Method and composition for detecting bacterial contamination in food products

• US 6,387,650 B1
• Filed: 06/07/1995
• Issued: 05/14/2002
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: BioControl Systems, Inc. (Merck & Co., Inc.)
• Original Assignee: BioControl Systems, Inc. (Merck & Co., Inc.)
• Inventors: Chen, Chun-ming, Townsend, David E.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US08/484,593
• Time in Patent Office: 2,533 Days
• Field of Search: 435/34, 435/18, 435/19, 435/24, 435/29, 435/38, 435/39, 435/968, 436/172
• US Class Current: 435/34
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/045 Culture media therefor

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 hydrolyzed 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.

Claims

1. A method for detecting the presence or measuring the concentration of bacteria in a food product, comprising the steps of:

providing a bacterial growth medium formulated for food testing, which comprises a first enzyme substrate for a phosphatase, a second enzyme substrate for a glycosidase, and a third enzyme substrate for a peptidase, wherein said first, second and third enzyme substrates cause or produce an identical type of detectable signal when hydrolyzed by their respective enzymes;

inoculating said medium with said food product and incubating said medium under conditions suitable for bacterial growth for a period of time; and

,

detecting or measuring the identical type of detectable signal as an indication of the presence or the concentration of bacteria in said food product.

2. The method of claim 1, wherein said first, second and third substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate produces a separate detectable moiety when hydrolyzed and said separate detectable moiety causes or produces said identical type of detectable signal.

3. The method of claim 2, wherein said detectable moiety is a fluorescent moiety and said identical type of detectable signal is a fluorescent signal.

4. 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.

5. The method of claim 1, wherein said first enzyme substrate comprises 4-methylumbelliferyl phosphate, said second enzyme substrate comprises 4-methylumbelliferyl-β

-D-glucoside and said third enzyme substrate comprises L-alanine-7-amido-4-methyl coumarin.

6. The method of claim 1, wherein said food product is ground beef.

7. The method of claim 1, wherein said food product is chicken.

8. The method of claim 1, wherein said food product is drinking water.

9. The method of claim 1, wherein said medium is liquid.

10. The method of claim 1, wherein said period of time is no more than 24 hours.

11. A method for detecting the presence or measuring the concentration of bacteria in a food product, comprising the steps of:

providing a bacterial growth medium formulated for food testing, which comprises a first enzyme substrate for a glycosidase and a second enzyme substrate for a peptidase, wherein said first and second enzyme substrates cause or produce an identical type of detectable signal when hydrolyzed by their respective enzymes;

inoculating said medium with said food product and incubating said medium under conditions suitable for bacterial growth for a period of time; and

,

detecting or measuring the identical type of detectable signal as an indication of the presence or the concentration of bacteria in said food product.

12. The method of claim 11, wherein said first and second substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate produces a separate detectable moiety when hydrolyzed, and said separate detectable moiety causes or produces said identical type of detectable signal.

13. The method of claim 12, wherein said detectable moiety is a fluorescent moiety and said identical type of detectable signal is a fluorescent signal.

14. The method of claim 11, 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.

15. The method of claim 11, wherein said first enzyme substrate comprises 4-methylumbelliferyl-β

-D-glucoside and said second enzyme substrate comprises L-alanine-7-amido-4-methyl coumarin.

16. The method of claim 11, wherein said food product is ground beef.

17. The method of claim 11, wherein said food product is chicken.

18. The method of claim 11, wherein said food product is drinking water.

19. The method of claim 11, wherein said medium is liquid.

20. The method of claim 11, wherein said period of time is no more than 24 hours.

41. The method of claim 11 or 38, wherein said peptidase enzyme is an aminopeptidase enzyme.

21. A bacterial growth medium formulated for food testing, comprising a first enzyme substrate for a phosphatase, a second enzyme substrate for a glycosidase, and a third enzyme substrate for a peptidase, wherein said first, second and third enzyme substrates cause or produce an identical type of detectable signal when hydrolyzed by their respective enzymes.

22. The medium of claim 21, wherein said first, second and third substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate produces a separate detectable moiety when hydrolyzed, and said separate detectable moiety causes or produces said identical type of detectable signal.

23. The method of claim 22, wherein said detectable moiety is a fluorescent moiety and said identical type of detectable signal is a fluorescent signal.

24. The medium of claim 21, wherein said first enzyme substrate comprises 4-methylumbelliferyl phosphate, said second enzyme substrate comprises 4-methylumbelliferyl-β

-D-glucoside and said third enzyme substrate comprises L-alanine-7-amido-4-methyl coumarin.

25. The medium of claim 21, wherein said food product is ground beef.

26. The medium of claim 21, wherein said food product is chicken.

27. The medium of claim 21, wherein said food product is drinking water.

28. The medium of claim 21, wherein said medium is liquid.

37. The medium of claim 21 or 29, wherein said peptidase enzyme is an aminopeptidase enzyme.

29. A bacterial growth medium formulated for food testing, comprising a first enzyme substrate for a glycosidase and a second enzyme substrate for a peptidase, wherein said first and second enzyme substrates cause or produce an identical type of detectable signal when hydrolyzed by their respective enzymes.

30. The medium of claim 29, 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 detectable signal.

31. The medium of claim 30, wherein said detectable moiety is a fluorescent moiety and said identical type of detectable signal is a fluorescent signal.

32. The medium of claim 29, wherein said first enzyme substrate comprises 4-methylumbelliferyl-β

-D-glucoside and said second enzyme substrate comprise L-alanine-7-amido-4-methyl coumarin.

33. The medium of claim 29, wherein said food product is ground beef.

34. The medium of claim 29, wherein said food product is chicken.

35. The medium of claim 29, wherein said food product is drinking water.

36. The medium of claim 29, wherein said medium is liquid.

38. A method for detecting the presence or measuring the concentration of eukaryotic microbes in a food product, comprising the steps of:

providing a growth medium formulated for food testing, which comprises a first enzyme substrate for a phosphatase, a second enzyme substrate for a glycosidase, and a third enzyme substrate for a peptidase wherein said first, second and third enzyme substrates cause or produce an identical type of detectable signal when hydrolyzed by their respective enzymes;

inoculating said medium with said food product and incubating said medium under conditions suitable for microbial growth for a period of time; and

,

detecting or measuring the identical type of detectable signal as an indication of the presence or the concentration of eukaryotic microbes in said food product.

39. The method of claim 38, wherein said first, second and third substrates each having both a nutrient moiety and a detectable moiety linked together by a covalent bond, and each said substrate produces a separate detectable moiety when hydrolyzed, and said separate detectable moiety causes or produces said identical type of detectable signal.

40. The method of claim 38, wherein said eukaryotic microbes comprise a yeast.