 2022-11-09 Chr. Hansen A/S Produit laitier fermenté obtenu par un procede ameliore
JP7190773B1 (ja) 2021-12-22 2022-12-16 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1990005459A1 (fr) * 1988-11-21 1990-05-31 Genencor, Inc. Production de produits alimentaires fermentes
US5198351A (en) * 1990-04-23 1993-03-30 The United States Of America As Represented By The Secretary Of Agriculture Lactose hydrolysis by mutant Streptococcus thermophilus

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1990005459A1 (fr) * 1988-11-21 1990-05-31 Genencor, Inc. Production de produits alimentaires fermentes
US5198351A (en) * 1990-04-23 1993-03-30 The United States Of America As Represented By The Secretary Of Agriculture Lactose hydrolysis by mutant Streptococcus thermophilus

Non-Patent Citations (2)

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Title
LIN MEEI-YN ET AL: "Management of lactose maldigestion by consuming milk containing lactobacilli.", DIGESTIVE DISEASES AND SCIENCES, vol. 43, no. 1, January 1998 (1998-01-01), pages 133 - 137, XP000960905, ISSN: 0163-2116 *
MUSTAPHA A ET AL: "IMPROVEMENT OF LACTOSE DIGESTION BY HUMANS FOLLOWING INGESTION OF UNFERMENTED ACIDOPHILUS MILK: INFLUENCE OF BILE SENSITIVITY, LACTOSE TRANSPORT, AND ACID TOLERANCE OF LACTOBACILLUS ACIDOPHILUS", JOURNAL OF DAIRY SCIENCE,US,AMERICAN DAIRY SCIENCE ASSOCIATION. CHAMPAIGN, ILLINOIS, vol. 80, no. 8, 1 August 1997 (1997-08-01), pages 1537 - 1545, XP000701948, ISSN: 0022-0302 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8758841B2 (en) 2002-04-12 2014-06-24 Meiji Co., Ltd. Cheese capable of disinfecting Helicobacter pylori
CN1305384C (zh) * 2002-04-12 2007-03-21 明治乳业株式会社 能够抵抗幽门螺旋杆菌的乳酪
KR101149285B1 (ko) 2002-04-12 2012-05-24 가부시키가이샤 메이지 헬리코박터 파일로리를 살균하는 치즈 첨가물
JP2005522202A (ja) * 2002-04-12 2005-07-28 明治乳業株式会社 Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ除菌性チーズ
WO2003086093A1 (fr) * 2002-04-12 2003-10-23 Meiji Dairies Corporation Fromage ayant un effet de desinfection contre l'helicobacter pylori
AU2003223118B2 (en) * 2002-04-12 2008-06-26 Meiji Dairies Corporation Cheese capable of disinfecting helicobacter pylori
US8399238B2 (en) 2004-03-24 2013-03-19 Nestec S.A. Shelf-stable product with living micro-organisms
WO2007096510A3 (fr) * 2006-02-20 2007-12-13 Gervais Danone Sa Souches de lactobacillus helveticus ne fermentant pas le lactose
US8323951B2 (en) 2006-02-20 2012-12-04 Compagnie Gervais Danone Strains of Lactobacillus helveticus which do not ferment lactose
EP1820851A1 (fr) * 2006-02-20 2007-08-22 Compagnie Gervais Danone Souches de Lactobacillus helveticus ne fermentant pas le lactose
WO2010139333A1 (fr) * 2009-06-04 2010-12-09 Chr. Hansen A/S Prématuration de lait
CN102367428A (zh) * 2011-10-24 2012-03-07 甘肃农业大学 低温发酵的干酪乳杆菌及用其制备牦牛乳硬质干酪的方法
CN102367428B (zh) * 2011-10-24 2012-10-03 甘肃农业大学 低温发酵的干酪乳杆菌及用其制备牦牛乳硬质干酪的方法
EP3821712B1 (fr) 2017-01-13 2022-11-09 Chr. Hansen A/S Produit laitier fermenté obtenu par un procede ameliore
WO2019206754A1 (fr) * 2018-04-24 2019-10-31 Chr. Hansen A/S Composition et procédé destinés à la production d'un produit laitier fermenté comprenant l'application d'une souche s thermophilus déficiente en lactose, d'une souche l bulgaricus déficiente en lactose et d'une souche probiotique
JP7190773B1 (ja) 2021-12-22 2022-12-16 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳
JP2023093232A (ja) * 2021-12-22 2023-07-04 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳
JP7089818B1 (ja) 2022-01-11 2022-06-23 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳
JP2023102164A (ja) * 2022-01-11 2023-07-24 オハヨー乳業株式会社 乳酸菌スターター及びそれを用いて得られる発酵乳

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