WO2000077186A2 - Protection bacterienne - Google Patents

Protection bacterienne Download PDF

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Publication number
WO2000077186A2
WO2000077186A2 PCT/EP2000/005403 EP0005403W WO0077186A2 WO 2000077186 A2 WO2000077186 A2 WO 2000077186A2 EP 0005403 W EP0005403 W EP 0005403W WO 0077186 A2 WO0077186 A2 WO 0077186A2
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WO
WIPO (PCT)
Prior art keywords
stress
cells
bacterial cell
lethal
bacteria
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Ceased
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PCT/EP2000/005403
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English (en)
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WO2000077186A3 (fr
Inventor
Gudrun Schmidt
Ralf Zink
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Societe des Produits Nestle SA
Nestle SA
Original Assignee
Societe des Produits Nestle SA
Nestle SA
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.)
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Publication date
Application filed by Societe des Produits Nestle SA, Nestle SA filed Critical Societe des Produits Nestle SA
Priority to JP2001503631A priority Critical patent/JP3774146B2/ja
Priority to EP00949176A priority patent/EP1190038A2/fr
Priority to BR0011742-0A priority patent/BR0011742A/pt
Priority to NZ515765A priority patent/NZ515765A/en
Priority to AU62629/00A priority patent/AU771519B2/en
Priority to US10/018,492 priority patent/US6939705B1/en
Publication of WO2000077186A2 publication Critical patent/WO2000077186A2/fr
Publication of WO2000077186A3 publication Critical patent/WO2000077186A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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; 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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; 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

Definitions

  • the present invention relates to a bacterial cell having protection against stress including the affects of extreme temperature change and osmotic shock; a nutritive or medicinal composition comprising the protected bacterial cell; and a method of protecting bacteria against stress.
  • the word “comprises” is taken to mean “includes, among other things”. It is not intended to be construed as “consists of only”.
  • stress is used interchangeably with the term “adverse conditions”. It includes, but is not limited to, adverse conditions of temperature (heat shock, cold shock), salt (osmotic shock), pH (pH shock), chemical stresses (antibiotics, alcohol, H202, etc.), nutritional stress, UV-stress, cold stress and oxygen concentration (oxidative stress).
  • Standard amino acid, RNA and DNA codes are used within this specification which are defined by the IUB Biochemical Nomenclature Commission.
  • bacteria such as lactic acid bacteria (LAB) are ubiquitously found in the environment and they are largely used for the production of fermented products.
  • bacteria are used in fermentation of milk products and production of starter cultures.
  • the bacteria that are employed must deal with different kinds of adverse conditions which generally have the effect of dramatically reducing their viability, stability and activity.
  • adverse conditions vary with production requirements and include thermal shock (freeze-drying or spray-drying), osmotic shock (drying) and pH shock (fermentation). It will be appreciated that the susceptibility or inability of bacteria to cope with these stresses is a problem in cases where bacteria are used on a large scale.
  • Bifidobacteria or lactobacilli in the human intestine, primarily the small and large intestine, is generally accepted as a contributing factor for a healthy well-being.
  • Bifidobacteria and lactobacilli may be useful in prophylaxis or treatment of ailments including gastrointestinal infections.
  • large populations of Bifidobacteria and lactobacilli in the intestine should be maintained and products comprising the bacteria should be administered. Often these products comprise different species of Bifidobacteria or lactobacilli.
  • the stresses that Bifidobacteria and lactobacilli are exposed to during manufacture and storage of the products can significantly reduce their viability and/or physiological activity.
  • stress-proteins The natural response by bacterial cultures to sublethal temperature shifts or other sublethal stresses (including exposure to oxygen and osmotic shock) includes rapid expression of a distinct set of polypeptides called "stress-proteins". These proteins have been shown to enable Gram-positive bacteria such as for example Lactococcus lactis, Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus sakei, Enterococcus faecalis, and Lactobacillus johnsonii to adapt to otherwise growth-limiting conditions.
  • the heat shock proteins or chaperones are generally involved in the maturation of newly synthesised proteins, and they assist in refolding of denatured proteins.
  • Numerous stress- response genes have been characterised in LAB, including those encoding the two major chaperone machines (groES/groEL and hrcA/g E/dnaK/dnaJ) involved in the proper folding of newly synthesised proteins and the repair of those that are denatured.
  • bacteria including Bifidobacteria and lactobacilli
  • levels of stress that are lethal in unprotected bacteria.
  • this can be done by subjecting the bacteria to a sublethal level of stress treatment.
  • a higher level of stress is required to adversely affect the bacteria. This is unexpected because it was thought that cells which are damaged by stress would be less likely to cope with additional stress. In fact, the converse has been found - pre-stressed cells are able to bear a higher stress level compared to control cells which have not been pre-stressed.
  • cross-protection Protection against one form of stress acquired by treatment with a dissimilar form of stress has been referred to as "cross-protection”. This is unexpected because it was thought that cells damaged by treatment with one stress should render them more sensitive against an additional sublethal or lethal stress.
  • the invention provides a bacterial cell having protection against conditions which are lethal to an unprotected bacterial cell wherein, the protected cell is obtained by subjecting a bacterial cell to treatment with a sublethal level of stress.
  • the invention provides a nutritive composition which comprises bacteria having protection against conditions which are lethal to unprotected bacteria wherein, the protected bacteria are obtained by subjecting bacteria to treatment with a sublethal level of stress and allowing them to recover.
  • the invention provides a method of protecting a bacterial cell against stress which comprises the steps of treating a bacterial cell with a sublethal level of stress selected from the group which comprises thermal shock, osmotic shock, pH shock, oxidative stress, chemical stress, nutritional stress, UV-stress, cold stress.
  • a sublethal level of stress selected from the group which comprises thermal shock, osmotic shock, pH shock, oxidative stress, chemical stress, nutritional stress, UV-stress, cold stress.
  • the method includes the additional step of allowing the cell to recover.
  • chemical stress is provided by treatment with antibiotics, alcohol or H 2 0 2 .
  • the bacterial cell is selected from the group which comprises Bifidobacteria, lactic acid bacteria, enterococci, streptomyces, and bacilli.
  • the bacterial cell is Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve or Lactobacillus johnsonii.
  • An advantage provided by these bacteria is that they have the ability to rapidly acidify their substrate, therefore producing microbiologically safe products. In addition they contribute to a healthy well-being in humans and animals. Furthermore, they display a protective role against attack by enteric pathogens and are associated with anti-carcinogenic, anti-mutagenic and anti-tumorgenic activities. Without wishing to be bound by theory, recent reports suggest that they might act directly in the intestinal tract through antimicrobial activity, indirectly through immunomodulation via intestinal cells or by modifying the function of the normal indigenous microflora.
  • bacteria more preferably Bifidobacteria and lactobacilli
  • the cells are treated with sublethal thermal stress to protect them against otherwise lethal temperatures.
  • treatment with salt e.g. NaCl
  • the invention alternatively includes the steps of treating cells with salt to protect against thermal stress or treating the cells with adverse temperature conditions to protect against salt stress.
  • the bacterial cells are selected from Bifidobacterium longum,
  • Bifidobacterium adolescentis or Lactobacillus johnsonii More preferably the bacterial cells are selected from Bifidobacterium longum NCC481, Bifidobacterium adolescentis NCC251 or Lactobacillus johnsonii Lai .
  • protection against lethal salt concentrations eg of between 0.1% and 0.1%
  • the salt is bile salt.
  • protection against lethal thermal stress eg of between about 50°C to about 60°C
  • protection against freeze-thawing is carried out by treatment of the cells with salt concentration of between 1% and 4%.
  • Bifidobacterium longum NCC481 cells are protected. More preferably, protection of Bifidobacterium longum NCC481 cells is carried out in the logarithmic phase of their growth cycle against lethal bile salt concentrations (eg of between about 0.2% and about 0.3% for 30 min) by subjecting the cells to about 0.1% bile salt for about 30 min before lethal challenge. Preferably, protection of Bifidobacterium longum NCC481 cells is carried out in the stationary phase of their growth cycle against lethal bile salt concentrations (eg of about 0.075% and about 0.15% for about 30 min) by treatment of the cells with about 0.05% bile salt for about 30 min before lethal challenge.
  • lethal bile salt concentrations eg of between about 0.2% and about 0.3% for 30 min
  • lethal bile salt concentrations eg of about 0.075% and about 0.15% for about 30 min
  • Bifidobacterium adolescentis NCC251 cells are protected. More preferably, protection of Bifidobacterium adolescentis NCC251 cells is carried out in the logarithmic phase of their growth cycle against lethal bile salt concentrations (eg of between about 0.3% and about 0.4% for about 30 min) by subjecting the cells to about 0.1% bile salt for about 30 min before lethal challenge.
  • lethal bile salt concentrations eg of between about 0.3% and about 0.4% for about 30 min
  • protection of Bifidobacterium adolescentis NCC251 cells is carried out in the stationary phase of their growth cycle against a lethal bile salt concentration (eg of about 0.15% for about 30 min) by subjecting the cells to about 0.1% bile salt for about 30 min before lethal challenge
  • a lethal bile salt concentration eg of about 0.15% for about 30 min
  • protection of Bifidobacterium adolescentis NCC251 cells is carried out in the stationary phase of their growth cycle against the otherwise lethal effect of (eg about 3 to about 4 cycles) freeze-thawing (about -80°C to about room temperature (preferably about 20°C to about 30°C, more preferably 25°C)) by subjecting the cells to about 2% of NaCl for about 1 h.
  • freeze-thawing about -80°C to about room temperature (preferably about 20°C to about 30°C, more preferably 25°C)
  • protection of Bifidobacterium adolescentis NCC251 cells is carried out in the logarithmic phase of their growth cycle against an otherwise lethal temperature of 55°C for 20 min by treatment of the cells for about 30 min at about 45°C, about 15 min at about 47°C or for about 1 h with 1% or 2% NaCl.
  • Lactobacillus johnsonii Lai cells are protected. More preferably, protection of Lactobacillus johnsonii Lai cells is carried out in the logarithmic phase of their growth cycle against an otherwise lethal temperature of 55°C for up to lh by treatment of the cells with about 3.5% NaCl for about 15 min or about 48°C for about 15 min. Preferably, protection of Lactobacillus johnsonii Lai cells is carried out in the stationary phase of their growth cycle against an otherwise lethal temperature of 55°C for up to lh by treatment of the cells with a temperature of about 48°C for about 15 min or with about 3.5% NaCl for about 15 min.
  • Figure 1 shows results of a dot blot hybridization of RNA from cells of Bifidobacterium longum NCC481 and Bifidobacterium adolescentis NCC251 after 10 min exposure to different kinds of stress. Hybridization was performed using the specific probes GSR8 and GSR5 for NCC481 and NCC251, respectively.
  • Figure 2 shows a graph of survival of Bifidobacterium adolescentis NCC2 1 at 55°C after different pre-inductions in the logarithmic phase.
  • Cells were grown in MRS and cysteine at 37°C to an OD600 of between 0.4 and 0.7. Aliquots were taken and subjected for 15 min to 47°C, for 30 min to 45°C, or 1 h to 1.5% NaCl or 2% NaCl; the control remained at 37°C. The samples were shifted to 55°C and after 10 and 20 min the viable cell counts were determined.
  • Figure 3 shows a graph of survival of Bifidobacterium adolescentis NCC251 after three and four cycles of freeze-thawing. Stationary phase cells were taken and subjected for 1 h to 2% NaCl, the control remained without salt addition. The samples were shifted to -80°C and thawed at room temperature. This cycle was repeated three and four times before the viable cell counts were determined.
  • Figure 4 shows a graph of survival of Bifidobacterium longum NCC481 under lethal bile salt conditions in the logarithmic phase.
  • Cells were grown to an OD600 (optical density at 600nm) between 0.4 and 0.7 and subjected for 30 min to 0.1%) Oxgall.
  • the control remained without Oxgall addition.
  • the samples were aliquoted and shifted to 0.2%, 0.25%, and 0.3% Oxgall for 30 min, and the viable cell counts were determined.
  • Figure 5 shows a graph of survival of Bifidobacterium longum NCC481 under lethal bile salt conditions in the stationary phase. Cells were subjected for 30 min to an 0.05% Oxgall-treatment. The control remained without any Oxgall addition.
  • the samples were aliquoted and shifted to 0.075%, 0.1%, and 0.15% Oxgall for 30 min, and the viable cell counts were determined.
  • Figure 6 shows a graph of survival of Bifidobacterium adolescentis NCC251 under lethal bile salt conditions in the logarithmic phase.
  • Cells were grown to an OD600 (optical density at 600nm) between 0.4 and 0.7 and subjected for 30 min to an 0.1% Oxgall-treatment. The control remained without any Oxgall addition. The samples were aliquoted and shifted to 0.3% and 0.4% Oxgall for 30 min, and the viable cell counts were determined.
  • Figure 7 shows a graph of survival of Bifidobacterium adolescentis NCC251 under lethal bile salt conditions in the stationary phase.
  • Cells were subjected for 30 min to an 0.1% Oxgall-treatment.
  • the control remained without any Oxgall addition.
  • the samples were aliquoted and shifted to 0.15% Oxgall for 30 min, and the viable cell counts were determined.
  • Figure 8 shows a graph of survival of Lactobacillus johnsonii Lalunder lethal thermal conditions.
  • Cells were grown in MRS at 37°C to an OD600 (optical density at 600nm) between 0.4 and 0.7. Samples were taken and subjected to 3.5% NaCl or 48°C for 15 min. The control remained at 37°C. Afterwards the samples were shifted to 55°C and the viable cell counts were determined after 30 min and 60 min.
  • Figure 9 shows a graph of survival of Lactobacillus johnsonii Lai in the stationary phase of their growth cycle under lethal thermal conditions. Samples were taken and subjected to 3.5% NaCl or 48°C for 15 min. The control remained at 37°C. Afterwards the samples were shifted to 55°C and the viable cell counts were determined after 60 min.
  • Bifidobacterium adolescentis NCC251 , Bifidobacterium longum NCC481 , Bifidobacterium longum NCC490, Bifidobacterium longum NCC585, and Bifidobacterium breve NCC298 were cultivated in MRS medium supplemented with 0.5 g/1 cysteine at 37°C under anaerobic conditions (98% nitrogen and 2% hydrogen). Lactococcus lactis MG1363 was grown in MRS medium at 30°C. Escherichia coli TGI (Amersham) was cultivated in Luria-Bertani medium at 37°C. Lactobacillus johnsonii Lai was grown in MRS at 37°C.
  • Cells were grown to an OD600 (optical density at 600nm) between 0.4 and 0.7 or taken in the stationary phase and subjected for different times to various stress conditions. Cells used for freeze-thawing experiments were concentrated in saline solution before being subjected to -80°C. Salt stress was exerted by adding sodium chloride to the samples while for bile-salt stress OXGALL (Trade Mark) (Difco) was used.
  • OXGALL Trade Mark
  • the stress treatment of Bifidobacteria was performed under anaerobic conditions while the determination of viable cells was carried out under aerobic conditions.
  • Cells of lactobacilli were grown under microaerophil conditions, stress treatments and determination of viable cell counts was performed under aerobic conditions.
  • pH e.g. HC1
  • Bile e.g. Oxgall
  • Oxgall 0.01%-0.1% 0.075%-0.4%
  • Time of pre- induction and lethal challenge can vary dependent on strain and stress conditions between 5 min to 2 h.
  • Ranges for inductions and lethal challenges re-induction lethal challenge pH e.g. HC1, lactic acid pH 6.0-4.5 pH 4.0-2
  • Salt e.g. NaCl 0.5%-3.5% 4%-8%
  • Time of pre-induction and lethal challenge can vary dependent on strain and stress conditions between 5 min to 2 h
  • RNA was isolated, denatured and transferred to uncharged nylon membranes (GeneScreen, NEN) according to standard methods.
  • the membranes were pre-hybridised (lh, 40°C) and subsequently hybridised for 4h with 100 pmol DIG-labelled probes (Boehringer).
  • the membranes were washed twice for 5 min in 2x SSC containing 0.1 % SDS at 40°C and once at the probe-dependent temperature, which was 46°C and 48°C, respectively for the two dnaK specific probes GSR5 (5 '-CATCGAAGGTGCCGCCAC-3 ') and GSR8 (5'-TCGTCACCACCGAGGTG-3'), and 51°C for the universal probe 1028R (5'-CCTTCTCCCGAAGTTACGG-3 r ). Detection was performed according to the manufacturers instructions.
  • the core dnaK region was amplified using the degenerate primers HS1 (5'- ATIACIGTICCIGCITA (T/C)TT(T/C)AA(T/C)GA-3') and HS2 (5'- CATIGT(T/C)TCIATICCIA(A/G)IGAIA(G/A)IGG-3 ') as well as 1 ⁇ g of chromosomal DNA as template.
  • Amplification reactions were performed in a total volume of 100 ⁇ l (containing 200 ⁇ M each of dATP, dCTP, dGTP, and dTTP, 50 pmol of each primer, 2.5 U of Super-Taq DNA Polymerase (HT Biotechnology), and the corresponding lx PCR buffer).
  • dnaK The transcriptional induction of dnaK was investigated with cells exposed to heat shock and to additional general stress conditions.
  • Bifidobacterium longum NCC481 and Bifidobacterium adolescentis NCC251 cells of the logarithmic phase were subjected to 0.1% bile salt, 1.5% NaCl or a heat shock for 10 min at 42°C and 45°C. Maximum temperatures of 47°C and 50°C were tested for NCC481 and NCC251, respectively. Uninduced cells from the logarithmic and stationary phase were always used as controls.
  • Total RNA was isolated and subjected to dot blot hybridization.
  • the dnaK specific probes GSR8 and GSR5 were used for NCC481 and NCC251, respectively.
  • the universal probe 1028R was chosen to verify the amount and quality of RNA on the membrane.
  • An increased concentration of dnaK specific mRNA was observed when subjecting the cells to increasing temperatures ( Figure 1).
  • dnaK of NCC481 was only slightly induced in cells entering the stationary phase.
  • a slight induction of dnaK was observed in NCC251 after bile-salt and NaCl treatment. No significant induction under identical conditions was obtained for NCC481.
  • Lactobacillus johnsonii Lai Bifidobacterium adolescentis NCC251 and Bifidobacterium longum NCC481 at different temperature, bile-salts and salt conditions were tested.
  • logarithmic phase NCC251 showed an increased resistance to the generally lethal temperature of 55°C after being treated with sublethal heat stress.
  • An almost 24- fold and 128-fold higher thermotolerance was observed after subjecting the cells to 47°C for 15 min prior to a heat shock for 10 min and 20 min, respectively (Figure 2).
  • Figure 2 show how that, unexpectedly effective pre-induction of cells can be to protect them against otherwise lethal challenges.
  • a 9-fold and 15-fold cross-protection of cells against 55°C was achieved by pretreatment for lh with 1.5% NaCl. An equal protection against thermal stress could also be observed by pre-inducing at
  • the core region of dnaK of Bifidobacterium longum NCC481, Bifidobacterium longum NCC490, Bifidobacterium longum NCC585, Bifidobacterium adolescentis NCC251 , and Bifidobacterium breve NCC298 were PCR-amplified and identified. Subsequent mRNA analyses revealed that in NCC251 and NCC481 the induction of dnaK is regulated at the transcriptional level. Transcription is generally induced by heat and for NCC251 also by treatment with salt and bile-salts.

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Abstract

L'invention concerne une cellule bactérienne présentant une protection contre des états létaux pour des cellules bactériennes non protégées. Cette cellule protégée est obtenue par soumission d'une cellule bactérienne à un traitement provoquant un niveau de stress sublétal.
PCT/EP2000/005403 1999-06-11 2000-06-09 Protection bacterienne Ceased WO2000077186A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2001503631A JP3774146B2 (ja) 1999-06-11 2000-06-09 細菌の防御
EP00949176A EP1190038A2 (fr) 1999-06-11 2000-06-09 Protection bacterienne contre le stress
BR0011742-0A BR0011742A (pt) 1999-06-11 2000-06-09 Proteção bacteriana
NZ515765A NZ515765A (en) 1999-06-11 2000-06-09 Method of protecting lactobacillus johnsonii La1 from stress or shock
AU62629/00A AU771519B2 (en) 1999-06-11 2000-06-09 Bacterial protection
US10/018,492 US6939705B1 (en) 1999-06-11 2000-06-09 Method of protecting L. johnsonii La1 against stress

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13894699P 1999-06-11 1999-06-11
US60/138,946 1999-06-11

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WO2000077186A2 true WO2000077186A2 (fr) 2000-12-21
WO2000077186A3 WO2000077186A3 (fr) 2001-06-28

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PCT/EP2000/005403 Ceased WO2000077186A2 (fr) 1999-06-11 2000-06-09 Protection bacterienne

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EP (1) EP1190038A2 (fr)
JP (1) JP3774146B2 (fr)
CN (1) CN1330748C (fr)
AU (1) AU771519B2 (fr)
BR (1) BR0011742A (fr)
NZ (1) NZ515765A (fr)
WO (1) WO2000077186A2 (fr)
ZA (1) ZA200109670B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2839855A1 (fr) * 2002-05-27 2003-11-28 Biosaveurs Procede de preparation d'une flore d'affinage comportant une etape de fragilisation cellulaire
WO2009053030A1 (fr) * 2007-10-23 2009-04-30 Nestec S.A. Bifidobactérie tolérante au stress

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6216507B2 (ja) * 2012-12-20 2017-10-18 雪印メグミルク株式会社 酸化ストレス耐性が向上した微生物およびその製造方法
CN103275921B (zh) * 2013-05-16 2015-04-15 江南大学 一种提高乳杆菌耐胆盐能力的方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2839855A1 (fr) * 2002-05-27 2003-11-28 Biosaveurs Procede de preparation d'une flore d'affinage comportant une etape de fragilisation cellulaire
WO2003100071A3 (fr) * 2002-05-27 2005-03-10 Biosaveurs Procédé de préparation d'une flore d'affinage comportant une étape de fragilisation cellulaire
WO2009053030A1 (fr) * 2007-10-23 2009-04-30 Nestec S.A. Bifidobactérie tolérante au stress
US8426190B2 (en) 2007-10-23 2013-04-23 Nestec S.A. Stress tolerant Bifidobacteria
US8741622B2 (en) 2007-10-23 2014-06-03 Nestec S.A. Stress tolerant Bifidobacteria

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AU771519B2 (en) 2004-03-25
EP1190038A2 (fr) 2002-03-27
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BR0011742A (pt) 2002-03-19
JP3774146B2 (ja) 2006-05-10
AU6262900A (en) 2001-01-02

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