Method and composition for detecting bacterial contamination in food products

Abstract

This invention relates to a method for detecting the existence or measuring the concentration of total viable bacteria in a test sample from a food product. A medium is provided which contains three or more different enzyme substrates each having a nutrient moiety and a detectable moiety linked together. When a substrate is hydrolysed by a bacterial enzyme to create a separate detectable moiety, it causes or produces a detectable signal. These substrates produce detectable signals when any one of a phosphatase enzyme, a glycosidase enzyme or a peptidase enzyme is present in the medium.

What is claimed is:  
     
         1 . Method for detecting the existence or measuring the concentration of bacteria in a test sample, comprising the steps of: 
 providing a bacterial growth medium comprising three or more different enzyme substrates, wherein each said substrate is hydrolysed by a different bacterial enzyme, and thereafter, causes or produces a detectable signal;    inoculating said medium with said test sample and incubating said medium under a condition suitable for bacterial growth for a certain time period; and    detecting or measuring the detectable signal as an indication of the existence or the concentration of bacteria in said test sample.    
     
     
         2 . The method of  claim 1 , wherein said different substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety, and said separate detectable moiety causes or produces a detectable signal.  
     
     
         3 . The method of  claim 1 , wherein said bacteria are selected from the group consisting of  Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcus hominis, Streptococcus constellatus, Streptococcus anginosus, Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum , and  Klebsiella pneumonia.    
     
     
         4 . The method of  claim 1 , wherein said bacterial enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase, sulfatase, β-D-xylosidase, peptidase, (L or D amino acid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin, and phosphohydrolase.  
     
     
         5 . The method of  claim 1 , wherein one of said substrates is hydrolysed by a phosphatase enzyme, another of said substrates is hydrolysed by a glycosidase enzyme, and a third said substrate is hydrolysed by a peptidase enzyme.  
     
     
         6 . The method of  claim 1 , wherein said detectable moiety is a fluorescent moiety and said detectable signal is a fluorescent signal.  
     
     
         7 . The method of  claim 1 , wherein said substrates comprise 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methyl coumarin.  
     
     
         8 . The method of  claim 1 , wherein said test sample is taken from a food product.  
     
     
         9 . The method of  claim 8 , wherein said food product is ground beef.  
     
     
         10 . The method of  claim 8 , wherein said food product is chicken.  
     
     
         11 . The method of  claim 8 , wherein said food product is water.  
     
     
         12 . The method of  claim 1 , wherein said medium is liquid.  
     
     
         13 . The method of  claim 1 , wherein said time period is no more than 24 hours.  
     
     
         14 . Method for detecting the existence or measuring the concentration of bacteria in a test sample, comprising the steps of: 
 providing a bacterial growth medium comprising two or more different substrates, wherein each said substrate is hydrolysed by a different bacterial enzyme and thereafter causes or produces an identical type of detectable signal;    inoculating said medium with said test sample and incubating said medium under a condition suitable for bacterial growth for a certain time period; and    detecting or measuring the detectable signal as an indication of the existence or the concentration of bacteria in said test sample.    
     
     
         15 . The method of  claim 14 , wherein said different substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety, and said separate detectable moiety causes or produces an identical type of detectable signal.  
     
     
         16 . The method of  claim 14 , wherein said bacteria are selected from the group consisting of  Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcus hominis, Streptococcus constellatus, Streptococcus anginosus, Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum , and  Klebsiella pneumonia.    
     
     
         17 . The method of  claim 14 , wherein said bacterial enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase, sulfatase, β-D-xylosidase, peptidase, (L or D amino acid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin, and phosphohydrolase.  
     
     
         18 . The method of  claim 14 , wherein said enzyme is selected from the group consisting of a phosphatase enzyme, a glycosidase enzyme and a peptidase enzyme.  
     
     
         19 . The method of  claim 14 , wherein said detectable moiety is a fluorescent moiety and said detectable signal is a fluorescent signal.  
     
     
         20 . The method of  claim 18  or  19 , wherein said substrates are selected from the group consisting of 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methyl coumarin.  
     
     
         21 . The method of  claim 14 , wherein said test sample is taken from a food product.  
     
     
         22 . The method of  claim 21 , wherein said food product is ground beef.  
     
     
         23 . The method of  claim 21 , wherein said food product is chicken.  
     
     
         24 . The method of  claim 21 , wherein said food product is water.  
     
     
         25 . The method of  claim 14 , wherein said medium is liquid.  
     
     
         26 . The method of  claim 14 , wherein said time period is no more than 24 hours.  
     
     
         27 . A bacterial growth medium comprising three or more different substrates, wherein each said substrate is hydrolysed by a different bacterial enzyme, and thereafter, causes or produces a detectable signal.  
     
     
         28 . The medium of  claim 27 , wherein said different substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety, and said separate detectable moiety causes or produces a detectable signal.  
     
     
         29 . The medium of  claim 27 , wherein said bacterial enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase, sulfatase, β-D-xylosidase, peptidase, (L or D amino acid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin, and phosphohydrolase.  
     
     
         30 . The medium of  claim 27 , wherein one of said substrates is hydrolysed by a phosphatase enzyme, another of said substrates is hydrolysed by a glycosidase enzyme, and a third said substrate is hydrolysed by a peptidase enzyme.  
     
     
         31 . The medium of  claim 27 , wherein said detectable moiety is a fluorescent moiety and said detectable signal is a fluorescent signal.  
     
     
         32 . The medium of  claim 27 , wherein said substrates comprise 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methyl coumarin.  
     
     
         33 . The medium of  claim 27 , further comprising a test sample from a food product.  
     
     
         34 . The medium of  claim 33 , wherein said food product is ground beef.  
     
     
         35 . The medium of  claim 33 , wherein said food product is chicken.  
     
     
         36 . The medium of  claim 33 , wherein said food product is water.  
     
     
         37 . The medium of  claim 27 , wherein said medium is liquid.  
     
     
         38 . A bacterial growth medium comprising two or more different substrates, wherein each said substrate is hydrolysed by a different bacterial enzyme and thereafter causes or produces an identical type of detectable signal.  
     
     
         39 . The medium of  claim 38 , wherein said different substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety, and said separate detectable moiety causes or produces an identical type of detectable signal.  
     
     
         40 . The medium of  claim 38 , wherein said bacterial enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, β-D-fucosidase, α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, pyrophosphatase, sulfatase, β-D-xylosidase, peptidase, (L or D amino acid)-aminopeptidase, L-alanine aminopeptidase, trypsin, chymotrypsin, and phosphohydrolase.  
     
     
         41 . The medium of  claim 38 , wherein said enzyme is selected from the group consisting of a phosphatase enzyme, a glycosidase enzyme and a peptidase enzyme.  
     
     
         42 . The medium of  claim 38 , wherein said detectable moiety is a fluorescent moiety and said detectable signal is a fluorescent signal.  
     
     
         43 . The medium of  claim 41  or  42 , wherein said substrates are selected from the group consisting of 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucoside and L-alanine-7-amido-4-methyl coumarin.  
     
     
         44 . The medium of  claim 38 , further comprising a test sample from a food product.  
     
     
         45 . The medium of  claim 44 , wherein said food product is ground beef.  
     
     
         46 . The medium of  claim 44 , wherein said food product is chicken.  
     
     
         47 . The medium of  claim 44 , wherein said food product is water.  
     
     
         48 . The medium of  claim 38 , wherein said medium is liquid.  
     
     
         49 . Method for detecting the existence or measuring the concentration of eukaryotic microbes in a test sample, comprising the steps of: 
 providing a growth medium comprising three or more different substrates, wherein each said substrate is hydrolysed by a different eukaryotic microbial enzyme and thereafter causes or produces a detectable signal;    inoculating said medium with said test sample and incubating said medium under a condition suitable for microbial growth for a certain time period; and    detecting or measuring the detectable signal as an indication of the existence or the concentration of eukaryotic microbes in said test sample.    
     
     
         50 . The method of  claim 49 , wherein said different substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate is hydrolysed by a different eukaryotic microbial enzyme to produce a separate detectable moiety, and said separate detectable moiety causes or produces a detectable signal.  
     
     
         51 . The method of  claim 49 , wherein said eukaryotic microbes comprise a yeast.  
     
     
         52 . The method of  claim 5  or  18 , wherein said peptidase enzyme is an aminopeptidase enzyme.  
     
     
         53 . The medium of  claim 30  or  41 , wherein said peptidase enzyme is an aminopeptidase enzyme.