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  • 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
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  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
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• • A—HUMAN NECESSITIES
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  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • A—HUMAN NECESSITIES
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  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
  • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
• • A—HUMAN NECESSITIES
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  • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
  • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
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  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present invention relates to probes and primers for quantification of Lactobacillus species.
• the intestinal flora of human milk-fed infants is primarily composed of the genus Bifidobacterium and Lactobacillus .
• the genera Bifidobacterium and Lactobacillus are considered to be important in maintaining a well-balanced intestinal microbiota and it has been postulated that they have several health-promoting effects.
• Lactobacillus in general, i.e. on the genus level.
• the genus Lactobacillus consists of many different species, which differ in metabolism, metabolites, enzyme activities, oligo- and polysaccharide utilisation, cell wall composition, and interaction with the host's immune system. It therefore can be expected that not every species of the genus Lactobacillus has the same functional effect on the infant.
• Examples of different Lactobacillus species are L. rhamnosus, L. casei, L. paracasei, L. reuteri, L. fermentum, L. plantarum, L. acidophilus , and L. delbrueckii .
• EP0674650 relates to nucleic acid sequences which preferentially bind to the rRNA or rDNA of microorganisms which cause the spoilage of beer.
• the beer spoilage microorganisms are predominantly of the genera Lactobacillus and Pediococcus .
• the nucleic acids may be used as probes in assays to detect the presence of these microorganisms. Kits containing two or more probes are also described.
• a method of species-specifically detecting and quantitatively assaying species of the genus Lactobacillus found in humans, particularly human infants, as well as a diagnostic kit for the detection and quantification of Lactobacillus species is provided.
• a method of species-specifically detecting and quantitatively assaying species of the genus Lactobacillus found in human faeces and/or intestine, particularly human infants, as well as a diagnostic kit for the detection and quantification of Lactobacillus species is provided.
• Lactobacillus species especially those found in humans, i.e. L. acidophilus, L. casei, L. delbrueckii, L. fermentum. L. paracasei, L. plantarum, L. reuteri and L rhamnosus using species specific oligonucleotide primes and probes.
• primers and probes can be used to identify species of Lactobacillus and Lactobacillus -like species via FISH, PCR, DGGE, TGGE, dot blot hybridisation and real time PCR methods. All these techniques have in common that it involves a hybridisation step with nucleotides. Preferably real time PCR is used to quantify Lactobacillus species.
• Each of the sequences described below may have additional bases bonded to the 5′- or 3′-terminal thereof as long as it functions as a probe.
• oligonucleotides can be prepared by conventional means for chemical synthesis, for example by an automated DNA synthesiser. DNA fragments containing the above-mentioned sequences can be prepared by enzymatic cleavage of genes from the corresponding Lactobacillus species.
• Duplex 5′ nuclease assays were developed for L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantarum, L. reuteri and L rhamnosus as a proportion of the total cells of the genus Lactobacillus .
• the choice for the intergenic spacer greatly depended on the fact that contamination and sensitivity issues were described for Real-Time PCR when 16S rDNA was used.
• the overall conserved regions of the sequences were used to design primers and probes for the Lactobacillus genus.
• TaqMan Minor groove Binding (MGB) probes were used.
• conserved regions in the sequences of the different kind of subspecies, which showed little homology with other species were used to design primers and probes for respectively L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantaurum, L. rhamnosus and L. reuteri.
• the primers and TaqMan MGB probes were designed with help of Primer Express 1.5a (Applied Biosystems, Nieuwerkerk a/d IJssel, N L). We applied the following criteria: The probe and primers should have a GC content of 30 to 80% and runs of more than 3 identically nucleotides (especially for guanidine (G)) should be avoided.
• the melting temperature (Tm) of the probe should be between 68° C. and 70° C., whereas the primers should have a melting temperature 10° C. below the melting temperature of the probe. Furthermore, no G on the 5′ end of the probe should be present and the strand with more cytosine (C) than G was selected.
• the last 5 nucleotides at the 3′ end of the primers should have no more than two G and/or C bases. Finally, the amplicon length should be less than 150 base pairs.
• the designed primers and TaqMan MGB probes are shown in table 1 and were tested on specificity using the Basic Local Alignment Search Tool (BLAST).
• the probe designed for the detection of the genus Lactobacillus consists of an oligonucleotide with the 5′ reporter dye VICTM (Applied Biosystems, NL) and the 3′ quencher NFQ-MGBTM (Applied Biosystems, NL) and the probes for the different Lactobacillus species of oligonucleotides with the 5′ reporter dye 6-carboxy-fluorescein (FAMTM) and the 3′ quencher NFQ-MGBTM (Applied Biosystems, NL).
• FAMTM 6-carboxy-fluorescein
• NFQ-MGBTM Applied Biosystems, NL
• the universal probe consists of oligonucleotides with the 5′ reporter dye 6-carboxy-fluorescein (FAMTM) and the 3′ quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRATM).
• FAMTM reporter dye 6-carboxy-fluorescein
• TAMRATM 3′ quencher dye 6-carboxy-tetramethyl-rhodamine
• Table 1 Designed primers and probes for use in the 5′ nuclease assays. % Primers & Tm G BLAST ID Amplicon SEQ Target Probes Sequence (5′ ⁇ 3′) (° C.) C number length ID No L.
• Labelled preparations are prepared by labelling the oligonucleotide with a detectable marker by conventional means.
• Labelling markers which may be used include radioisotopes, fluorescent substances, enzymes, biotin and haptens.
• Hybridisation between the labelled preparation and a sample can be performed in accordance with known techniques, such as dot blot hybridisation and northern hybridisation. The hybrid which are formed can be confirmed through the detection of the labelled preparation by known means, for example, autoradiography using radioisotopes, enzyme-labelled antibody techniques using enzyme or biotin, and the like.
• the DNA fragments represented by SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, and SEQ ID No 26 respectively can be used as a primer in the PCR method for identification of species.
• microbial cells to be identified are subjected to bacteriolysis, and any of the DNA fragments of SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 25, respectively and SEQ ID selected from SEQ ID No 2, SEQ ID No 5, SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26, respectively is added thereto as a primer, followed by treatment with a DNA polymerase. If DNA amplification is observed using electrophoresis, etc., this means that the cells possess a gene portion which corresponds to the DNA fragment used, i.e. the cells are identified to be of the same species as the origin of the DNA fragment primer.
• oligonucleotides comprising SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, SEQ ID No 26, and sequences complementary thereto.
• oligonucleotide probe for detection of a nucleic acid target sequence which is characteristic of the species of the genus Lactobacillus , said probe being selected from:
• a labelled oligonucleotide which specifically hybridises to Lactobacillus fermentum DNA represented by SEQ ID No 12 or a sequence complementary thereto;
• the present invention also encompasses a method of species-specifically detecting species of the genus Lactobacillus found in human, particularly human infants, comprising the steps of:
• the oligonucleotide probe above mentioned hybridises to the nucleic acid target sequence preferably the oligonucleotide probe is of SEQ ID No selected from SEQ ID No 3, SEQ ID No 6, SEQ ID No 9, SEQ ID No 12, SEQ ID No 15, SEQ ID No 18, SEQ ID No 21, SEQ ID No 24, SEQ ID No 27.
• a diagnostic kit is herein provided for the detection in a sample of Lactobacillus species selected from L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantarum, L. reuteri and L. rhamnosus , by means of hybridisation analysis, comprising at least a DNA probe as mentioned above as well as one or more further means required for hybridisation analysis, such as denaturation liquid, a hybridisation liquid, a washing liquid, a solid carrier, a hybridisation vessel and label detecting means.
• a diagnostic kit for the detection in a sample of the above-mentioned Lactobacillus species by means of PCR analysis comprising a set of DNA primers as mentioned above as well as one or more further means required for PCR analysis, such as a polymerase, a polymerisation liquid, an oil overlay, a reaction vessel and means for detecting the amplified DNA.
• the diagnostic kit comprises at least a DNA probe as described above and a set of primers as described above and optionally further means as mentioned above.
• the present probes and methods are particularly suitable for quantifying Lactobacillus on a species level in the intestine and/or faeces.
• the probe and primers with SEQ ID 25, 26, and 27 are not related to specific Lactobacillus species but to the genus Lactobacillus .
• SEQ ID 25, 26, 27 are used to quantify the genus Lactobacillus and to relate the quantity of specific Lactobacillus species to all lactobacilli .
• SEQ ID NOs 1-24 are used for detecting and identifying a species and that SEQ ID NOs 25-27 are used for quantifying this identified species.
• Lactobacillus species are quantified in infant faeces, since lactobacilli play an important role in the infants' health.
• the results obtained from such an analysis can be used for selecting suitable Lactobacillus species as a probiotic supplement or probiotic food or component in a (helath) nutritional composition and/or selecting non-digestible saccharides selectively stimulating Lactobacillus species to be incorporated into a (health) nutritional composition.
• These probiotics and/or prebiotics are preferably to be administered to the subject low in specific Lactobacillus species in order to rebalance the intestinal flora on a Lactobacillus species level and prevent or treat disorders associated with this imbalance.
• the disorder is selected from the group consisting of allergy, atopic disease, eczema, diarrhoea, intestinal inflammation and infections.
• the present probes and methods are preferably applied for selecting probiotics and/or non-digestible saccharides for administration to animals in order to promote animal health, more preferably the gastro-intestinal health of animals.
• a method for preparing a nutritional composition comprising selecting suitable Lactobacillus species and/or selecting non-digestible saccharides selectively stimulating Lactobacillus species by applying the oligonucleotide probes according to the present invention and preferably the oligonucleotide primers according to the present invention, preferably in a method according to the present invention and incorporating said selected Lactobacillus species and/or non-digestible saccharides into a (health) nutritional composition.
• the present probes and methods are preferably used for quantification and species determination of systemic infections by species from the genus Lactobacillus.
• the present probes and methods are preferably used in controlling and/or monitoring food fermentation by Lactobacillus species.
• Lactobacillus Strains Origin a Other Strains Origin a L. acidophilus ATCC 4356 Bacillus cereus ATCC 11778 ATCC 521 Bacteroides fragilis LMG 10263 T DSM 20079 T Brevibacterium casei ATCC 35513 T L. amylovorus DSM 20552 Clostridium difficile ATCC 9689 T L. bavaricus JCM 1128 Enterococcus feacalis DSM 20478 T L.
• each duplex 5′ nuclease assay was tested by performing a 25 ⁇ l amplification of the different strains. These 25 ⁇ l PCR reactions were performed using 2.5 ⁇ l DNA template, 12.5 ⁇ l TaqMan Universal Master Mix (Applied Biosystems), optimised concentrations of the primers and probes (see table 3), followed by running the TaqMan Universal Temperature Profile, on the ABI Prism 7700 (Applied Biosystems, Nieuwerkerk a/d IJssel, N L).
• 5′ nuclease assays were specific for the Lactobacillus species for which they were developed and the 5′nuclease assay for determination of the genus Lactobacillus detected all Lactobacillus species tested, but no other bacteria like Propionibacterium, Enterococcus or Pediococcus species.
• the optimised concentrations of the primers and probes are given in table 3.
• the CV values for reproducibility and repeatability for the different kind of 5′ nuclease assays were determined and can be found in table 4.
• TABLE 3 Optimised final primer and probe concentrations used in the different duplex 5′ nuclease assays Forward Reverse Probe Target 5′ nuclease assay Primer (nM) Primer (nM) (nM) L. acidophilus L. acidophilus 900 900 200 Genus Lactobacillus 900 900 200 L. casei L. casei 900 900 200 Genus Lactobacillus 300 300 50 L. delbrueckii L. delbrueckii 300 300 100 Genus Lactobacillus 900 900 200 L.
• fermentum L. fermentum 300 300 100 Genus Lactobacillus 300 300 100 L. paracasei L. paracasei 300 300 100 Genus Lactobacillus 300 300 100 L. plantarum L. plantarum 300 300 100 Genus Lactobacillus 300 300 100 L. reuteri L. reuteri 300 300 100 Genus Lactobacillus 900 900 200 L. rhamnosus L. rhamnosus 900 450 200 Genus Lactobacillus 150 100 100 Genus Genus Lactobacillus 600 600 100 Lactobacillus All bacteria 300 300 100 100
• the developed and validated duplex 5′ nuclease assays show a good specificity, sensitivity, reproducibility and repeatability and can therefore be seen as a good tool for enumeration of the different Lactobacillus species in faeces or intestinal biopsies.

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