Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-02-24
2002-10-29
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S019000, C435S024000, C435S039000
Reexamination Certificate
active
06472167
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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° 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° C.; thus, the concentrations of total viable bacteria measured by the above methods may be underestimated.
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.
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.
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
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.
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.
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.
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 (abandoned in favor of C-I-P application 08/423,134 filed Apr. 18, 1995 and issued as U.S. Pat. No. 5,610,029 on Mar. 11, 1997), and Ser. No. 08/335,149, (issued as a U.S. Pat. No. 5,620,865 on Apr. 15, 1997), both filed on Nov. 4, 1994, incorporated by reference herein. In a preferred embodiment, the medium is liquid.
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.
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.
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).
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 &mgr;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 &mgr;g); ferric chloride (e.g., about 150.0 to 250.0 &mgr;g); manganese sulfate (e.g., about 300.0 to 500.0 &mgr;g); sodium molybdate (e.g., about 150.0 to 250.0 &mgr;g); zinc sulfate (e.g. about 300.0 to 500.0 &mgr;g); a
Chen Chun-ming
Townsend David E.
BioControl Systems, Inc.
Gitomer Ralph
Seed Intellectual Property Law Group PLLC
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