WO1999014359A1 - Method of monitoring the competitiveness of a microbial strain - Google Patents
Method of monitoring the competitiveness of a microbial strain Download PDFInfo
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- WO1999014359A1 WO1999014359A1 PCT/IB1998/001411 IB9801411W WO9914359A1 WO 1999014359 A1 WO1999014359 A1 WO 1999014359A1 IB 9801411 W IB9801411 W IB 9801411W WO 9914359 A1 WO9914359 A1 WO 9914359A1
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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
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- 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
- A23C19/0321—Propionic acid bacteria
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- 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
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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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
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- 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
- A23C2220/00—Biochemical treatment
- A23C2220/20—Treatment with microorganisms
- A23C2220/202—Genetic engineering of microorganisms used in dairy technology
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to the field of monitoring the development and competitiveness of microbial strains in a pre-selected environment, e.g. in a food processing environment or in a food product.
- a pre-selected environment e.g. in a food processing environment or in a food product.
- the invention makes use of genetically labelled microbial strains that can be selectively monitored in the pre-selected envi- ronment in respect of growth, survival and/or metabolic activity whereby it is e.g. made possible to select strains having in that particular environment desirable functional and competitive characteristics.
- Micro-organisms play important roles in the food industry.
- starter cultures in particular of organisms belonging to the large group of lactic acid bacteria and strains of Propionibacterium species, Bifidobacterium species and fungal species are widely used in the manufacturing of a large variety of food products such as dairy products, meat products, bakery products and beverages including wine.
- the use of such desirable and useful organisms constitutes an integrated element of the food manufacturing process
- other micro-organisms are highly undesirable in food products as they cause spoilage of the products or cause food poisoning and other food-borne diseases and the presence of such organisms must therefore be kept as low as possible.
- ONFIR ATION COPY cally complex and varied environments of food starting materials in the process of manufacturing and in the finished food products.
- most food materials and products comprise a large variety of micro-organisms of which some are added deliberately as starter cultures to confer desirable sensory and dietary characteristics to the food product, whereas others are contaminating spoilage or pathogenic organisms, the presence and activity of which is to be controlled to the largest possible extent.
- a range of selective culturing methods are available to monitor the presence of specific organisms or groups of organisms in food materials and products.
- such methods are available for detecting the presence and/or the numbers of pathogenic organisms such as e.g. Salmonella spp., toxinogenic E. coll, Shigella spp., Vibrio spp., Listeria spp., Campy/obacter spp. and Staphylococcus spp.
- pathogenic organisms such as e.g. Salmonella spp., toxinogenic E. coll, Shigella spp., Vibrio spp., Listeria spp., Campy/obacter spp. and Staphylococcus spp.
- pathogenic organisms such as e.g. Salmonella spp., toxinogenic E. coll, Shigella spp., Vibrio spp., Listeria spp., Campy/obacter spp. and Staphylococc
- a particularly difficult problem associated with the use of starter cultures as mentioned above in food manufacturing is that generally applicable selective culturing methods are not available that can discriminate between individual strains of the same or related species or group of species.
- selective culturing methods are not available that can discriminate between individual strains of the same or related species or group of species.
- lactic acid bacteria there are relatively selective culturing methods available that permit the enumeration of viable lactic acid bacteria as a group based on counting the number of typical colonies of such bacteria on solid media.
- starter culture compositions comprising two or more strains of lactic acid bacteria, Bifidobacterium and/or Propionibacterium as the morphology of colonies of such species are so similar that reliable selective enumerations of individual species or strains are not possible.
- the present invention relates in a first aspect to a method of selectively monitoring the development and/or competitiveness of a microbial strain in a sample environment, the method comprising that a selectively detectable genetic label is inserted into a replicon of said strain whose development and/or competitiveness is to be monitored, adding the thus labelled strain to the sample environment and isolating DNA or RNA of said strain from the sample environment and detecting the amount of the genetic label.
- a method of selecting, among a multiplicity of microbial strains, the strain that in a pre-selected environment and under pre-selected conditions has the highest capability to survive, multiply and/or express a pre-selected gene comprising providing in any one of said microbial strains a selectively detectable genetic label, introducing said labelled strains into the particular environment and keeping said environment under the pre-selected conditions for a period of time during which at least one sample is collected and measuring in said sample the amount of cells of each individual microbial strain or the amount of gene expression as determined by the amount of their detectable genetic label, and selecting the strain of which the highest amount of cells or gene expression is present.
- the invention pertains in a still further aspect to a microbial culture composition com- prising a multiplicity of microbial strains where at least one of said strains contains a selectively detectable genetic label permitting the development and/or competitiveness of said labelled strain(s) to be monitored selectively in a sample environment to which the composition is added.
- the invention relates to the use in the above methods of a PCR method that is capable of detecting a genetic label consisting of a mutational substitution of one or more nucleotides in a coding sequence of a replicon, the PCR including the use of a probe that is capable of binding to a DNA or RNA template comprising the genetic label and which is labelled with at least two fluorescent labels, the probe is hydrolysed during each PCR cycle resulting in emission of a fluorescence signal, the amount of which is proportionate to the amount of genetic label detected, including such a use wherein the first PCR cycle involves the use of a reverse transcriptase whereby a labelled mRNA and/or cDNA template is generated.
- the expression “selectively monitoring” indicates that at least one of the presence, number, physiological state or metabolic activity of a specific microbial strain can be determined selectively in a pre-selected environment including such an environment where other microbial strains are present.
- microbial strain refers to a strain of any type of micro-organisms including prokaryotic and eukaryotic organisms such as bacteria, fungi including yeasts, viruses, bacteriophages, plant cells and animal cells.
- sample environment is used herein to designate any environment that can be described in terms of space, time, physical and chemical parameters including chemical composition, temperature, pH, osmolality, water activity, presence/absence of preservatives, and presence/- absence of other micro-organisms.
- a food processing line containing the materials which form the starting material for the finished product constitutes a sample environment according to the invention.
- the above method is based on the insertion into the microbial strain, the development and/or competitiveness of which is to be monitored a selectively detectable genetic label which permits that the cell number, physiological state and/or metabolic activity of the thus labelled microbial strain can be monitored in the sample environment by isolating DNA and/or RNA of said strain from the sample environment and detecting the presence and/or amount of the genetic label.
- the expression "genetic label” refers to any selectively detect- able modification of the DNA of the microbial strain to be labelled.
- the modification consists in inserting into the selected microbial strain a nucleotide sequence including an isolated, naturally occurring sequence or a synthetically made nucleotide sequence.
- the inserted nucleotide sequence label may comprise naturally occurring nucleotides, non-naturally occurring nucleotides, a PNA sequence or a nucleotide sequence consisting of any mixture of such sequences.
- the inserted nucleotide sequence comprises an identification 5 code containing identifiable information such as e.g. on the origin of the strain or its batch number.
- an information carrying code is e.g. in the form of an alphanumeric code.
- the size of such labels may vary considerably, depending on the informational content which it is desirable to incorporate in the labels, a suitable size being in the range of 1 5 10 to 1000 nucleotides such as in the range of 50 to 500 nucleotides including the range of 100 to 250 nucleotides.
- the above genetic labels can be inserted into the microbial strain according to any conventional technique for the insertion of nucleotide sequences.
- the insertion of nucleotide sequences can be any conventional technique for the insertion of nucleotide sequences.
- bacteriophage 20 can be made by a site-specific recombination method, one particularly useful specific method being that disclosed in WO 94/1 9460 to which there is referred.
- This method is based on the use of bacteriophage ⁇ LC3 which comprises integration functions permitting that the bacteriophage vector is integrated at specific sites in a bacterial strain.
- This bacteriophage-based integration vector comprises a site for insertion of
- the insertion of the genetic label can be made by integrating the label into the chromosome of the strain. This is generally considered to be advan- tageous as the label will be maintained stably and passed on to the progeny of the cells.
- the label is introduced into an episomal replicon of the strain including a plasmid, a cosmid or a vector, in particular in cases where such extrachromosomal elements are modified to become stably maintained in their host cell.
- An important aspect of the manner whereby the genetic label is inserted is that the insertion occurs at a site of the DNA of the microbial strain which does not result in any interruption or reduction in the expression of genes, the gene products of which is required for non-limited function of the cells of the microbial strain. It is possible to a large extent to avoid such an undesirable effect by using a site-specific integration method as referred to above, although it cannot be completely ruled out that the insertion of foreign nucleotide sequence may render the resulting recombinant strain slightly less competitive as the progeny of the initially labelled cells will be encum- bered by the synthesis of the labelling nucleotide sequence.
- An alternative approach to avoiding any adverse effects on gene expression in the microbial strain is to provide the genetic label according to the invention in the form of a substitution of one or more, preferably a few, nucleotides in a DNA sequence naturally occurring in the microbial strain.
- Such a labelling can, in contrast to the above insertion of a foreign sequence, even be made in a coding sequence of a gene without impairing the expression of the gene, in particular when the substitution is "silent", i.e. it does not result in a codon for a different amino acid.
- substitutions of nucleotides in a DNA sequence of a micro- bial strain can be made by any conventional techniques for that purpose including site- directed mutagenesis using appropriate oligonucleotide primers. It will, however, be understood that the substitutional event may also be provided by using random mutagenesis followed by selecting cells where a substitution as defined above has occurred or by selecting spontaneously occurring mutants having such a substitution.
- the strain if competitive, is capable of growth and metabolic activity for any appropriate period of time during which at least one sample of the environment is collected to determine to what extent the labelled strain has been capable of developing and competing in the selected environment.
- the sample material for determining the amount of labelled DNA is either the sample as such, a preparation of microbial cells separated from the sample or DNA preparations provided by isolating DNA from the sample itself or from more or less concentrated preparations of cells previously separated from the sample material.
- the amount of the label is detected by any suitable PCR method which permits the amount and optionally, the sequence of the label to be determined e.g. using the methods as described in WO 96/1 7954.
- the determination of the amount of target nucleotide sequences is at the best semi-quantitative, as it has to be based e.g. on a "most probable amount” concept, i.e. the amount is determined by serially diluting the sample containing the target sequence and determining the dilution at which the target DNA can no longer be detected. Based on this end point determination, the most probable amount of genetic label in the sample can be assessed at least semi-quantitatively.
- the TaqManTM -based PCR differs from a standard PCR technique i.a. in that a DNA probe is used that hybridises selectively to the target DNA but which during the PCR cycle is hydrolysed by the 5'-3' nuclease activity of the Taq DNA polymerase.
- the probe is labelled with two different fluorogenic dyes, a reporter dye and a quencher dye and as long as the probe is intact, such that both dye moieties are attached to the same molecule, the quencher dye will absorb the excited state energy of the reporter dye moiety.
- the present inventors have found that the above PCR technique is highly suitable in the methods of the invention as it permits rapid and reliable determination of the amount of genetic label to be made on a large number of samples simultaneously. Additionally, and importantly, the techniques are highly selective for specific sequences, as the labelled probe cannot be hydrolysed if just one single mismatch between the probe and its target sequence occurs.
- RNA as target template and carrying out the first amplification step using a reverse transcriptase whereby is generated a mRNA or a cDNA template, or by using a thermostable polymerase that can use both DNA and RNA as template, optionally after a treatment of the sample with DNase, only genetic labels of viable cells are detected.
- this approach is referred to as RT-PCR (RT: Re- verse Transcriptase).
- the RT-PCR technique makes it possible to measure the level of translation of any pre-selected, genetically labelled gene by using the corre- sponding mRNA as template in the RT-PCR method.
- a significant aspect of the above use of the TaqManTM -based PCR is the high degree of specificity. This implies that there can be made nucleotide substitutions as explained above in several genes of the same microbial strain or in the same gene in a number of different strains.
- any of the above techniques for monitoring the development or competitiveness of a particular microbial strains can be applied in any sample environment including food product starting materials or finished food products, feed product materials and feed compositions, pharmaceutical products, sample environments of animal bodies including human bodies, water bodies, soil and any production process environment where microorganisms are involved as production strains or contaminating pathogenic or spoilage organisms.
- any of such environments there is a need to monitor the survival, growth, gene expression and metabolic activity and interaction with other mi- crobial strains including strains that are indigenous for a particular environment. It will be appreciated that such monitoring can be made over a period of time by collecting samples from the environment at appropriate time intervals.
- a particular useful application of the above method is for monitoring the growth and/or metabolic activity of a starter culture microbial strain during processing of food materials inoculated with the starter culture strain, typical examples being milk, vegetables, meat products, wine musts or wine inoculated with a lactic acid bacterial strain to obtain a fermentation of the food product starting materials.
- a strain that is genetically labelled in accordance with the invention it is possible to collect samples of the inoculated food materials at appropriate time intervals and detect in these samples the amount of genetic label using any of the methods described above.
- the results of such detection tests can be provided in short time making it possible to adjust the processing conditions if the detection tests show that the growth or activity of the starter culture strain is not at an optimum level.
- the genetically labelled microbial strain is a pathogenic organism. It is evident that there is a need to monitor the survival and competitiveness of such organisms in any of the above environments.
- the present method is useful for studying the survival and growth of pathogenic organisms in food starting materials during manufacturing of food products and in the resulting products during distribution and storage.
- the above RT-PCR technique e.g. provides the means for detecting under which environmental conditions genes coding for toxic gene products are expressed and under which conditions they are not.
- Another exam- pie of the usefulness of the above method of the invention is its application in studying the ability of a given pathogenic or beneficial microbial strain to colonise in an animal body, e.g. in the gastro-intestinal tract.
- “beneficial microbial strains” include so-called probiotically active strains of e.g. lactic acid bacteria that are administered to animals or humans in order to improve the general heath of the ani- mals or humans e.g. by inhibiting pathogenic organisms in the gastro-intestinal tract, by stimulating the immune system or by exerting a growth promoting effect. The ability of such strains to become established in the body can be monitored by the present method.
- the method can, as it is mentioned above, be used for monitoring any strain of pro- karyotic or eukaryotic micro-organisms including gram-positive bacterial species and gram-negative bacterial species.
- interesting gram-positive species are lactic acid bacterial species, Propionibacterium species and Bifidobacterium species.
- Lactic acid bacterial species include Lactococcus species such as Lactococcus lactis, Streptococcus species such as Streptococcus thermophilus, Lactobacillus species including Lactobacilllus acidophilus, Leuconostoc species such as Leuconostoc oenos and Pediococcus species including Pediococcus acidilactici.
- the availability of such a method provides an extremely useful tool in strain selection which presently is based on isolating a range of strains that are potentially useful and test any of these strains individually and in combination for their ability to grow and perform a certain activity in a pre-selected environment.
- the method comprises providing in any one of the microbial strains to be tested a selectively detectable genetic la- bel followed by introducing said labelled strains into the particular environment and keeping the environment under the pre-selected conditions for a period of time during which at least one sample is collected and measuring in said sample or samples the amount of cells of each individual microbial strain or the amount of gene expression as determined by the amount of their detectable genetic label. Based on the results of these measurements, the strain of which the highest amount of cells and/or the highest level of gene expression is present can be selected.
- Such a method is particularly useful in the selection of a food starter culture strain e.g. of a lactic acid bacterial species, a Propionibacterium species or a Bifidobacterium species which are widely used in the manufacturing of fermented food products such as dairy products including cheese, yoghurt and butter. It is recognized in the art that within the same species there are considerable variation among individual strains with respect to their ability to perform a food fermentation process and hence, it is required when improved starter culture strains are to be selected to subject a relative large number of strain of the selected species to a comprehensive and costly screening scheme including testing the performance of the strains under industrial fermentation conditions.
- the pre-selected strains can now be genetically labelled, using for each individual strain a unique selectively detectable label as described above. This individual labelling then permits the growth and metabolic activity of each strain to be determined selectively. It will be appreciated that it is possible to add several of such individually labelled strains to the same environment and test their performance simultaneously.
- a label as described above consisting of a mutational substitution of one or more nucleotides in a coding sequence of the strain, as this makes it possible to monitor not only the number of viable and/or dead cells at a given point in time but also the level of expression of a la- belled gene by measuring the amount of labelled mRNA or cDNA in accordance with the RT-PCR method as also described above.
- Yet a further objective of the invention is to provide a microbial culture composition comprising a multiplicity of microbial strains where at least one of said strains contains a selectively detectable genetic label as described above. As also described above, this labelling will permit the identification and origin of said labelled strain(s) to be determined or the development and/or competitiveness of said labelled strain(s) to be monitored selectively in a sample environment to which the composition is added.
- the strain/strains is/are labelled with a genetic label that is selected from the group consisting of a DNA sequence comprising naturally occurring nucleotides, a RNA sequence comprising naturally occurring nucleotides, a DNA sequence comprising non-naturally occurring nucleotides, a RNA sequence comprising non-naturally occurring nucleotides, a PNA sequence and a nucleotide sequence consisting of any mixture of such sequences.
- the genetic label consists of a mutational substitution, including a silent substitution, of one or more nucleotides in a coding sequence of the labelled strain(s).
- the culture composition according to the invention is a food starter culture composition such as a dairy starter culture composition, comprising two strains or more of a lactic acid bacterium, a Propionibacterium species and a Bifidobacterium species.
- a starter culture composition for the manufacturing of yoghurt may comprise a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus.
- Another example is a mixed strain culture for acidifying cheese milk that comprises strains of Lactococcus lactis subspecies cremoris, Lactococcus lactis subspecies lactis, Leuconostoc mesenteroides subsp. cremoris and Lactococcus lactis subspecies diacety lactis.
- Other useful species, which are used in dairy starter cultures include Propionibacterium species, Lactobacillus helveticus and Lactobacillus delbrueckii subspecies bulgaricus.
- Dairy starter culture compositions and starter culture compositions for other food fermentation purposes such as fermentation of doughs, meat products, vegetables, fruit musts and wine are generally provided as concentrates of the culture in freeze-dried or frozen form.
- such compositions comprises a high number of viable cells, calculated as colony forming units (CFU) e.g. in the range of 10 6 to 10 12 CFU per g.
- CFU colony forming units
- the compositions may, in addition to the bacterial cultures, comprise further components, including nutrient components and cryoprotectants.
- Such compositions comprising one or more strains that are genetically labelled in accordance with the inven- tion constitutes a further aspect of the invention.
- the invention provides the use of a PCR method that is capable of detecting a genetic label consisting of a mutational substitution of one or more nucleotides in a coding sequence of a replicon
- the PCR includes the use of a probe that is capable of binding to a DNA or RNA template comprising the genetic label and which is labelled with at least two fluorescent labels, the probe is hydrolysed during each PCR cycle resulting in emission of a fluorescence signal, the amount of which is proportionate to the amount of genetic label in any of the method according to the invention, including such use wherein the first PCR cycle involves the use of a reverse transcriptase whereby a labelled mRNA and/or cDNA template is generated.
- Fig. 1 illustrates the construction of the replacement vector pTAG 1 .
- Fig. 2 shows the integration of a genetic label DNA sequence into the Bacillus subtilis chromosome; the cloned chromosomal DNA fragment (open box) is interrupted by the label sequence (filled box).
- Two 1 kb DNA fragments homologous to a 2 kb Bacillus subtilis chromosomal sequence were produced by PCR using primers containing the recognition sites for Xba ⁇ , EcoR ⁇ , Bam ⁇ and Kpn ⁇ , respectively at their 5' ends. After digesting these PCR-gener- ated fragments and a genetic label fragment with the appropriate endonucleases, the three fragments were mixed and joined by ligation.
- the recombining cassette comprising the 2 kb B. subtilis chromosomal sequence interrupted by the label sequence was amplified from the ligase reaction mixture as shown in Fig. 1 .
- the cassette was then ligated between the Xba ⁇ and Sail restriction sites of the thermosensitive plasmid pG + host4 (Appligene, lllkirch, France) to give the replacement vector pTAG 1 .
- pTAG 1 Following introduction of the pTAG 1 vector into B. subtilis, a Campbell-like single crossing-over event between the chromosome and the homologous region in pTAG1 will result in plasmid integration and duplication of the homologous sequence at the vector-chromosome junction regions. Subsequently, recombination between these DNA repeats will lead to excision of the plasmid. Integration through a followed by excision through b (or vice versa) will result in integration of the label sequence (Fig. 2).
- pTAG1 was introduced into B. subtilis cells by electrotransformation as described by McDonald et al., J. Appl. Bacteriol.79:21 3-21 8, 1 995.
- Electroporated cells were diluted 10-fold in 2xLB medium and incubated with shaking for 2.5 hours at 30°C and subsequently mixed with 1 0 ml of LB containing 10 ⁇ g of erythromycin per ml.
- the temperature was raised to 37.5°C (a temperature that is non-permissive for plasmid-directed replication) and the culture was incubated overnight to obtain a population of integrants.
- the culture was then diluted 1 : 1 0 5 in LB medium without antibiotic and the tempera- ture shifted to 28°C to stimulate homologous recombination and excision of the integrated plasmid through plasmid-directed replication.
- the cell culture was plated at various cell concentrations at 37.5°C without erythromycin selection. Colonies were transferred by use of tooth sticks to plates containing 10 ⁇ g of erythromycin per ml. Erythromycin sensitive colonies were analysed for presence of genetic label sequence by PCR, using PCR primers hybridising to the chromosomal DNA sequence on each side of the label sequence. Finally, the integrated state of the genetic label sequence was verified by Southern hybridisation.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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AU88809/98A AU8880998A (en) | 1997-09-18 | 1998-09-11 | Method of monitoring the competitiveness of a microbial strain |
CA002334007A CA2334007A1 (en) | 1997-09-18 | 1998-09-11 | Method of monitoring the competitiveness of a microbial strain |
EP98940492A EP1015626A1 (en) | 1997-09-18 | 1998-09-11 | Method of monitoring the competitiveness of a microbial strain |
NO20001420A NO20001420L (en) | 1997-09-18 | 2000-03-17 | Procedure for controlling the competitiveness of a microbial strain |
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Application Number | Priority Date | Filing Date | Title |
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NO974299A NO974299D0 (en) | 1997-09-18 | 1997-09-18 | Method for stable labeling of microorganisms |
NO974299 | 1997-09-18 |
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WO1999014359A1 true WO1999014359A1 (en) | 1999-03-25 |
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PCT/IB1998/001411 WO1999014359A1 (en) | 1997-09-18 | 1998-09-11 | Method of monitoring the competitiveness of a microbial strain |
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EP (1) | EP1015626A1 (en) |
AU (1) | AU8880998A (en) |
CA (1) | CA2334007A1 (en) |
NO (1) | NO974299D0 (en) |
WO (1) | WO1999014359A1 (en) |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6849430B2 (en) | 2001-04-23 | 2005-02-01 | Monsanto Technology Llc | PCR-based monitoring in wastewater biotreatment systems |
WO2014015925A1 (en) | 2012-07-26 | 2014-01-30 | Sartorius Stedim Biotech Gmbh | Method for differentiating between living and dead cells |
CN108982430A (en) * | 2017-05-31 | 2018-12-11 | 北京大学 | Mark kit, method, the bacteria flora with fluorescent marker and its application of bacteria flora sample |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6849430B2 (en) | 2001-04-23 | 2005-02-01 | Monsanto Technology Llc | PCR-based monitoring in wastewater biotreatment systems |
WO2014015925A1 (en) | 2012-07-26 | 2014-01-30 | Sartorius Stedim Biotech Gmbh | Method for differentiating between living and dead cells |
CN108982430A (en) * | 2017-05-31 | 2018-12-11 | 北京大学 | Mark kit, method, the bacteria flora with fluorescent marker and its application of bacteria flora sample |
CN108982430B (en) * | 2017-05-31 | 2020-09-29 | 北京大学 | Kit and method for marking bacterial flora sample and application |
Also Published As
Publication number | Publication date |
---|---|
AU8880998A (en) | 1999-04-05 |
NO974299D0 (en) | 1997-09-18 |
EP1015626A1 (en) | 2000-07-05 |
CA2334007A1 (en) | 1999-03-25 |
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