ES2336534B1 - USE OF PEPTIDES FOR THE DETECTION OF CLOSTRIDIUM TYROBUTYRICUM SPORTS. - Google Patents

Abstract

Use of peptides for the detection of spores of Clostridium tyrobutyricum .

The present invention falls within the sector of food control and specifically, within the sector of methods of detection of microorganisms in foods based on the use of specific ligands, specifically based on the use of peptides that specifically recognize the Clostridium tyrobutyricum spurs present in a sample. The use of these peptides allows the rapid and specific detection and / or quantification of said spores.

Description

Use of peptides for the detection of spores of Clostridium tyrobutyricum .

The present invention falls within the sector of food control and specifically, within the sector of methods of detection of microorganisms in foods based on the use of specific ligands, specifically based on the use of peptides that specifically recognize the Clostridium tyrobutyricum spurs present in a sample. The use of these peptides allows the rapid and specific detection and / or quantification of said spores.

Prior art

Butyric fermentation is a serious problem in the manufacture of hard and semi-hard cheese, which causes losses of millions of euros in the sector every year. This problem manifests itself throughout the ripening of the cheese and causes a defect known as "late swelling." In Spain, an important part of cheese production is potentially affected by this problem (Rilla et al ., 2003. International Journal of Food Microbiology 85 (1): 23-33 .; González et al ., 2007. Food Control 18 (6): 716-722 .; Anonymous (2007a). Resolution of July 11, 2007, of the National Institute of Agricultural and Food Technology and Research. Official Gazette of Spain 208: 36181-36182). The main agent responsible for butyric fermentation is Clostridium tyrobutyricum (Bergére and Sivelä, 1990. Bulletin of the International Dairy Federation 251: 15-23), a Gram positive bacterium, spore-forming, strict anaerobic and non-pathogenic for humans and animals ( Wiedmann et al ., 2000. Encyclopedia of Food Microbiology Robinson RK, Batt CA and Patel PD (Editors), Academic Press, London, pp: 451-458), which comes primarily from silage. The spores of Clostridium tyrobutyricum contaminate the milk and remain in it until it is used for cheese making, since the pasteurization heat treatment the milk receives, previously, is insufficient to inactivate the spores (Franciosa et al ., 1999. Journal of Food Protection 62: 867-871).

Throughout the maturation of the cheese, germination of the spores occurs, followed by a multiplication of the vegetative forms, which metabolize the lactate and release as fermentation products, gas (H2 and CO2) and butyric acid (Rosen et al ., 1989. Milchwissenschaft . 44 (6): 355-357; Herlin and Christiansson, 1993. Milchwissenschaft . 48 (12): 686-690). As a consequence, organoleptic defects can also occur in the cheese as a bad smell and taste, due to the presence of butyric acid (Bergére and Favreau, 1987. Sciences des Aliments . 7 (No. hors-series VII): 89-98).

The current analysis methods used for the detection of Clostridium tyrobutyricum spores are microbiological and take 7 days to obtain a result, time in which milk has already been used in cheese making. Currently, as there is not a sufficiently fast method, milk lysozyme, which is a high-cost enzyme product, is added to milk destined for the production of mature cheeses.

In patent document US / 2007/141628, discloses a method to bind peptides to surfaces coated with polyethylene, comprising active domains with a binding site for agents capable of bonding on the surface of the polyethylene. In This document mentions the sequence SEQ ID NO: 1 but is not mentions the use claimed in the present invention.

Patent document US / 2007/065387 discloses the use of beneficial agents (conditioners and dyes) and methods to prolong the binding time of these agents, coated with polymers, to a body surface such as hair or skin, in the presence of a composition that encompasses a peptide that It has affinity for the polymer. The sequences of the peptides SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 herein invention but, as in the previous document, it is not mention the claimed use.

The peptides SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 have been cited in various documents but in none of the cases cites its use for the detection of spores of Clostridium tyrobutyricum .

On the other hand, in a report by Doctoranda María Lavilla Martín on the project of her doctoral thesis: "Development of an immunochemical method for the detection and quantification of Clostridium tyrobutyricum in milk spores" refers to the description of the objectives and methodology to be developed to detect and quantify Clostridium tyrobutyricum spores but in no case are the peptide sequences of the present invention included.

The use of these peptides expressed in phages, in methods of detection and quantification of Clostridium tyrobutyricum spores, is a great advantage over the usual method of quantification of this microorganism (most likely number method) since it takes 1 day versus to incubations of up to 7 days of the conventional method.

Explanation of the invention.

The present invention is based on the use of peptides that allow rapid and specific detection of Clostridium tyrobutyricum spores present in a sample. The detection of the spores of this microorganism is carried out in 1 day compared to the 7 days needed when using the conventional method (method of the most probable number). The conventional method is not very specific, since it does not allow differentiation between different species of the Clostridium genus. In addition, the peptides of the invention advantageously avoid non-specific serological reactivity with other microorganisms that can be detected by immunochemical methods using polyclonal antibodies. Likewise, the laboriousness of obtaining monoclonal antibodies by creating hybridomas and the lack of affinity that these antibodies usually present are avoided. Another advantage of the peptides with respect to the use of both polyclonal and monoclonal antibodies, is the possibility of their chemical synthesis, which avoids the need to work with experimental animals.

The term spores refers to the forms of resistance adopted by bacteria, in the case of this invention, of the species Clostridium tyrobutyricum under unfavorable environmental conditions. This term is used as a synonym for spores.

The peptides are isolated by an affinity selection procedure, from a library of random peptides expressed on the surface of a bacteriophage (hereinafter phage) (Phage display). The DNA sequences of said peptides are included in phage M13KE. Thus, the "Ph.D.-12 ™ Phage Display Peptide Library Kit" contains a library of peptides fused with a bacteriophage M13 surface protein. In this way, those peptides that bind specifically to the Clostridium tyrobutyricum spores can be selected , their nucleotide sequence can be known and the resulting amino acid sequence of the different amplifications obtained can be determined.

In this sense, a first aspect of the The present invention relates to a peptide selected from the list consisting essentially of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 or any of its combinations

A second aspect of the present invention relates to the use of a peptide for the detection of Clostridium tyrobutyricum spores. The peptide is selected; from the above list, from the list consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or any combination of the peptides from the two previous lists.

The term essentially, in the present invention refers to the sequences described above and to the following sequences derived from them in the sense that outlined below:

- Sequences resulting from the elimination of amino acids from the N-terminal and / or regions C-terminal of any of the SEQ ID sequences NO: 1 to SEQ ID NO: 13;

- Sequences resulting from the addition of amino acids to the N-terminal and / or regions C-terminal of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13 or;

- Sequences resulting from elimination and / or addition of amino acids, modified or unmodified, at sites internal sequence of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13.

In the free text of the sequence list, describe the following characteristics;

Sequences SEQ ID NO: 1 to SEQ ID NO: 13; peptide expressed on the surface of phage M13KE.

Sequences SEQ ID NO: 14 to SEQ ID NO: 26; sequence of biological interest encoding peptides that recognize Clostrydium tyrobutyricum spores.

In a third aspect of the invention reference is made to the use of a nucleotide sequence, which constitutes the coding sequence of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13 for the detection of Clostridium tyrobutyricum spores.

The coding sequences of SEQ peptides ID NO: 1 to SEQ ID NO: 13 would comprise those sequences nucleotides with all possible combinations of triplets that give rise to the same amino acid since the genetic code is degenerated and a certain amino acid can be encoded by More than a triplet. Triplet is understood to be the group of 3 nucleotides that determine an amino acid.

In a preferred embodiment for the detection of Clostridium tyrobutyricum spores, the peptide is selected from SEQ ID NO: 1 to SEQ ID NO: 4 or any combination thereof.

In another more preferred embodiment, the peptide of The previous selection is SEQ ID NO: 1.

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These preferences in the above embodiments are justified, as mentioned below in Example 1, because the sequences of the specific Clostridium tyrobutyricum spore-binding peptides obtained by translation of the nucleotide sequences have different occurrence frequencies. Thus, those with the highest representation in the total set of sequences amplified by PCR with specific primers, correspond to the peptides characterized by SEQ ID NO: 1 (57%), SEQ ID NO: 2 (8%), SEQ ID NO: 3 (5%) and SEQ ID NO: 4 (5%). The rest of the sequences have a representation of 3%. The translation of the sequences defined in SEQ ID NO: 14 to SEQ ID NO: 26 corresponds one by one with the sequences SEQ ID NO: 1 to SEQ ID NO: 13, that is, the translation of SEQ ID NO: 1 corresponds to SEQ ID NO: 14 and so on until SEQ ID NO: 13, which results in a sequence defined by SEQ ID NO: 26.

A fourth, more concrete aspect of the present invention is the use of the peptides described above, that is, any of the peptides defined by SEQ ID NO: 1 to SEQ ID NO: 13 or any combination thereof for the detection of Clostridium tyrobutyricum spurs in a food intended for humans or any
animal.

In a preferred embodiment the food is milk or any dairy derivative.

On the one hand, in farms linked to grazing and production of preserved crop fodder Herbaceous two forms of conservation of these forages are used: henified and silage. Mishandling of silos both in the realization process as in conservation, can cause contamination thereof with undesirable microorganisms that later, to through feeding, they would pass into the milk of cattle.

On the other hand, as mentioned in the state of the art, butyric fermentation is a serious problem in the manufacture of hard and semi-hard cheese. The main agent responsible for butyric fermentation is Clostridium tyrobutyricum , a Gram positive bacterium, spore-forming and strict anaerobic that comes primarily from silage. The spores of Clostridium tyrobutyricum contaminate the milk and remain in it until it is used to make the cheese in which the germination of the spores occurs, followed by a multiplication of the vegetative forms, which metabolize the lactate and release as products of fermentation, gas (H2 and CO2) and butyric acid. As a consequence, organoleptic defects such as bad smell and taste, due to the presence of butyric acid, can also occur.

A fifth aspect of the present invention is the detection and / or quantification of Clostridium tyrobutyricum spores by a method comprising:

to)

Selection of at least one peptide from Enter SEQ ID NO: 1 to SEQ ID NO: 13;

b)

immobilization of the peptide / s, labeled or not, on a solid support;

C)

incubation of a sample with the peptides immobilized in (b) for the binding of the spores to the peptides;

d)

detection and / or quantification, using a marked element, from the spores joined in (c) to the immobilized peptides.

The peptides are fixed to the solid support through different processes of covalent union that recognize the peptides that are to be immobilized or to the element that has been incorporated into them through the process of marking.

The marking consists of the union of an element which is specifically incorporated into another, which allows the location and quantification of a phenomenon or process.

Solid support is understood as a great variety of materials, such as ion exchange resin or adsorption, glass, plastic, latex, nylon, gel, esters cellulose, paramagnetic spheres or the combination of some of them, not excluding other types of media not mentioned previously.

In a preferred embodiment of the described method previously, the marking is done with a marker selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly.

In another preferred embodiment the solid support They are paramagnetic spheres. The paramagnetic spheres are covered by active groups that in appropriate situations react and covalently bind the selected peptide. Further, the paramagnetic spheres can be upholstered on the surface by streptavidin and, in that case, the peptides are conjugated with biotin to be able to attach to the solid support through the Streptavidin

In another more preferred embodiment the detection of step (d) of the method described above is carried out through:

to)

the derivatization binding of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13 with a molecule or enzyme by way of marker;

b)

incubation of the peptides obtained in (a) with the spores attached to the immobilized peptides obtained according to the method defined in the fifth aspect of this invention (section c);

C)

incubation of the product obtained in (b) with a complex formed by a marker binding element used in (a) and an element that can be detected and / or quantified;

d)

detection of spores by reaction of the incubation of the product obtained in (c) with a composition containing a chromogenic indicator substrate, Fluorogenic and / or chemiluminescent.

The term derivatization refers to a technique that transforms a chemical compound to a product of similar chemical structure that changes its reactivity, state of aggregation, etc ..., resulting in a product with new properties chemicals that can be used to join different groups reagents present on surfaces or other chemical compounds.

The binding element to the complex marker defined in (c) is a compound that shows chemical affinity enough to bind the peptide marker. As an example, if the peptide is labeled with biotin, the binding element can be Avidina

The element that can be detected and / or quantified must be a component that is capable of reacting with some substrate, so that a detection is derived from it chromogenic, fluorogenic and / or chemiluminescent. This item you can join the element of the previous paragraph (in the example, avidin) directly, or through another component such as biotin An example of an element that can be detected is the peroxidase enzyme. So, in a preferred embodiment, the marker is biotin and the complex is avidin-biotin peroxidase and, In addition, the indicator substrate is chromogenic, such as the tetramethylbenzidine (TMB), the acid azino-bis (3-ethylbenzothiazoline 6-sulfonic acid) or phenylenediamine, without limitation of Use other substrates.

In the case of using a chromogenic substrate, the presence of spores is evidenced by the appearance of a color whose intensity varies directly with the number of Clostridium tyrobutyricum spores present in the sample and their quantification can be carried out, for example, by means of spectrophotometry. If a fluorogenic substrate is used the detection and quantification is carried out by means of fluorimetry and if the substrate is chemiluminescent, the signal can be quantified by means of a luminometer.

In addition, the detection of step (d) above described can be carried out by:

to)

the obtaining anti-spore antibodies;

b)

antibody conjugation obtained in (a) with an enzyme;

C)

antibody incubation conjugates of (b) with the peptide-spore complexes obtained according to the method defined in the fifth aspect of the present invention (section c);

d)

incubation of the complexes obtained in (c) with a composition containing an indicator substrate chromogenic, fluorogenic and / or chemiluminescent.

In this case, it is necessary to inject in an animal, such as a rabbit, Clostridium tyrobutyricum sputum to obtain immunization in order to obtain it . For this purpose, subcutaneous injections are performed with a suspension of previously heated Clostridium tyrobutyricum spores. Three weeks later, rabbits are reinjected with an equal amount of spores. Two weeks after the second inoculation, a test bleed is performed and the presence of antibodies is checked by the immunodotting method. For successive bleeding, the animals are allowed to recover for 15 days, repeating the inoculations every approximately 30 days.

The peptide-spore complexes fixed on the solid support (for example paramagnetic spheres) are incubate with a solution of rabbit antisporos antibodies conjugated with an enzyme (for example peroxidase) and finally, after wash to remove unbound antibodies, they are transferred (the new complexes anchored in the solid support) to a plate of 96 wells (or other container) and incubated with an indicator substrate chromogenic, fluorogenic and / or chemiluminescent.

Another aspect of the present invention is a Clostridium tyrobutyricum spore detection kit comprising:

to)

a peptide selected from SEQ ID NO: 1 to SEQ ID NO: 13, or any of its combinations, marked or not, and immobilized in a solid support and

b)

means for the detection and quantification of Clostridium tyrobutyricum spores containing the complex formed by a spore-binding element labeled with an element capable of being detected and / or quantified as well as a chromogenic, fluorogenic and / or chemiluminescent substrate for that last item.

The peptides are immobilized on the solid support and labeled as defined in the previous aspect which deals with a method for the detection and / or quantification of Clostridium tyrobutyricum spores.

Also, the solid support has also been defined above as well as the different joining elements, markers, binding elements and modes of detection of Different substrates

In a preferred embodiment of the defined kit previously, the immobilization of the peptide of section (a) is carries out covalently, the detection of the spores of the section (b) is performed with the biotin-labeled peptide and the avidin-biotin-peroxidase complex and the indicator substrate is chromogenic.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the figures

Fig 1. Percentage of bacteriophages eluted during the different selection phases of the Clostridium tyrobutyricum spore binding peptides .

The X number indicates the number of the selection phase and on the Y axis the percentage of Recovery. (Recovery (%) = (Eluted Phages / Added Phages) x 100).

Fig 2. Detection of different concentrations of Clostridium tyrobutyricum spores , with 50 100 twenty 101 and 10 ({{117}) mug / mL of the biotinylated SEQ ID NO: 1 peptide.

Fig 3. Percentage of spores recovered with paramagnetic spheres upholstered with different concentrations of the peptide SEQ ID NO: 1 .

The theoretical number is indicated on the X axis of peptide molecules per sphere and on the Y axis the number of spores recovered (Recovery (%) = (Spore attached to the spheres / spurs added) x 100).

Fig 4. Detection of different concentrations of Clostridium tyrobutyricum spores .

The detection was carried out after concentration with spheres upholstered with 100 ({{110}), 1000 102 5000 103 and 10,000 104 peptide units SEQ ID NO: 1 per sphere, and incubating with 50 µg / mL of the same biotinylated peptide.

Examples

The invention will now be illustrated by tests describing the method of obtaining the peptides as well as the detection of Clostridium tyrobutyricum spores.

Example 1 Isolation of specific peptides against the spores of Clostridium tyrobutyricum by using a phage display library (Phage Display)

The phages from the library were incubated, to eliminate cross-reactions, with a mixed suspension of Clostridium acetobutylicum, Geobacillus stearothermophilus and Lactobacillus plantarum and subsequently, phages that were not eliminated in said subtraction, were incubated with the Clostridium tyrobutyricum spurs to select those who were related. After the process of binding the phages to the spores, they were washed 8 times with a buffer with 0.1% Tween-20 in order to eliminate those non-specific bound phages. After amplification of the selected phages, by incubating them in a culture with their host bacteria, the same protocol was repeated from the beginning 3 more times (in total, 4 cycles of subtraction and selection). In the last phase of selection the wash buffer was replaced with 6 M urea, in order to increase the washing force and thus select the phages with greater affinity. (Fig 1).

Once the phage selection was made that bind to the spores, agar plates were seeded LB-IPTG / Xgal with consecutive decimal dilutions of the phage suspension from the last selection cycle. Of these plates, and individually, several were transferred colonies from said plates to tubes containing 1 mL of a phage host bacteria culture, to amplify its number.

Subsequently, the phages were concentrated adding PEG / NaCl and its DNA was extracted by the addition of phenol. The DNA was concentrated by adding 0.1 volumes of acetate. 3M sodium, pH 5.2, and 2.5 volumes of absolute ethanol, leaving incubate at 4 ° C overnight. Finally, the DNA washed two times with 70% ethanol previously cooled in ice. Behind the Last wash, the rest of the ethanol left in the tubes was left evaporate in an oven at 40 ° C and the DNA was resuspended in 50 µL of a Tris-EDTA buffer. For optimum conservation, The extracted DNA was frozen at -80 until analysis.

After extraction, an amplification was performed of the DNA fragment in which the peptide was encoded, by a Polymerase Chain Reaction (PCR) with the specific primers After the reaction the size of the inserts amplified by agarose gel electrophoresis. The band that corresponded with the correct fragment was purified to starting the gel by means of a column system for centrifugation (GenElute ™ Agarose Spin Columns). Subsequently, the DNA was sequenced

Example 2 Sequence determination of isolated peptides

From the sequence obtained from all the amplified fragment, by locating the sequences flanking, the DNA sequence encoding the peptide expressed in the phage. Below are the sequences obtained:

SEQ ID NO: 14 (21).

SEQ ID NO: 15 (3).

SEQ ID NO: 16 (2).

SEQ ID NO: 17 (2).

SEQ ID NO: 18 (1).

SEQ ID NO: 19 (1).

SEQ ID NO: 20 (1).

SEQ ID NO: 21 (1).

SEQ ID NO: 22 (1).

SEQ ID NO: 23 (1).

SEQ ID NO: 24 (1).

SEQ ID NO: 25 (1).

SEQ ID NO: 26 (1).

The numbers in brackets refer to the number of DNA samples matching that sequence. These samples came from the 37 colonies of E.coli infected with the phages isolated from the final phase of the selection process.

From the nucleotide sequence, they deduced the sequences of 12 amino acids in each of them. The sequences obtained (ordered according to their frequency of appearance, from highest to lowest) were:

SEQ ID NO: 1 (57%).

SEQ ID NO: 2 (8%).

SEQ ID NO: 3 (5%).

SEQ ID NO: 4 (5%).

SEQ ID NO: 5 (3%).

SEQ ID NO: 6 (3%).

SEQ ID NO: 7 (3%).

SEQ ID NO: 8 (3%).

SEQ ID NO: 9 (3%).

SEQ ID NO: 10 (3%).

SEQ ID NO: 11 (3%).

SEQ ID NO: 12 (3%).

SEQ ID NO: 13 (3%).

Of all the peptide sequences obtained, the one that was repeated more frequently was synthesized, that is, SEQ ID NO: 1, in a "Fmoc" chemistry peptide synthesizer both without derivatizing, and including some modification amino-terminal with biotin or another molecule or enzyme as a marker (fluorescein, peroxidase, etc ...).

Example 3 Determination of the binding capacity of the peptide synthesized with the spores of Clostridium tyrobutyricum

Incubation of several suspensions was performed of different concentration of spores with 10, 20 and 50 µg / mL of peptide SEQ ID NO: 1 derivatized with biotin. Binding was detected using the complex avidin-biotin-peroxidase and the tetramethylbenzidine (TMB) substrate of peroxidase (Fig 2).

Example 4 Immobilization of the SEQ ID NO: 1 peptide in aminated paramagnetic spheres and detection of Clostridium tyrobutyricum spores

In the case of synthesized peptides without derivatize, were immobilized in paramagnetic spheres aminated to 10 2 -10 4 peptide / sphere ratio. For estimate the ability to bind sporo to spheres in depending on the amount of peptide bound, the spheres were incubated with a suspension of 10 3 spores and the percentage of recovery by enumeration in culture in a medium specific (Fig 3).

Paramagnetic spheres upholstered with peptides were used for the concentration of the spores of the sample to be analyzed. The sample was incubated in an Eppendorf-type vial with a concentration of spheres of at least 10 9 spheres / mL and subsequently, the spheres with the bound spurs were concentrated with the aid of a magnetic separator. Once washed to remove unspecifically bound microorganisms or components of the sample, the spherical-complexes were incubated with a solution of 50 µg / ml of the biotinylated peptide (Fig 4) and finally, after washing to remove unbound peptides, incubated with the avidin-biotin-peroxidase complex and then the spheres were transferred to a 96-well plate and a peroxidase substrate was added, such as tetramethylbenzidine (TMB), azino-bis (3-ethylbenzothiazoline 6- sulfonic) or phenylenediamine. In the case of TMB being used as a substrate, the color development reaction is stopped after 15 minutes of incubation with said substrate, by adding 50 µL of 2M sulfuric acid to each well. The presence of spores was evidenced by the appearance of a yellow color whose intensity varied directly with the number of Clostridium tyrobutyricum spores present in the milk sample.

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<223> surface expressed peptide of phage M13KE

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         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ hskip1cm
      

9

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<223> surface expressed peptide of phage M13KE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ hskip1cm
      

10

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<223> surface expressed peptide of phage M13KE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

         \ hskip1cm
      

12

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<223> surface expressed peptide of phage M13KE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

         \ hskip1cm
      

eleven

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<223> surface expressed peptide of phage M13KE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

         \ hskip1cm
      

13

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<223> surface expressed peptide of phage M13KE

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

         \ hskip1cm
      

14

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

         \ hskip1cm
      

fifteen

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

         \ hskip1cm
      

16

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

         \ hskip1cm
      

17

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ hskip1cm
      

18

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ hskip1cm
      

19

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

         \ hskip1cm
      

twenty

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

         \ hskip1cm
      

twenty-one

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

         \ hskip1cm
      

22

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

         \ hskip1cm
      

2. 3

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> sequence of biological interest encoding peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

         \ hskip1cm
      

24

         \ vskip0.400000 \ baselineskip
      

<210> 24

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 24

         \ hskip1cm
      

25

         \ vskip0.400000 \ baselineskip
      

<210> 25

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 25

         \ hskip1cm
      

26

         \ vskip0.400000 \ baselineskip
      

<210> 26

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of biological interest coding for peptides that recognize Clostrydium tyrobutyricum spores

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 26

         \ hskip1cm
      

27

Claims (15)

1. Peptide selected from the list consisting essentially in SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 or any combination thereof. 2. Use of a peptide selected from

to)

a peptide according to claim 1,

b)

a peptide selected from the list consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or

C)

any combination of the peptides according to (a) and / or (b). for the detection of spores of Clostridium tyrobutyricum .

         \ vskip1.000000 \ baselineskip
      

3. Use of a nucleotide sequence, which constitutes the coding sequence of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13 for the detection of Clostridium tyrobutyricum spores. 4. Use according to claims 2 and 3 wherein the Peptide is selected from SEQ ID NO: 1 to SEQ ID NO: 4 or Any of your combinations. 5. Use according to claim 4 wherein the Peptide is SEQ ID NO: 1. 6. Use according to any of claims 2 to 5 for the detection of Clostridium tyrobutyricum spores in a food. 7. Use according to claim 6 wherein the Food is milk or any dairy derivative. 8. Method of detection and / or quantification of Clostridium tyrobutyricum spores comprising:

to.

Selection of at least one peptide from Enter SEQ ID NO: 1 to SEQ ID NO: 13,

b.

immobilization of the peptide / s of (a), marked / s or not, on a solid support,

C.

incubation of a sample with the peptides immobilized in (b) for the binding of the spores to the peptides

d.

detection and / or quantification, using a marked element, from the spores joined in (c) to the immobilized peptides.

         \ vskip1.000000 \ baselineskip
      

9. Method according to claim 8 wherein the marking is done with a bookmark selected from the list that comprises radioisotopes, enzymes, fluorophores or any molecule liable to be conjugated with another molecule or detected and / or quantified directly. 10. Method according to claim 9 wherein the Mareaje is done with biotin. 11. Method according to claim 8 wherein the Solid support are paramagnetic spheres. 12. Method according to claim 8 wherein the Step 8 (d) detection is carried out by:

to)

the derivatization binding of any of the peptides SEQ ID NO: 1 to SEQ ID NO: 13 with a molecule or enzyme by way of marker.

b)

incubation of the peptides obtained in (a) with the spores bound to the immobilized peptides obtained in 8 (c).

C)

incubation of the product obtained in (b) with a complex formed by a marker binding element used in (a) and an element that can be detected and / or quantified,

d)

detection of spores by reaction of the incubation of the product obtained in (c) with a composition containing a chromogenic indicator substrate, Fluorogenic and / or chemiluminescent.

         \ vskip1.000000 \ baselineskip
      

13. Method according to claim 12 wherein the marker of step (a) is biotin, the complex of step (c) is avidin-biotin-peroxidase and the indicator substrate of step (d) is chromogenic. 14.Kittridium tyrobutyricum spore detection kit comprising:

to)

a peptide selected from SEQ ID NO: 1 to SEQ ID NO: 13, or any of its combinations, marked or not, and immobilized in a solid support and

b)

means for the detection and quantification of Clostridium tyrobutyricum spores containing the complexes defined in 12 (c) and a chromogenic, fluorogenic and / or chemiluminescent substrate.

         \ vskip1.000000 \ baselineskip
      

15. Kit according to claim 15 wherein the immobilization of the peptide of section (a) is carried out in a manner covalently, the detection of the spores of section (b) is carried out with the biotin-labeled peptide and the complex avidin-biotin-peroxidase and the Indicator substrate is chromogenic.