阅读说明：本技术 用于检测食物腐败微生物的方法 (Method for detecting food spoilage microorganisms ) 是由 朱尔津·咖泽恩丹 于 2018-06-06 设计创作，主要内容包括：用于检测食物样品中的食物腐败微生物的方法,包括使食物样品与肽底物接触,所述肽底物包含具有650-900nm的发射波长的荧光剂、用于淬灭所述第一荧光剂的所述发射的具有650-900nm的吸收波长的非荧光剂,以及位于所述荧光剂和所述非荧光剂之间的切割位点,b)监测步骤a)中的含有所述肽底物的样品的荧光,其中荧光的增加指示存在食物腐败微生物。(A method for detecting a food spoilage microorganism in a food sample, comprising contacting a food sample with a peptide substrate comprising a fluorescent agent having an emission wavelength of 650- & 900nm, a non-fluorescent agent having an absorption wavelength of 650- & 900nm for quenching said emission of said first fluorescent agent, and a cleavage site located between said fluorescent agent and said non-fluorescent agent, b) monitoring fluorescence of the sample containing said peptide substrate in step a), wherein an increase in fluorescence is indicative for the presence of a food spoilage microorganism.)

1. A method for detecting food spoilage microorganisms in a food sample, comprising:

a) adding a first pH adjusting agent to the food sample to provide a food sample having a pH in the range of pH 1-5, separating any solid precipitate present in the pH adjusted food sample to provide a pH adjusted food sample,

adding a second pH adjusting agent to the pH adjusted food sample to provide a food sample having a pH in the range of pH6.5-9 to be used in step b).

b) Contacting a food sample with a peptide substrate, wherein the peptide substrate comprises a peptide comprising: a fluorescent agent having an emission wavelength of 650-,

c) monitoring the fluorescence of the sample containing the peptide substrate in step b),

wherein an increase in fluorescence is indicative of the presence of a food spoilage microorganism belonging to the first group.

2. The method of claim 1, wherein the first pH adjusting agent is selected from the group consisting of: hydrochloric acid, acetic acid, trichloroacetic acid and citric acid.

3. The method of claim 1 or 2, wherein the second pH adjusting agent is selected from the group consisting of: sodium hydroxide, sodium acetate, tris buffer and phosphate buffer.

4. The method of any one of the preceding claims, wherein the food sample is selected from the group consisting of: dairy products, fruit-based beverages, soft drinks, beer and wine.

5. The method of any one of the preceding claims, wherein the dairy product is a yoghurt, cheese, butter, curd, cream or milk, preferably milk.

6. The method of any one of the preceding claims, wherein the cleavage site is cleavable by a protease provided by a bacterium from the genus: pseudomonas (Pseudomonas), Alicyclobacillus (Alicyclobacillus), Bacillus (Bacillus), Clostridium (Clostridium), Corynebacterium (Corynebacterium), Arthrobacter (Arthrobacter), Lactobacillus (Lactobacillus), Listeria (Listeria), Microbacterium (Microbacterium), Micrococcus (Micrococcus) and Streptococcus (Streptococcus).

7. The method of any one of the preceding claims, wherein the peptide comprises a cleavage site selected from the group consisting of: AAAFALAC (SEQ ID NO:1), AAFAALAC (SEQ ID NO:2), AAAAFLAC (SEQ ID NO:3), FAAAALAC (SEQ ID NO:4), FAAFALAC (SEQ ID NO: 5).

8. The method of any one of claims 1-7, wherein the cleavage site is cleavable by a protease provided by a bacterium from the genus Bacillus, and wherein the peptide comprises a cleavage site AAAFALAC (SEQ ID NO: 1).

9. The method of any one of claims 1-6, wherein the first cleavage site is cleavable by a protease provided by Listeria monocytogenes (Listeria monocytogenes), and the peptide comprises a cleavage site selected from: AANAKTNC (SEQ ID NO:6), AANKVTNC (SEQ ID NO:7), ALNKVTNC (SEQ ID NO:8), ALNAKTNC (SEQ ID NO: 9).

10. The method as claimed in any one of the preceding claims, wherein the fluorescent agent is a cyanine dye having an emission wavelength of 650-900nm, and wherein the non-fluorescent agent is a cyanine dye having an absorption wavelength of 650-900 nm.

11. The method of any one of the preceding claims, wherein the method does not comprise a step of enriching a microbial population.

12. A kit for detecting a food spoilage microorganism, comprising:

a) a tube containing a peptide substrate comprising a fluorescent agent having an emission wavelength of 650-900nm, a non-fluorescent agent having an absorption wavelength of 650-900nm for quenching said emission of said fluorescent agent, and a cleavage site located between said fluorescent agent and said non-fluorescent agent, said cleavage site being cleavable by a protease specifically provided by a food spoilage microorganism belonging to a first group of food spoilage microorganisms consisting of a limited number of microorganism strains, species or genera, and being not cleavable by any compound provided by any microorganism not belonging to said first group of food spoilage microorganisms, preferably wherein said cleavage site is cleavable by a protease provided by a bacterium from the genera: pseudomonas, Alicyclobacillus, Bacillus, Clostridium, Corynebacterium, Arthrobacter, Lactobacillus, Listeria, Microbacterium, Micrococcus, and Streptococcus.

b) A tube comprising a pH adjusting agent, preferably selected from the group consisting of hydrochloric acid, acetic acid, trichloroacetic acid and citric acid,

c) a tube comprising a second pH adjusting agent, preferably selected from the group consisting of sodium hydroxide, sodium acetate, tris buffer and phosphate buffer.

13. The kit of claim 12, comprising a device for monitoring fluorescence, wherein the device is adapted to receive a tube containing a food sample and the peptide substrate.

14. The kit of claim 12 or 13, wherein the peptide comprises a cleavage site selected from: AAAFALAC (SEQ ID NO:1), AAFAALAC (SEQ ID NO:2), AAAAFLAC (SEQ ID NO:3), FAAAALAC (SEQ ID NO:4), FAAFALAC (SEQ ID NO:5), AANAKTNC (SEQ ID NO:6), AANKVTNC (SEQ ID NO:7), ALNKVTNC (SEQ ID NO:8), ALNAKTNC (SEQ ID NO: 9).

15. The kit of any one of claims 12-14, wherein the fluorogenic agent is a cyanine dye having an emission wavelength of 650-900nm, and wherein the non-fluorogenic agent is a cyanine dye having an absorption wavelength of 650-900 nm.

Technical Field

The present invention relates to methods for detecting food spoilage microorganisms. The invention also relates to kits for carrying out the methods of the invention.

Background

Despite the greatest efforts made in the food industry, food products such as dairy products and beverages such as beer and fruit juices can still be contaminated with food spoilage microorganisms, resulting in shortened shelf life, reduced palatability of the product, and in some cases health risks.

In the dairy industry, raw milk is subjected to various heating and other treatments to remove pathogenic microorganisms and increase the shelf life of dairy products. Pasteurization is mainly applied to certain food products, such as milk, to reduce the risk of microorganisms and to improve their preservation. Pasteurization is not intended to kill all pathogenic microorganisms in food or liquids. UHT (ultra high temperature treatment) is also used for milk treatment. UHT treatment milk was held at a temperature of 138 ℃ for a fraction of a second. In countries such as india where milk is produced in a non-standardized way, pasteurization and/or UHT treatment are not always effective in inactivating spoilage microorganisms, resulting in a reduced shelf life of milk. Currently, there is no rapid test to determine whether pasteurization and/or UHT treatment is effective. The sample must be incubated to enable bacterial enumeration in order to determine whether the sample is contaminated.

WO2016/018798 relates to an in vitro method for detecting biomarkers of inflammation, infection and/or bacterial activity in a milk sample from a cow, which indicates problems with the milk itself (i.e. spoilage) or problems with the health of the cow (i.e. mastitis).

In the brewing industry, it is necessary to determine whether a batch is contaminated with beer spoilage bacteria such as Lactobacillus lindneri, Lactobacillus brevis and Pediococcus damnosus. PCR has been used to detect beer spoilage microorganisms (S.borg,2016,229, Proceedings of World Brewing Congress), however, PCR still takes 24-48 hours to determine the presence of spoilage bacteria. There is still a need for rapid testing to minimize delays in the production process.

MicroSnap^TMListeria spp. (Hygenia) is a test for the detection and enumeration of Listeria. The test uses a bioluminescent test reaction that produces light when an enzyme specific to escherichia coli (e. Then, the user can use the device to perform the operation,in EnSURE^TMThe signal is generated by quantifying the light in a luminometer. Results were obtained in 24 hours. However, this method relies on enrichment for listeria. In order to be able to make a quick decision in a production environment, a quick field test needs to be performed in order not to unnecessarily delay the manufacturing process of the food product.

It is an object of the present invention to provide a rapid method for determining food spoilage microorganisms.

Summary of The Invention

The present invention seeks to provide a method for detecting food spoilage microorganisms in a food sample, comprising:

a) adding a first pH adjusting agent to the food sample to provide a food sample having a pH in the range of pH 1-5, separating any solid precipitate present in the pH adjusted food sample to provide a pH adjusted food sample, and adding a second pH adjusting agent to the pH adjusted food sample to provide a food sample having a pH in the range of pH6.5-9 to be used in step b),

b) contacting a food sample with a peptide substrate, wherein the peptide substrate comprises a peptide comprising: a fluorescent agent having an emission wavelength of 650-,

c) monitoring the fluorescence of the sample containing the peptide substrate in step b),

wherein an increase in fluorescence is indicative of the presence of a food spoilage microorganism belonging to the first group of food spoilage microorganisms.

According to the present invention, a method as defined above is provided which enables a rapid and specific detection of microorganisms causing food spoilage. Cleavage of the peptide at the cleavage site in the presence of a protease that recognizes and cleaves the cleavage site results in release of the first non-fluorescent agent from the peptide substrate, and thus a fluorescent signal can be detected by a suitable detector. An advantage of the present method is that it can be carried out in raw food samples without the need to enrich the microbial population by e.g. culturing the microorganisms. Thus, the method of the invention enables detection of bacterial contamination within, for example, 10 minutes of contacting the peptide substrate with the food sample.

Without wishing to be bound by theory, the inventors postulate that the emission wavelength of the fluorescent agent enables fluorescence to be detected in the region of minimal background fluorescence. The prior art methods rely on monitoring the region below 600nm by, for example, bioluminescence or by simple colorimetric detection. However, monitoring in the region below 600nm provides insufficient sensitivity due to background emission caused by spontaneous luminescence and/or autofluorescence of amino acids such as tryptophan, tyrosine and phenylalanine. The method according to the invention enables the detection of the presence of food spoilage microorganisms in a food sample without the need to enrich the microbial population.

The present invention also provides a kit for detecting a food spoilage microorganism, comprising:

a) a tube containing a peptide substrate comprising a fluorescent agent having an emission wavelength of 650-900nm, a non-fluorescent agent having an absorption wavelength of 650-900nm for quenching said emission of said fluorescent agent, and a cleavage site located between said fluorescent agent and said non-fluorescent agent, said cleavage site being cleavable by a protease specifically provided by a food spoilage microorganism belonging to a first group of food spoilage microorganisms consisting of a limited number of strains, species or genera of microorganisms and being cleavable by any compound provided by any microorganism not belonging to said first group of food spoilage microorganisms,

b) a tube comprising a first pH adjusting agent, preferably selected from the group consisting of hydrochloric acid, acetic acid, trichloroacetic acid and citric acid, and

c) a tube containing a second pH adjusting agent, preferably selected from the group consisting of sodium hydroxide, sodium acetate, tris buffer and sodium phosphate buffer.

Description of the embodiments

The present invention relates to a method for detecting food spoilage microorganisms, comprising:

a) adding a first pH adjusting agent to the food sample to provide a food sample having a pH in the range of pH 1-5, separating any solid precipitate present in the pH adjusted food sample to provide a pH adjusted food sample, and adding a second pH adjusting agent to the pH adjusted food sample to provide a food sample having a pH in the range of pH6.5-9 to be used in step b),

b) contacting a food sample with a peptide substrate, wherein the peptide substrate comprises: a fluorescent agent having an emission wavelength of 650-,

c) monitoring the fluorescence of the sample containing the peptide substrate in step b),

wherein an increase in fluorescence is indicative of the presence of a food spoilage microorganism belonging to the first group of food spoilage microorganisms.

The term "food spoilage" as used herein means an unsatisfactory change in the organoleptic properties of a food product due to mold, yeast and bacterial contamination.

The term "food spoilage microorganism" as used herein means a mold, yeast, or bacteria that causes food spoilage.

The term "food sample" as used herein means a food or beverage. The food product may be solid or fluid. Preferably, the food product is fluid, more preferably liquid.

The term "peptide" as used herein means an oligomer comprising at least 3 amino acids. Preferably, the peptide comprises no more than 20 amino acids. Preferably, the peptide comprises 4-20 amino acids, more preferably 6-15 amino acids. The amino acid used may be any amino acid, preferably selected from naturally occurring amino acids or synthetic amino acids, in particular derivatives of natural amino acids.

The term "cleavage site" as used herein means an amino acid motif that can be cleaved by a particular compound, whereby the cleavage site comprises one or more amide bonds. The cleavage site may have the structure XYZ, wherein X is at least one amino acid, Y is a part of a molecular structure consisting of at least two amino acids, and Z is at least one amino acid. The amino acids are preferably those that allow binding to the compound that effects cleavage at the first cleavage site.

The term "protease" as used herein means a protein secreted by a microorganism or present in the membrane of a microorganism, such as an enzyme, in other words a protease or a transpeptidase, capable of cleaving an amino acid motif.

The term "a first group of food spoilage microorganisms consisting of a limited number of microbial strains, species or genera" as used herein means a class of microorganisms that share the ability to cleave cleavage sites present in a peptide substrate.

The term "limited" means that such a group will not contain all microorganisms or all bacteria, but rather a small, i.e. limited, number, such that the release of non-fluorescent agents is indeed indicative of one or more food spoilage microorganisms, but not all or any microorganisms or bacteria, etc.

It has been found that by lowering the pH of a fluid food sample to pH 1-5, proteinaceous matter having an isoelectric point in the range of pH 1-5 can be easily removed as a precipitate, and thus proteins that may cause background noise in food sample measurements can be easily removed without affecting the activity of proteases retained in solutions of pH 1-5. The pH of the resulting solution is then adjusted to bring the pH to a pH in the range of pH 6.5-9. Preferably, such removal of proteinaceous matter is carried out when the food sample is a dairy product, such as yoghurt, cream or milk.

A first pH modifier is added to the fluid food sample to provide a food sample having a pH in the range of pH 2-5, more preferably pH 3-5, even more preferably pH 4-5. A second pH adjusting agent is added to the resulting fluid sample to provide a fluid food sample having a pH in the range of pH7.5-9, more preferably pH 8-9.

The separation of the solid precipitate from the remaining sample can be performed by, for example, centrifugation, filtration or decantation. Preferably, the solid precipitate is separated by centrifugation.

In a preferred embodiment, the first pH adjusting agent is selected from: hydrochloric acid, acetic acid, trichloroacetic acid and citric acid.

In embodiments, the second pH adjusting agent is selected from: sodium hydroxide, sodium acetate, tris buffer and phosphate buffer.

Preferably, the food sample is selected from: dairy products, fruit-based beverages, soft drinks, beer and wine.

In a preferred embodiment, the dairy product is a yoghurt, cheese, butter, curd, cream or milk, preferably milk.

The food sample may have been pasteurized or UHT treated. Sampling food samples after pasteurization or UHT process treatment provides an indication as to whether the treatment step has successfully eliminated food spoilage microorganisms from the samples.

Preferably, the cleavage site is cleavable by a protease provided by a bacterium from the genus: pseudomonas (Pseudomonas), Bacillus (Bacillus), Clostridium (Clostridium), Corynebacterium (Corynebacterium), Arthrobacter (Arthrobacter), Alicyclobacillus (Alicyclobacillus), Lactobacillus (Lactobacillus), Listeria (Listeria), Microbacterium (Microbacterium), Micrococcus (Micrococcus) and Streptococcus (Streptococcus). Preferably, the bacteria are selected from: alicyclobacillus, bacillus and listeria. Preferably, the bacteria are selected from: alicyclobacillus acidoterrestris (a), alicyclobacillus thermophilus (a), cyclobacillus pear (a pomorum), alicyclobacillus fastidiosus (afastoidosis).

In some preferred embodiments, the cleavage site is a substrate for a serine protease or a serine transpeptidase. Preferably, the serine protease belongs to group ec.3.4.21.62, such as subtilisin (subtilisin), or group ec.3.4.24.26, such as pseudolysin.

Preferably, the cleavage site has the structure XYZ, wherein X is at least one amino acid, Y is part of a molecular structure consisting of at least two amino acids, and Z is at least one amino acid. X and Z may be any amino acid. Preferably, Y comprises a dipeptide consisting of an aliphatic, hydrophobic amino acid and an aromatic or cyclic amino acid or a basic amino acid and a hydrophilic amino acid. The aliphatic hydrophobic amino acids are preferably selected from: glycine, alanine, leucine, valine and derivatives thereof. The aromatic or cyclic amino acids are preferably selected from: phenylalanine, tyrosine, tryptophan, proline and derivatives thereof. The basic amino acid is preferably lysine, and the hydrophilic amino acid is preferably threonine or serine.

Preferably, Y comprises a dipeptide that is a combination of alanine-phenylalanine, alanine-tryptophan, alanine-proline or lysine-threonine, more preferably, Y is a combination of alanine-phenylalanine, alanine-tryptophan, alanine-proline.

Preferably, the peptide comprises a sequence selected from: AAAFALAC (SEQ ID NO:1), AAFAALAC (SEQ ID NO:2), AAAAFLAC (SEQ ID NO:3), FAAAALAC (SEQ ID NO:4), FAAFALAC (SEQ ID NO: 5).

In a preferred embodiment, the peptide has a sequence selected from the group consisting of: AAAFALAC (SEQ ID NO:1), AAFAALAC (SEQ ID NO:2), AAAAFLAC (SEQ ID NO:3), FAAAALAC (SEQ ID NO:4), FAAFALAC (SEQ ID NO:5), wherein the peptide substrate is cleavable by a protease from Pseudomonas, Bacillus, Clostridium, Corynebacterium, Arthrobacter, Microbacterium, Micrococcus, and Streptococcus. In this embodiment, the method according to the invention enables monitoring of the contamination of the most prevalent food spoilage organisms. Such monitoring is important in, for example, the dairy industry, where any food spoilage bacteria present in the dairy product can shorten shelf life, resulting in poor mouthfeel of the product or health risks upon consumption.

In another preferred embodiment, the cleavage site is cleavable by a protease provided by a bacterium from the genus Bacillus and wherein the peptide comprises the cleavage site AAAFALAC (SEQ ID NO: 1).

Preferably, the peptide is a substrate for a protease provided by a bacterium from the genus listeria, such as listeria monocytogenes (listeria monocytogenes), and the peptide comprises a sequence selected from the group consisting of: AANAKTNC (SEQ ID NO:6), AANKVTNC (SEQ ID NO:7), ALNKVTNC (SEQ ID NO:8), ALNAKTNC (SEQ ID NO: 9). When the peptide has a sequence selected from the group consisting of SEQ ID NO 6-SEQ ID NO 9, the peptide contains a cleavage site specific for Listeria monocytogenes such that the peptide substrate is cleavable by a protease specifically provided by Listeria monocytogenes but not by a food spoilage microorganism belonging to said species of food spoilage microorganism.

In another preferred embodiment, the substrate comprises a second cleavage site, which is cleavable by a protease specifically provided by a food spoilage microorganism belonging to a second group consisting of a limited number of microorganism strains, species or genera, and which is not cleavable by any compound provided by any microorganism not belonging to said second and/or first group.

In another embodiment, the second cleavage site is different from the first cleavage site.

Preferably, the fluorescent agent is a cyanine dye having an emission wavelength of 650-900nm, and the non-fluorescent agent is a cyanine dye having an absorption wavelength of 650-900 nm.

In embodiments, the first fluorescent agent is a cyanine dye having a general formula as shown in formula I, wherein R is¹Selected from: H. halogen and

wherein R is¹⁷Selected from: : carboxy, amino and sulfo (sulfonato); x is selected from: o, S, NH and N-hydrocarbyl; r²、R³、R⁹、R¹⁰Each independently selected from: h and a hydrocarbyl group; r⁴、R⁵、R¹¹、R¹²Each independently selected from: H. hydrocarbyl radicalAnd a sulfo group, or together with the atoms to which they are bound form an aromatic ring; r⁶、R⁷、R¹³、R¹⁴Each independently selected from: h and a hydrocarbon radical, R⁸And R¹⁵Each independently selected from: hydrocarbyl, (CH)₂) qFG or (CH)₂)_PLN, wherein R⁸And R¹⁵Is (CH)₂) qFG, wherein q is an integer from 1 to 20 and FG is a functional group that does not react directly with a carboxyl, hydroxyl, amino or thiol group, wherein p is an integer from 1 to 20 and LN is a linker group that reacts with a carboxyl, hydroxyl, amino or thiol group; r¹⁶Is H or a hydrocarbyl group.

Preferably, the fluorescer is an agent wherein R is¹Is that

Wherein X is O, and R¹⁷Is SO₃Na；R²、R³、R⁹、R¹⁰Is a hydrocarbyl group, preferably methyl; r⁴And R¹¹Is H, and R⁵And R¹²Is H or sulfo; r⁶、R⁷、R¹³、R¹⁴Is H; r8 is (CH)₂) qFG, wherein q is 4 and FG is sulfo; r¹⁵Is (CH)₂)_PLN, wherein p is 5, and LN is carboxy, R¹⁶Is H.

Even more preferably, the fluorescer is an agent wherein R is¹Is that

Wherein X is O, and R¹⁷Is SO₃Na；R²、R³、R⁹、R¹⁰Is methyl; r⁴And R¹¹Is H, and R⁵And R¹²Is a sulfo group; r⁶、R⁷、R¹³、R¹⁴Is H; r8 is (CH)₂) qFG, wherein q is 4 and FG is sulfo; r¹⁵Is (CH)₂)_PLN, wherein p is 5, and LN is carboxy, R¹⁶Is H. Preferably, the fluorescent agent is an agent corresponding to formula II.

Non-fluorescent agents having an absorption wavelength of 650-900nm are compounds which have little or no intrinsic fluorescence and which can effectively quench fluorescence from adjacent fluorophores with little background. In embodiments, the non-fluorescent agent is a cyanine molecule. Cyanine molecules, also known as cyanine dyes, include compounds having two substituted or unsubstituted nitrogen-containing heterocycles connected by a polymethine chain.

In a preferred embodiment, the non-fluorescent agent is an agent wherein R is¹Is chlorine, R²、R³、R⁹、R¹⁰Is methyl; r⁴Is H, and R⁵Is an N-hydrocarbyl radical, preferably N [ (CH)₂)₃SO₃Na]₂；R¹¹And R¹²Forming an aromatic ring monosubstituted with sulfo; r⁶、R⁷、R¹³、R¹⁴Is H; (ii) a R⁸Is (CH)₂) qFG, wherein q is 3 and FG is sulfo; r¹⁵Is (CH)₂)_PLN, wherein p is 5, and LN is carboxy; r¹⁶Is H.

In another embodiment, the fluorescer and the non-fluorescent agent are the same agent, preferably wherein R is¹Is that

X is O, and R¹⁷Is SO₃Na、R²、R³、R⁹、R¹⁰Is a hydrocarbon radical, preferably methyl, R⁴、R⁵、R¹¹、R¹²Is H, R⁶、R⁷、R¹³、R¹⁴Is H, R⁸Is (CH)₂) qFG, wherein q is 4 and FG is sulfo, R¹⁵Is (CH)₂) PLN, wherein p is 5 and LN is carboxy, R¹⁶Is H.

The non-fluorescent agent may also be a quenching moiety, such as BHQ3, (Biosearch) QC-1(Li-cor. com) or particles comprising such compounds, such as gold nanoparticles and iron nanoparticles. In embodiments, the peptide substrate is a nanoparticle comprising a peptide as defined herein.

Examples of fluorescent agents that may be used in the present invention include, but are not limited to: : alexa

660、Alexa

680、Alexa

700、Alexa

750ATTO 680、ATTO 700、DY-647、DY-650、DY-673、DY-675、DY-676、DY-680、DY-681、DY-682、DY-690、DY-700、DY-701、DY-730、DY-731、DY-732、DY-734、DY-750、DY-751、DY-752、DY-776、DY-781、DY-782、DY-831、La Jolla Blue、Cy5、Cy5.5、Cy7、

800CW、

38、

800RS、

700DX、

680, etc. The "Alexa Fluor" dye is available from Molecular Peptides inc, Eugene, OR, u.s.a. (www.peptides.com). "ATTO" dyes are available from ATTO-tec GmbH, Siegen, Germany (www.atto-tec. "DY" dyes are available from Dyomics GmbH, Jena, Germany (www.dyomics.com). LaJollaBlue is available from Hyperion Inc. "Cy" dyes are available from Amersham Biosciences, Piscataway, NJ, U.S. A. (www.amersham.com).

Can be selected fromBioscience,Inc.Lincoln,N E,U.S.A(www.licor.com) And (4) obtaining.

Preferably, the method does not comprise a step of enriching the microbial population. Advantageously, the present invention can detect the presence of food spoilage microorganisms without first enriching the number of microorganisms present by incubating the sample for several hours.

In embodiments, the method comprises contacting the sample with a peptide substrate comprising a second peptide comprising a second cleavage site that is cleavable by a protease specifically provided by a food spoilage microorganism belonging to a second group consisting of a limited number of strains, species or genera of microorganisms and that is not cleavable by any compound provided by any microorganism not belonging to said second or first group.

In another aspect, the invention relates to a kit for detecting a food spoilage microorganism, comprising: a) a tube containing a peptide substrate comprising a fluorescent agent having an emission wavelength of 650900 nm, a non-fluorescent agent having an absorption wavelength of 650-900nm for quenching said emission of said fluorescent agent, and a cleavage site located between said fluorescent agent and said non-fluorescent agent, said cleavage site being cleavable by a protease specifically provided by a food spoilage microorganism belonging to a first group of food spoilage microorganisms consisting of a limited number of strains, species or genera of microorganisms and being cleavable by any compound provided by any microorganism not belonging to said first group of food spoilage microorganisms,

b) a tube comprising a first pH adjusting agent, preferably selected from the group consisting of hydrochloric acid, acetic acid, trichloroacetic acid and citric acid,

c) a tube comprising a second pH adjusting agent, preferably selected from the group consisting of sodium hydroxide, sodium acetate, tris buffer and phosphate buffer.

In a preferred embodiment, the kit comprises a device for monitoring fluorescence, wherein said device is adapted to receive a tube containing a food sample and a peptide substrate.

The embodiment descriptions for the method may be applied mutatis mutandis to the kit according to the invention.

Advantageously, the kit enables detection of food spoilage microorganisms in a simple manner without first incubating the sample. The kit can be used in a production environment and does not require specialized laboratory equipment.

The invention has been described above with reference to a number of exemplary embodiments as shown in the accompanying drawings. Modifications and alternative implementations of some parts or elements are possible and are included in the scope of protection as defined in the appended claims.

Fig. 1 shows a plot of RFU versus time for milk samples contaminated with Pseudomonas aeruginosa (circles), Bacillus Cereus (crosses) and human Ochrobactrum anthropi (triangles).