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WO2008003811A1 - Procédé de détection et d'identification simultanées et spécifiques de bactéries lactiques et de bifidobactéries dans des laits fermentés et dans des cultures de départ pour laits fermentés - Google Patents

Procédé de détection et d'identification simultanées et spécifiques de bactéries lactiques et de bifidobactéries dans des laits fermentés et dans des cultures de départ pour laits fermentés Download PDF

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Publication number
WO2008003811A1
WO2008003811A1 PCT/ES2007/070124 ES2007070124W WO2008003811A1 WO 2008003811 A1 WO2008003811 A1 WO 2008003811A1 ES 2007070124 W ES2007070124 W ES 2007070124W WO 2008003811 A1 WO2008003811 A1 WO 2008003811A1
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WIPO (PCT)
Prior art keywords
identification
seq
fermented
milks
simultaneous
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PCT/ES2007/070124
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English (en)
Spanish (es)
Inventor
Raquel Tabasco Rentero
Torsten Paarup
Carolina Janer Otero
Carmen PELÁEZ MARTÍNEZ
Teresa REQUENA ROLANÍA
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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Publication of WO2008003811A1 publication Critical patent/WO2008003811A1/fr
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Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • 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
    • 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
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the technique is part of the Agroalimentaha Area, in the Dairy Sector, and develops a procedure for the detection and identification of different species of lactic bacteria and specific bifidobacteria of mixed populations in yogurts and fermented milks.
  • sequences of the 16S rRNA gene of bifidobacteria reflects a high analogy between species (similarity that ranges between 92 and 99%) that hinders their differentiation based on the analysis of said sequence, so that other genes have been sought polymorphic for the identification of these species, as is the one that codes for the transaldolase enzyme [Requena, T., Burton, J., Matsuki, T., Munro, K., Simon, MA, Tanaka, R., Tannock, GW (2002) Appl. Environ. Microbiol 68: 2420-2427].
  • PCR-DGGE polyacrylamide gel electrophoresis with denaturing gradient
  • the object of the present invention is the application of a method of rapid and specific detection and identification of different species of lactic bacteria and bifidobacteria present in mixed populations in fermented milks and in starter cultures for fermented milks, based on the design of unpublished primers and species specific for the PCR amplification of sequences identified in variable regions of the 16S rRNA of S. thermophilus, L. delbrueckii subsp. bulgaricus, L. acidophilus and L. casei subsp. casei and of the transaldolase gene in B. lactis and is complemented by the differentiation of the fragments obtained by DGGE separation.
  • the species under study are usually added as starter cultures in fermented probiotic milks marketed in Spain.
  • thermophilus (ii) patents JP1 1 151097 and JP1 1123093, published in 1999 and requested by the company Yakult Honska KK, which are characterized by the description of usable oligonucleotides for the generic identification of lactic bacteria and bifidobacteria, respectively; and (iii) WO2005103294 published in 2005, requested by the inventors Park, Y.-H., Bae, J. -W., Rhee, S. -K., Park, JR, Kim, B.-C. (Korea) characterized by the use of "microarray" technology for the detection and identification of lactic bacteria, bifidobacteria and propionibacteria. However, there is no patented procedure for the detection and identification of lactic bacteria and bifidobacteria in mixed cultures in fermented milks using the PCR-DGGE technology.
  • DESCRIPTION OF THE INVENTION - Brief description of the invention A procedure has been developed that allows the rapid detection and identification of different species of lactic bacteria (S. thermophilus, L. bulgaricus, L. casei, L. acidophilus) in mixed cultures with B. lactis, species usually present in yogurt and fermented milks that contain probiotics.
  • the method employs specific DNA sequences (primers) designed from the 16S rRNA gene for lactic bacteria species and the transaldolase gene for bifidobacteria and which are used for the identification of DNA from said species by specific PCR amplification.
  • the identification procedure is complemented by the separation of the amplified products by electrophoresis in polyacrylamide gel with denaturing gradient (DGGE). - Detailed description of the invention
  • the procedure for detecting and identifying different species of lactic bacteria (S. thermophilus, L. bulgaricus, L. casei, L. acidophilus) and B. lactis present in mixed cultures in fermented milks is characterized by the use of specific primers designed from sequences present in variable regions of the 16S rRNA gene for lactic bacteria species and the transaldolase gene for bifidobacteria and that the method used is the specific amplification by PCR.
  • the similarity in the length of the amplified fragments does not allow a differentiation of the PCR products in conventional agarose gels, so that in the present invention the application of the procedure in the simultaneous identification of the species when they are in mixed cultures is completed by means of the separation of the products amplified in polyacrylamide gels with denaturing gradient (DGGE).
  • DGGE denaturing gradient
  • the primers selected are derived from the nucleotide sequence present in the variable regions V1 and V2 identified in the 16S rRNA gene for each of the species of lactic bacteria under study; for this, the complete nucleotide sequence of said gene has been obtained by automated sequencing from a representative strain of each of the species.
  • the transaldolase gene sequence has been obtained from previous work [Requena, T., Burton, J., Matsuki, T., Munro, K., Simón, MA, Tanaka, R., Tannock, GW (2002 ) Appl. Environ. Microbiol 68: 2420-2427]].
  • the selected primers are characterized in that each specific pair of designed oligonucleotides, or those derived from the complementary chain, only generate an amplification product from the genomic DNA from the species for which they were designed (Fig. 1).
  • the sequence object of the amplification with the specific primers can also be represented by RNA, depending on the method of preparation of the sample.
  • the pairs of primers for each of the species object of the invention are described below: • S. thermophilus: Thermfor specific primers are used; SEQ ID N01 and Thermrev: SEQ ID N02 that generate a 157 bp PCR product.
  • Casrev SEQ ID N08 that generate a PCR product of 142 bp.
  • the GC clamp is added to one of the primers, that is, a nucleotide extension in position 5 'constituted by the sequence SEQ ID N011.
  • the selection of the optimal primer to contain the GC clamp is carried out experimentally from each of the species in order to obtain a ladder of perfectly separated amplified products, so that when mixed they constitute a standard of specific products ("ladder") of reference for the identification of the species in the problem samples (Fig. 2).
  • the main industrial application of the procedure developed is the detection and identification of different species of lactic bacteria and bifidobacteria in yogurts and fermented milks that contain mixed cultures of these bacteria, in order to determine the authenticity of the microbiological composition shown in the product labeling .
  • the described procedure has application both in research and in the industrial development of products and in their quality control. Description of the content of the figures.
  • Fig. 1 PCR Products shows an electrophoregram of a 2% agarose gel with the PCR products obtained by amplification of DNA from S. thermophilus and two colonies quantified as belonging to this species (lanes 2, 3 and 4) . Similarly, the equivalent products from DNA and colonies of L. bulgaricus (lanes 5, 6 and 7), L. acidophilus (lanes 8, 9 and 10), L. casei (lanes 12, 13 and 14) are shown. and B. lactis (lanes 15, 16 and 17). Lanes 1, 1, 1 and 18 are molecular size standards for DNA fragments.
  • Fig. 2 DGGE separation mixes PCR products shows an electrophoregram of a 9% polyacrylamide gel with a gradient between 40 and 60% of 7 M urea and 40% formamide with the mixture of specific PCR products obtained from DNA pure of each species ("ladder"; lanes 1 and 7) and those obtained from the total DNA extracted from fermented milks using the specific primers for S. thermophilus (lane 2), L. bulgaricus (lane 3), L. casei (lane 4), L. acidophilus (lane 5) and B. lactis (lane 6).
  • Example 1 Identification of colonies of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis obtained in differential and selective media for counting these species present in fermented milks. The procedure for the identification of the five mentioned species was applied to confirm the enumeration made in selective and differential media from samples of commercial fermented milk indicated on the label containing said species. The verification of the conformity of the counts was carried out in 10% of the colonies grown in the different differential or selective media described in the invention patent. Procedure for differentiating and quantifying lactic bacteria and bifidobacteria in fermented milks that employs culture media. antibiotic-free selective primers were used for this described in Table 1 and designed from the procedure described in the present invention.
  • the study colonies grown in the differential and selective media were suspended in 20 ⁇ l_ of sterile milliQ water and heated at 100 ° C for 5 min; then, the cell suspension was frozen at -20 ° C for 24 h.
  • 2 ⁇ l_ of the thawed suspension was used and mixed with the pair of primers corresponding to the species object of identification (Table 1), at the final concentration in the reaction of 0.4 ⁇ M for each primer.
  • the standard components were added to the reaction: dNTP (200 ⁇ M), MgCl 2 (1.5 mM) and the specific buffer for the reaction with Taq Polymerase (Roche).
  • PCR is common for all pairs of primers: 35 cycles with denaturation temperature at 94 ° C, hybridization at 60 ° C and elongation at 72 ° C. Prior to the first reaction cycle the samples were incubated at 94 5 C for 3 minutes and after the end of the last cycle the elongation of fragments was prolonged 5 minutes at 72 ° C, followed by cooling at 4 ° C. The products of the reaction (5 ⁇ L) were analyzed by 2% agarose gel electrophoresis for which samples were previously centrifuged at 10,000 rpm for 3 minutes in order to sediment the cell debris present in the reaction.
  • Colonies were identified by comparing the amplified products from the problem colonies with those of the DNA from each of the pure species (Fig. 1). The separation obtained is not sufficient in the case of having mixed cultures in which the strains are not previously identified.
  • Example 2 Detection and identification by PCR-DGGE of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis present in mixed cultures in fermented milks.
  • the process described in the present invention can also be used for the specific detection and identification of lactic bacteria and bifidobacteria without the need to carry out the previous separation of the species by selective or differential culture.
  • the detection and identification of the five species was applied in the analysis of samples of commercial fermented milk indicated on the label containing said species.
  • the total DNA of the samples (mixture of DNA from all species) was obtained from 3 ml of fermented milk, which was neutralized to pH 6 with 1 M NaOH, to which 10 ml of EDTA was added to the 0.2%, pH 12, to disperse caseins.
  • the cells were sedimented by centrifugation for 15 minutes at 6,000 rpm.
  • the cells were broken with glass beads (diameter 150-212 ⁇ m) and the DNA was precipitated with isopropanol, after removal of material remains cell with 10 M ammonium acetate and a 24: 1 mixture of chloroform: isoamyl alcohol.
  • Total DNA was finally purified by ethanol precipitation and resuspended in 10M Tris-HCI buffer, pH 8, and 1M EDTA. 500 ng of total DNA was used for the PCR and the reaction conditions described in the Example were followed. 1, using the primers shown in Table 1, but with the difference that the GC clamp was added in primers SEQ ID NO1, 4,5,7,10 ThermforGC, BulgrevGC, AcidforGC, CasforGC and RevIacGC.
  • PCR products obtained were added (1 ⁇ l_) to a 9% polyacrylamide gel with a gradient between 40 and 60% of 7 M urea and 40% formamide and separated by electrophoresis at 130 V for 4.5 hours.
  • a standard of specific products (“ladder") obtained from the mixture of 0.5 ⁇ l_ of each of the fragments from the amplification of the pure DNA of each species (Fig. 2, lanes 1 and 7).
  • the amplification of the total DNA obtained from the fermented milks with the pairs of specific primers allows the detection and identification of the species of lactic bacteria and bifidobacteria present in the sample (Fig. 2, lanes 2 to 6).

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Abstract

L'invention concerne un procédé destiné à la détection et à l'identification simultanées et spécifiques de bactéries lactiques et de bifidobactéries dans des laits fermentés et dans des cultures de départ pour laits fermentés. Le procédé permet de détecter et d'identifier rapidement et simultanément différentes espèces de bactéries lactiques (S. thermophilus, L. bulgaricus, L. casei, L. acidophilus) dans des cultures mixtes avec B. lactis, des espèces habituellement présentes dans le yoghourt et dans des laits fermentés contenant des probiotiques. Le procédé utilise des amorceurs d'ADN spécifiques obtenus à partir du gène 16S rRNA pour les bactéries lactiques et du gène de la transaldolase pour les bifidobactéries. Le procédé d'identification par amplification spécifique par PCR est complété par une séparation des produits amplifiés par électrophorèse sur gel de polyacrylamide à gradient dénaturant (DGGE). Le procédé se caractérise par le fait qu'il permet d'identifier ensemble et en une seule réaction des cultures mixtes présentes dans des laits fermentés.
PCT/ES2007/070124 2006-07-03 2007-07-02 Procédé de détection et d'identification simultanées et spécifiques de bactéries lactiques et de bifidobactéries dans des laits fermentés et dans des cultures de départ pour laits fermentés Ceased WO2008003811A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200601788A ES2322827B1 (es) 2006-07-03 2006-07-03 Procedimiento para la deteccion e identificacion simultanea y especifica de bacterias lacticas y bifidobacterias en leches fermentadas y en cultivos iniciadores para leches fermentadas.
ESP200601788 2006-07-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011039359A3 (fr) * 2009-10-01 2011-09-29 Agroscope Liebefeld-Posieux Alp Procédé d'authentification de produits laitiers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GUEIMONDE M. ET AL.: "Viability and diversity of probiotic Lactobacillus and Bifidobacterium populations included in commercial fermented milks", FOOD RESEARCH INTERNATIONAL, vol. 37, 2004, pages 839 - 850 *
RANDAZZO C. ET AL.: "Diversity, dynamics and activity of bacterial communities during production of an artisanal silician cheese as evaluated by 16S rRNA analysis", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, no. 4, April 2002 (2002-04-01), pages 1882 - 1892, XP009017967, DOI: doi:10.1128/AEM.68.4.1882-1892.2002 *
REQUENA T. ET AL.: "Identification, detection and enumeration of human Bifidobacterium species by PCR targeting the Transaldolase Gene", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, no. 5, May 2002 (2002-05-01), pages 2420 - 2427 *
SATOKARI R.M. ET AL.: "Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract", SYSTEMATIC AND APPLIED MICROBIOLOGY, vol. 26, no. 4, 2003, pages 572 - 584, XP004957350 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011039359A3 (fr) * 2009-10-01 2011-09-29 Agroscope Liebefeld-Posieux Alp Procédé d'authentification de produits laitiers
US8883422B2 (en) 2009-10-01 2014-11-11 Agroscope Liebefeld-Posieux Alp Authentication method of dairy products

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ES2322827B1 (es) 2010-04-23

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