U.S. patent application number 09/923695 was filed with the patent office on 2002-03-14 for method and composition for detecting bacterial contamination in food products.
This patent application is currently assigned to Biocontrol Systems, Inc.. Invention is credited to Chen, Chun-Ming, Townsend, David E..
Application Number | 20020031796 09/923695 |
Document ID | / |
Family ID | 23924786 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031796 |
Kind Code |
A1 |
Townsend, David E. ; et
al. |
March 14, 2002 |
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.
Inventors: |
Townsend, David E.;
(Scarborough, ME) ; Chen, Chun-Ming; (Falmouth,
ME) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Biocontrol Systems, Inc.
|
Family ID: |
23924786 |
Appl. No.: |
09/923695 |
Filed: |
August 6, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09923695 |
Aug 6, 2001 |
|
|
|
09038665 |
Feb 24, 1998 |
|
|
|
09038665 |
Feb 24, 1998 |
|
|
|
08484593 |
Jun 7, 1995 |
|
|
|
Current U.S.
Class: |
435/34 |
Current CPC
Class: |
C12Q 1/045 20130101;
C12Q 1/04 20130101 |
Class at
Publication: |
435/34 |
International
Class: |
C12Q 001/04 |
Claims
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-.beta.-D-galactosaminidase,
N-acetyl-.beta.-D-glucosamin- idase, Neuraminidase,
L-arabinopyranosidase, .beta.-D-fucosidase, .alpha.-L-fucosidase,
.beta.-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .alpha.-D-glucosidase,
.beta.-D-glucosidase, .beta.-D-glucuronidase,
.alpha.-D-mannosidase, pyrophosphatase, sulfatase,
.beta.-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-.beta.-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-.beta.-D-galactosaminidase,
N-acetyl-.beta.-D-glucosamin- idase, Neuraminidase,
L-arabinopyranosidase, .beta.-D-fucosidase, .alpha.-L-fucosidase,
.beta.-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .alpha.-D-glucosidase,
.beta.-D-glucosidase, .beta.-D-glucuronidase,
.alpha.-D-mannosidase, pyrophosphatase, sulfatase,
.beta.-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-.beta.-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-.beta.-D-galactosaminidase,
N-acetyl-.beta.-D-glucosamin- idase, Neuraminidase,
L-arabinopyranosidase, .beta.-D-fucosidase, .alpha.-L-fucosidase,
.beta.-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .alpha.-D-glucosidase,
.beta.-D-glucosidase, .beta.-D-glucuronidase,
.alpha.-D-mannosidase, pyrophosphatase, sulfatase,
.beta.-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-.beta.-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-.beta.-D-galactosaminidase,
N-acetyl-.beta.-D-glucosamin- idase, Neuraminidase,
L-arabinopyranosidase, .beta.-D-fucosidase, .alpha.-L-fucosidase,
.beta.-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .alpha.-D-glucosidase,
.beta.-D-glucosidase, .beta.-D-glucuronidase,
.alpha.-D-mannosidase, pyrophosphatase, sulfatase,
.beta.-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-.beta.-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.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods and compositions for
detecting the existence or measuring the concentration of bacterial
contamination in food products.
BACKGROUND OF THE INVENTION
[0002] Ground beef and chicken are susceptible to rapid spoilage by
psychotropic bacteria which thrive at refrigeration temperatures.
As a result, these products have very short shelf-lives which are
directly related to the initial concentration of contaminating
bacteria.
[0003] Current methods for measuring the concentrations of
bacterial contamination in ground beef and chicken include the
standard plate count (Difco Laboratories) as well as the Petri Film
system (3M) (see generally, Compendium of Methods for the
Microbiological Examination of Foods, Third Edition, Edited by Carl
Vanderzant and Don F. Splittstoesser, Compiled by the APHA
Technical Committee on Microbiological Methods for Foods). These
methods require around 48 hours of incubation in a 35.degree. C.
incubator before the results can be read. Both methods utilize a
solid nutrient base to support the growth of individual cells into
bacterial colonies. Many food-borne bacteria are incapable of
growing into colonies on these surfaces when incubated at
35.degree. C.; thus, the concentrations of total viable bacteria
measured by the above methods may be underestimated.
[0004] In addition, the long incubation periods of these methods
can cause these food products to remain in storage for several days
until the concentrations of contaminating bacteria are known. If
these tests could be completed in a shorter period of time it would
allow companies to release their products sooner so as to lower
costs, increase sales, and provide better product to the
consumer.
[0005] There have been attempts to measure the bacterial
concentration in food by measuring specific metabolic by-products
of individual microorganisms. These methods include: electrical
impedance assays, ATP assays, antibody-based assays, and carbon-14
labelled substrate assays. Indicators of microbial growth have also
been used to monitor the growth of target microbes which change
color only after growth of the target microbe is detected. These
indicators normally react chemically with a metabolic by-product
produced by the is target microbes resulting in a color change in
the medium. Examples of chemicals which change color in the
presence of pH changes associated with growth include phenol red,
bromocresol blue, and neutral red. For example, Golber, U.S. Pat.
No. 3,206,317, uses phenol red, a chemical which changes color in
the presence of acidic waste products produced by the target
microbe. Berger et al., U.S. Pat. No. 3,496,066, describes the use
of compounds which bacteria convert to dyestuffs, e.g., tropinones
and dioxanes, Bochner, U.S. Pat. No. 4,129,483 describes using a
non-biodegradable substance (tetrazolium) which is chemically
reduced to produce a color change. In all of these examples, the
indicator is a compound which does not serve as a source of a
required nutrient.
[0006] Edberg (U.S. Pat. No. 4,925,789), incorporated by reference
herein, describes a selective growth medium for a microbe
containing a nutrient indicator which can only be metabolized by a
target microbe. When metabolized by a target microbe, the nutrient
indicator releases a moiety which imparts a detectable change to
the medium.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a bacterial growth medium
and methods for detecting the existence or measuring the
concentration of bacteria in a test sample. The claimed medium and
methods measure viable bacteria as a function of the activities of
several classes of bacterial enzymes, including, but not limited
to, phosphatases, glycosidases (such as glucosidases), and
aminopeptidases. The presence of at least one of these groups of
enzymes in any given bacterial species will be detected by the
appearance of a detectable signal such as a fluorescent signal.
Therefore, this invention is useful in detecting the existence or
measuring the concentration of total viable bacteria or at least a
multitude of viable bacteria in a test sample in a single assay. In
specific examples, cocktails of enzyme substrates are made to
measure the concentration of bacterial contamination in food
products, such as ground beef and chicken.
[0008] Thus, in one aspect, the invention features a bacterial
growth medium containing three or more different enzyme substrates
each hydrolysed by a different bacterial enzyme to cause or produce
a detectable signal.
[0009] In a preferred embodiment, the three or more different
enzyme substrates each has both a nutrient moiety and a detectable
moiety linked together by a covalent bond. Each of these enzyme
substrates is hydrolysed by a different bacterial enzyme to produce
a separate detectable moiety which causes or produces a detectable
signal in the medium. In a further preferred embodiment, the
detectable signals caused or produced are of identical type.
[0010] By "medium" is meant a solid, powder or liquid mixture which
contains all or substantially all of the nutrients necessary to
support bacterial growth. Amino acids, minerals, vitamins and other
elements known to those skilled in the art to be necessary for
bacterial growth are provided in the medium, including, but not
limited to, those disclosed in U.S. application Ser. Nos.
08/334,788 and 08/335,149, both filed on Nov. 4, 1994, incorporated
by reference herein. In a preferred embodiment, the medium is
liquid.
[0011] For example, the following components are provided in the
medium in approximately the amounts indicated. Those in the art
will understand that not every component is required. Components
may also be substituted with other components of similar
properties. The amounts of components may also be varied.
[0012] Amino acids may be provided from a variety of sources. These
can be provided from natural sources (e.g., extracts of organisms),
as mixtures, or in purified form. The natural mixtures may contain
varying amounts of such amino acids and vitamins. Not all amino
acids must be provided, and the relative amount of each can vary.
For general guidance, specific amounts of such amino acids and
vitamins are indicated below. These amounts are for guidance only
and are not limiting in this invention. Those in the art will
recognize that many different combinations of amino acids and
vitamins can be used in the medium of this invention. The lists
provided below exemplify just one such example. Normally, only
amino acids which cannot be synthesized endogenously by the
microorganisms to be detected must be provided. However, other
amino acids may be provided without departing from the medium of
the invention.
[0013] The medium preferably includes at least the following amino
acids in approximately the following amounts (per liter of medium):
Alanine (0.015 to 0.60 grams), Arginine (0.080 to 3.2 grams),
Aspartic Acid (0.018 to 0.72 grams), Cystine (0.09 to 3.6 grams),
Glutamic Acid (0.030 to 1.20 grams), Glycine (0.050 to 2.00 grams),
Histidine (0.025 to 1.00 grams), Isoleucine (0.035 to 1.40 grams),
Leucine (0.040 to 1.60 grams), Lysine (0.050 to 2.00 grams),
Methionine (0.01 to 0.50 grams), Phenylalanine (0.01 to 0.90
grams), Proline (0.02 to 2.80 grams), Serine (0.01 to 0.40 grams),
Threonine (0.01 to 1.10 grams), Tryptophan (0.002 to 0.26 grams),
Tyrosine (0.01 to 1.20 grams), and Valine (0.02 to 1.10 grams).
[0014] Salts may be provided as a source of ions upon dissociation.
Such salts may include (per liter of medium): potassium chloride
(e.g., about 0.5 to 1.5 grams); copper sulfate (e.g., about 40 to
50 .mu.g); ammonium acetate or ammonium sulfate (e.g., about 4.0 to
6.0 grams); potassium iodide (e.g., about 50.0 to 150.0 .mu.g);
ferric chloride (e.g., about 150.0 to 250.0 .mu.g); manganese
sulfate (e.g., about 300.0 to 500.0 .mu.g); sodium molybdate (e.g.,
about 150.0 to 250.0 .mu.g); zinc sulfate (e.g. about 300.0 to
500.0 .mu.g); and sodium chloride (e.g. about 0.05 to 0.15 g).
[0015] Other inorganic moieties may be included to aid microbial
growth. These include the following (to the extent not already
provided in the above sources of various chemical entities and
described in amounts per liter): Phosphorus (about 0.5 mg),
Potassium (about 0.4 mg), Sodium (about 30 to 60 mg), and trace
amounts of Calcium, Magnesium, Aluminum, Barium, Chloride, Cobalt,
Copper, Iron, Lead, Manganese, Suffate, Sulfur, Tin and Zinc.
[0016] Vitamins required for growth and reproduction of the
microorganism sought to be detected may also be provided. These can
be provided in a pure form or as part of a more complex medium.
Such vitamins may be present in approximately the following amounts
(per liter of medium): Biotin (about 0.15 to 60 .mu.g), Pantothenic
Acid (about 15.0 to 65.0 .mu.g), Pyridoxine (about 2.0 to 9.0
.mu.g), Riboflavin (about 10.0 to 50.0 .mu.g), Folic acid (about
5.00 to 50.00 .mu.g), Thiamine (about 10.0 to 50.0 .mu.g), Vitamin
B12 (about 0.20 to 0.50 .mu.g), and Niacin (about 15.0 to 55.0
.mu.g).
[0017] By "bacterial enzyme" is meant an enzyme whose enzymatic
activity such as the ability to hydrolyse a substrate or a
plurality of substrates is characteristic of a bacterium or a
plurality of bacteria. In this invention, the enzymatic activities
of a bacterial enzyme or bacterial enzymes are used to detect or
measure the concentration of bacteria in a test sample. The
bacterial enzymes include all those known to one skilled in the
art, including, but not limited to, those listed in Enzymes, 3rd
edition, edited by Malcolm Dixson, Edwin C. Webb, C. J. R. Thorne,
and K. F. Tipton, 1979, Academic Press, U.S.A. In a preferred
embodiment, the bacterial enzyme is selected from the group
consisting of alkaline phosphatase, acid phosphatase, esterase,
lipase, N-acetyl-.beta.-D-galact- osaminidase,
N-acetyl-.beta.-D-glucosaminidase, Neuraminidase,
L-arabinopyranosidase, .beta.-D-fucosidase, .alpha.-L-fucosidase,
.beta.-L-fucosidase, .alpha.-D-galactosidase,
.beta.-D-galactosidase, .alpha.-D-glucosidase,
.beta.-D-glucosidase, .beta.-D-glucuronidase,
.alpha.-D-mannosidase, pyrophosphatase, sulfatase,
.beta.-D-xylosidase, peptidase (preferably an aminopeptidase, more
preferably an (L or D amino acid)-aminopeptidase), trypsin,
chymotrypsin, and phosphohydrolase.
[0018] By "substrate" is meant a molecule or substance on which a
bacterial enzyme acts. The enzymatic reaction usually involves
hydrolysing one or more covalent bonds, forming one or more
covalent bonds, or both. A covalent bond in the substrate between
the nutrient moiety and the detectable moiety is hydrolysed by a
bacterial enzyme to produce a separate detectable moiety. The
substrates include all those known to one skilled in the art,
including, but not limited to, those in the product listing of
AerChem, Inc. with detectable moieties attached thereto (see Table
I).
[0019] By "nutrient moiety" is meant a molecule or substance which
is a nutrient or metabolic source for a bacterium, including, but
not limited to, vitamins, minerals (e.g., phosphorus in the form of
phosphate), trace elements, amino acids (e.g., L-alanine), carbon
(e.g., glucose), or nitrogen.
[0020] By "detectable signal" is meant a characteristic change in a
medium or sample that is observable or measurable by physical,
chemical, or biological means known to those skilled in the art.
Such a detectable signal may be a change in emission or absorbance
of visible or invisible light or radio waves at a certain
wavelength, electrical conductivity, hybridization, enzymatic
reaction, emission of gas, or odor. A detectable signal may also be
a change in physical state such as between solid, liquid and gas.
In preferred embodiments, detectable signals include a change in
color or fluorescent emission of the medium.
[0021] By "identical type of detectable signal" is meant that the
separate detectable moieties hydrolysed from different enzyme
substrates cause or produce detectable signals that are measurable
by the same or substantially the same physical, chemical or
biological parameter, including, but not limited to, color,
fluorescent emission, odor, enzymatic reaction, hybridization, or
electric conductivity (although the intensity or quantity of
signals caused or produced by different separate detectable
moieties may be different). For example, yellow colors of different
intensity would be considered of the identical type. Color change
and fluorescence would not be considered to be identical type of
detectable signal.
[0022] By "detectable moiety" is meant a molecule or substance
which can be covalently linked to a nutrient moiety or exists as a
separate entity by itself. The detectable moiety does not cause or
produce a detectable signal when it is covalently bonded to a
nutrient moiety. However, when an enzyme from a bacterium
hydrolyses the substrate, a detectable moiety is released and
causes or produces a detectable signal. In preferred embodiments,
the detectable moieties are chromogens which produce a color change
observable in the visible wavelength range or fluoresces when
properly excited by an external energy source. Examples of
detectable moieties include, but are not limited to,
orthonitrophenyl, phenolphthalein, and 4-methylumbelliferone
moieties.
[0023] The invention also features a method of using the medium to
detect the existence or measure the concentration of bacterial
contamination in a test sample. The medium is inoculated with the
test sample and incubated under a condition suitable for bacterial
growth for a certain time period (preferably no more than 24 hours,
more preferably no more than 15 hrs, even more preferably no more
than 10 hours). Then the detectable signal is measured as an
indication of the concentration of bacteria in the test sample.
Using this method, a detectable signal is produced when at least
one of the three or more different bacterial enzymes is or are
present in the bacteria which are incubating in the medium.
[0024] By "test sample" is meant a piece, fraction, aliquot,
droplet, portion, fragment, volume, or tidbit taken from a food
product such as ground beef or chicken, a human or animal test
subject, a soil, water, air or other environmental source, or any
other source whose bacterial concentration is to be measured. A
test sample may be taken from a source using techniques known to
one skilled in the art, including, but not limited to, those
described or referred to in Compendium of Methods for the
Microbiological Examination of Foods, Third Edition, Edited by Carl
Vanderzant and Don F. Splittstoesser, Compiled by the APHA
Technical Committee on Microbiological Methods for Foods,
incorporated by reference herein.
[0025] By "bacteria" is meant one or more viable bacteria existing
or co-existing collectively in a test sample. The term may refer to
a single bacterium (e.g., Aeromonas hydrophilia, Aeromonas caviae,
Aeromonas sobria, Streptococcus uberis, Enterococcus faecium,
Enterococcus faecalis, Bacillus aphaericus, 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), a genus of bacteria (e.g.,
streptococci, pseudomonas and enterococci), a number of related
species of bacteria (e.g. coliforms), an even larger group of
bacteria having a common characteristic (e.g., all gram-negative
bacteria), a group of bacteria commonly found in a food product, an
animal or human subject, or an environmental source, or a
combination of two or more bacteria listed above. The bacteria
include those described or referred to in Bergey's Manual of
Systematic Bacteriology, 1989, Williams and Wilkins, U.S.A.,
incorporated by reference herein.
[0026] In preferred embodiments, one of the substrates is
hydrolysed by the enzyme alkaline phosphatase; another substrate is
hydrolysed by the enzyme glycosidase, including, but not limited
to, .beta.-D-glucosidase; and a third substrate is hydrolysed by a
peptidase (preferably an aminopeptidase, more preferably an (L or D
amino acid)-aminopeptidase), including, but not limited to,
L-alanine aminopeptidase; the detectable moiety is a fluorescent
moiety such that when the detectable moiety is hydrolysed from a
substrate, it causes or produces a fluorescent signal; the medium
contains at least the following three substrates:
4-methylumbelliferyl phosphate,
4-methylumbelliferyl-.beta.-D-glucoside and
L-alanine-7-amido-4-methyl coumarin; and the medium is inoculated
with a test sample from a food product, including, but not limited
to, ground beef, chicken, milk, dairy products, and drinking
water.
[0027] In another aspect, the invention features a bacterial growth
medium containing two or more different enzyme substrates each
hydrolysed by a different bacterial enzyme to cause or produce an
identical type of detectable signal.
[0028] In a preferred embodiment, the two or more different
substrates each has both a nutrient moiety and a detectable moiety
linked together by a covalent bond. Each of these substrates is
hydrolysed by a different bacterial enzyme to produce a separate
detectable moiety which causes or produces an identical type of
detectable signal.
[0029] The invention also features a method of using the medium to
detect the existence or measure the concentration of bacteria in a
test sample. The medium is inoculated with the test sample and
incubated under a condition suitable for bacterial growth for a
certain time period (preferably no more than 24 hours, more
preferably no more than 15 hrs, even more preferably no more than
10 hours). Then the detectable signal is measured as an indication
of the concentration of bacterial contamination in the test sample.
Using this method, a detectable signal is produced when at least
one of the two or more different bacterial enzymes is present in
the incubation medium.
[0030] In preferred embodiments, the substrates are hydrolysed by
an enzyme selected from the group consisting of alkaline
phosphatase, glycosidase (which includes, but is not limited to,
.beta.-D-glucosidase), and peptidase (preferably an aminopeptidase,
more preferably an (L or D amino acid)-aminopeptidase, including,
but not limited to, L-alanine aminopeptidase); and the detectable
moiety and the medium are analogous to those noted above.
[0031] In other embodiments, the invention uses the apparatus
described by Naqui et al. in U.S. patent application Ser. No.
08/201,110, incorporated by reference herein, to quantify the
concentration of bacterial contamination. An example of such an
apparatus is sold by Idexx Laboratories Inc. under the name of
Quanti Tray.TM.. The quantifying step involves providing a test
sample in a liquid form. The sample is placed or dispensed into the
sample holding bag described by Naqui et al., and mixed with a
medium to allow and promote growth of target bacteria within
individual compartments. The mixture is incubated and the quantity
and quality of the color or fluorescence change in each compartment
is detected. The quantity and quality of positive compartment
(i.e., a compartment having a detectable color or fluorescence
change) is compared to a most probable number table which relates
that value to the bacterial concentration of the test sample.
[0032] This invention has many advantages over the methods
currently used to measure bacterial contamination. One advantage is
its relatively short time to results. Certain psychotropic bacteria
grow very slowly and can take from 48 to 72 hours before their
colonies become large enough to count on an agar plate. However,
countable colonies need not be present for the results of
Applicant's test to be read. The fluorescent color produced by
these bacteria in the invention appears much faster than their
corresponding colonies which results in a much shorter detection
time. Applicant's test can reduce the incubation period to 24 hours
or less.
[0033] Another advantage of the invention has over standard methods
is the absence of interference by bacterial overgrowth. This is a
particular problem when Bacillus species are present because they
tend to grow over other bacterial colonies in such a way that the
plate is unreadable. The Bacillus species are common in food,
particularly those that have been heat treated, such as pasteurized
milk. This problem is avoided in the invention because it does not
depend on counting individual bacterial colonies.
[0034] This invention can be used in microbiology laboratories
involved in end product testing and/or quality control of food
products; the meat and poultry industries, the dairy industry, and
the water industry. The invention may be used to measure the
concentration of total viable bacteria in drinking water.
[0035] This invention also relates to a growth medium and methods
for detecting or measuring the concentration of yeasts, fungi, or
other eukaryotic microorganisms in a test sample using a formulated
medium and steps like those described above.
[0036] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] In the following description, reference will be made to
various methodologies known to those of skill in the chemical,
biological and microbiological arts. Publications and other
materials setting forth such known methodologies to which reference
is made are incorporated herein by reference in their entireties as
though set forth in full. The compositions, methods, and products
of this invention are applicable to biological and environmental
specimens, and are useful in the chemical, biological and
microbiological arts for the detection of bacterial
contamination.
[0038] Detecting Bacteria by Measuring Bacterial Enzyme
Activities
[0039] Bacteria derive their nutrients from an array of sources.
The ability to metabolize certain sources may be characteristic of
a particular bacterium or group of bacteria. Families, groups or
species of bacteria may share enzyme specificity for certain
nutrients which are lacking in other bacteria. By taking advantage
of the metabolic characteristics of bacteria, it is possible to
test for the presence of these enzyme systems, and thus, the
bacteria which display these enzyme systems themselves. See Edberg,
supra. Many enzymes have been identified which are specific to
particular groups of bacteria and others likely will be identified
in the future (see generally, Bergey's Manual of Systematic
Bacteriology, 1989, Williams and Wilkins, U.S.A.).
[0040] For example, most gram negative bacteria, as a group, have
L-alanine aminopeptidase enzyme activity. Substrates such as
L-alanine-.beta.-orthonitrophenyl,
.beta.-naphthalamide-.beta.-L-alanine,
.alpha.-naphthol-.beta.-L-alanine,
4-methylumbelliferyl-.beta.-L-alanine, and
L-alanine-7-amido-4-methyl coumarin may be used in the medium to
test for the presence of gram negative bacteria. The enzyme
.beta.-D-glucosidase is found in the Enterococcus group of
bacteria. The enzyme may catalyze the hydrolysis of 10 appropriate
substrates containing chromogenic or fluorogenic moieties linked to
a .beta.-glucoside. This property may be used to indicate the
presence or absence of enterococci in a sample. Substrates such as
4-methylumbelliferyl-.beta.-D-glucopyranoside may be used to
indicate the presence of enterococci. Staphylococcus aureus is
capable of hydrolysing orthonitrophenyl phosphate. Thus, if the
growth medium contains this substrate as a source of phosphate,
Staphylococcus aureus will grow and a color change will be produced
by the release of the orthonitrophenyl moiety. Mycobacterium
fortuitum requires SO.sub.4 as its source of sulfur, and this
species can hydrolyse phenolphthalein-sulfate. Thus, in a selective
medium whose only sulfur source is phenolphthalein-sulfate, this
species will grow and produce a characteristic color change by
release of the colored moiety. Furthermore, the enzyme
.beta.-D-glucuronidase is present in E. coli. Substrates such as
orthonitrophenyl-.beta.-D-glucuronide,
.beta.-naphthalamide-.beta.-D-gluc- uronide,
.alpha.-naphthol-.beta.-D-glucuronide or methylumbelliferyl-.beta-
.-D-glucuronide may be used in a medium for the detection of E.
coli.
[0041] Substrates and Detectable Moieties
[0042] Substrates including a chromogenic moiety have been
demonstrated to display a characteristic color change in samples
containing target bacteria having a bacterial enzyme capable of
hydrolysing the substrates. For example, in the presence of
.beta.-D-glucuronidase, orthonitrophenyl-.beta.-D-glucuronide
produces a color change to yellow,
4-methylumbelliferyl-.beta.-D-glucuronide produces fluorescence
after excitation at 366 nm, and
bromochloro-indole-.beta.-D-glucuronide produces a color change to
blue when E. coli is present. In the presence of
.beta.-D-galactosidase, orthonitrophenyl-.beta.-D-galactopyranoside
produces a color change to yellow and
4-methylumbelliferyl-.beta.-D-galac- topyranoside produces
fluorescence after excitation at 366 nm when E. coli is
present.
[0043] Two substrates producing different types of detectable
signals have been used for detecting the presence of E. coli among
total coliform bacteria. 4-methylumbelliferyl-.beta.-D-glucuronide
may be used together with
orthonitrophenyl-.beta.-D-galactopyranoside. If any E. coli is
present, the sample solution both changes color to yellow and emits
fluorescence after excitation at 366 nm.
[0044] Table I is a list of substrates from AerChem, Inc. that may
be used to detect bacterial enzyme activities.
[0045] A detectable moiety may be attached to a nutrient moiety by
methods known to those skilled in the art. The methods generally
feature coupling or conjugating a nutrient moiety to a detectable
moiety, such as a chromogenic moiety. Examples of such methods are
described by Edberg in U.S. Pat. No. 4,925,789, incorporated by
reference herein.
[0046] The following non-limiting example features a liquid based
bacterial growth medium used to quantify the total number of viable
bacteria present in ground beef and chicken. This medium comprises
4-methylumbelliferyl phosphate (MUP),
4-methylumbelliferyl-.beta.-D-gluco- side (MUD), and
L-alanine-7-amido-4-methyl coumarin (ala-AMC). An example of the
composition is described in Table II. The composition of defined
media is described in Table III. MUP, MUD, ala-AMC, and potassium
nitrate were purchased from Sigma. Bacto Proteose Peptone No. 3 was
purchased from DIFCO.
[0047] The substrate 4-methylumbelliferyl-.beta.-D-glucoside is
used to detect the presence of the enzyme .beta.-D-glucosidase
which is present in Streptococci, Enterococci, and other related
bacteria commonly found in fresh meat.
[0048] The substrate L-alanine-7-amido-4-methylcoumarin is used to
detect the presence of the enzyme L-alanine aminopeptidase which is
found in most pseudomonas species and other gram negative bacteria.
Applicant discovered that this substrate is particularly sensitive
to the presence of psychotropic bacteria which cause spoilage in
meat. Other substrates can be used in place of
L-alanine-7-amido-4-methylcoumarin to detect other types of
aminopeptidases in this group of bacteria without sacrificing
sensitivity.
[0049] The substrate 4-methylumbelliferyl phosphate is used to
detect the presence of phosphatases such as alkaline phosphatase
and acid phosphatase which are found in most bacterial species.
This enzyme substrate supports the detection of bacteria which lack
or have diminished L-alanine aminopeptidase and
.beta.-D-glucosidase activities.
[0050] Because phosphatase, .beta.-D-glucosidase, and L-alanine
aminopeptidase are present in the vast majority of bacteria which
contaminate ground beef and chicken, only one of these enzymes
needs to be functional in the food-borne bacteria for viability to
be detected. This test, therefore, has built-in redundant screens
which support a highly accurate measure of total viable bacteria in
ground beef and chicken.
[0051] The presence of bacteria is indicated by the appearance of a
blue fluorescent color in the medium after it is exposed to an
external ultra-violet lamp (366 nm wavelength). This test yields
result after no more than 24 hours of incubation at 35.degree.
C.
[0052] The substrates MUP, MUD, or ala-AMC are hydrolysed by
phosphatase, .beta.-D-glucosidase, or L-alanine aminopeptidase to
produce both nutrient and fluorescent moieties. The nutrient
moieties (i.e., phosphate, glucose, and L-alanine) are consumed by
the bacteria as a part of their normal metabolism. The fluorescent
is moieties (i.e., 4-methylumberiferone or 7-amino-4-methyl
coumarin) produce fluorescent signals (maximum emission at 450 nm)
which are used as indicators of bacterial viability.
[0053] The time required for the fluorescent color to appear is
dependent upon the concentration of bacteria present in the
reagent. Higher concentration of viable bacteria in the medium
results in a proportional decrease in the time required for color
development. Therefore, this test can be adapted to instrumentation
because of the linear relationship between bacterial concentration
and time to signal development, such as that described in Naqui et
al., U.S. application Ser. No. 08/201,110, hereby incorporated by
reference.
[0054] Naqui et al. describes an accurate method for quantifying
one number of bacteria in a liquid sample. The invention employs a
novel apparatus for holding a liquid sample. The apparatus features
a bag which is designed for receiving a liquid sample and
subsequently distributes the liquid sample into separate
compartments within the bag so that different aliquots of one or
more sizes may be tested. The invention described in that
application further allows quantifying the microorganisms present
in the sample by adding a medium to promote growth of
microorganisms, heat sealing the bag of the invention for about
five seconds at a temperature of about 250.degree. F. to
350.degree. F., incubating the sample at an appropriate temperature
for an appropriate length of time to allow growth of
microorganisms, and recording and analyzing the results. The
quantifying step involves detecting the quantity and quality of the
color change in each compartment, and comparing that quantity and
quality to a most probable number table which relates that value to
the bacterial concentration of the test sample.
[0055] For example, each 10 ml Quanti Tray.TM. system contains 50
individual wells capable of holding 0.2 ml of medium. A 51st well
is present which collects any "overfill" of medium not distributed
into the first 50 wells. To begin the test the powder containing
enzyme substrates is first dissolved in 10 ml of sterile water.
Next, the reagent is inoculated with a predetermined volume of
homogenized food material. Finally, the reagent is sealed in a 10
ml Quanti Tray.TM. and placed in a 35.degree. C. incubator for 24
hours. The number of fluorescent wells present after incubation is
compared against a most probable number (MPN) chart to determine
the original concentration of bacteria present in the sample of
food. Food containing higher than acceptable concentrations of
contaminating bacteria can be retested to verify the results and/or
disposed of to prevent distribution.
[0056] Because not all food is contaminated by the same bacteria
found in ground beef and chicken, other enzyme targets may need to
be selected to measure the total bacterial concentration of other
types of food.
[0057] To design a medium for measuring the concentration of
bacterial contamination in a test sample from another type of food
or other sources prone to bacterial contamination, methods known to
those skilled in the art (including, but not limited to, plating,
nucleic acid hybridization study, microscopic observation, etc.)
are used to identify bacteria species existing in the sample. Once
the bacteria species are identified, one skilled in the art would
be able to identify an enzyme or a group of enzymes that are
characteristic of the bacteria species, and substrates acted on by
the enzymes. Substrates having a nutrient moiety and a detectable
moiety linked together by a covalent bond that is hydrolysed by the
enzymes are produced to be used in the medium.
[0058] All publications referenced are incorporated by reference
herein, including the nucleic acid sequences and amino acid
sequences listed in each publication. All the compounds disclosed
and referred to in the publications mentioned above are
incorporated by reference herein, including those compounds
disclosed and referred to in articles cited by the publications
mentioned above.
[0059] Other embodiments of this invention are disclosed in the
following claims.
* * * * *