Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase

Definitions

• the invention relates to a chemiluminescent method of monitoring of products after heat treatment. More particularly, the invention concerns a rapid, sensitive and accurate method to examine products which are heat processed to reduce or eliminate microbiological or biological activity, so as to validate any heat treatment, and/or processing of the product and to verify that the product has been subjected to an adequate temperature within the time of the heat treatment process.
• the invention concerns a chemiluminescent method for monitoring or validating the processing of the heat- treatable product, by monitoring the concentration of a receptor or enzyme in the product by chemiluminescent means.
• the method comprises obtaining a sample of the heat-treated product directly from the product itself or from a surface contaminated with the product; and admixing this sample, typically and optionally, with a buffer-type solution such as saline buffer solution of pH 6-10 or water.
• the method includes combining the sample, either before or after incubation, with a room temperature (20- 25°C) , stable, chemiluminescent compound, usually in the presence of one or more chemiluminescent enhancers, which chemiluminescent compound is susceptible to the enzyme in the sample, which triggers the decomposition of the chemiluminescent compound to provide for chemiluminescence from the admixed sample and compound.
• the enzyme present in the compound would comprise phosphatase or lactoperoxidase, and in one preferred embodiment, the chemiluminescent compound would comprise a 1, 2-dioxetane compound, with an enhancer to render the chemiluminescent test method more sensitive.
• the test method includes incubating the sample with the chemiluminescent compound and/or enhancer for a selected incubation time period and incubation temperature, typically, for example, about 1-15 minutes and for example at 25-65°C, or more typically 1 to about 3 minutes at 30-45°C, to provide for the enzymatically cleavable labile substituent of the chemiluminescent compounds to be cleaved.
• the chemiluminescent compound may be added to the sample before or after the incubation period.
• the method also includes, preferentially, bringing the incubation to the selected incubation time period and employing a stopper solution, typically a buffer solution having a pH, for example, of about 3-12.
• a stopper solution typically a buffer solution having a pH, for example, of about 3-12.
• the method then includes observing and counting the chemiluminescent light emission over time (seconds/minutes) for an indication of the temperature and time profile of the sample that was subjected to heat processing.
• the invention relates to a method of monitoring of pharmaceutical, clinical, and food products, for instance and including dairy products such as milk, cheese and meat, or nonbiological products, to monitor the heat- treating and to validate the heat treatment of the heat- treatable product or a sample taken therefrom.
• the present invention provides a rapid and sensitive chemiluminescent method to examine products and heat processes to reduce or eliminate microbiological or biological activity, and particularly to prolong the shelf-life of a biological product.
• the present chemiluminescent methods have been demonstrated to be over 100 times more sensitive than current prior art methods and allow monitoring over a 12 log microbial reduction in pasteurization, ultrapasteurization and ultra short time, high temperature processes (UHT) of treating products.
• the present methods avoid many of the limitations of prior art test processes by incorporating chemiluminescent compounds with or without chemiluminescent enhancers to make the test more sensitive, and can detect enzymes, for example acid phosphatase (AP) or other enzymes or receptors, down to about 0.001U of AP in about 3 minutes.
• AP acid phosphatase
• lactoperoxidase has been suggested for monitoring UHT, but, due to sensitivity, only ultrapasteurization can be monitored, while the chemiluminescent test method enhances the sensitivity over 50 times and makes it possible to monitor UHT.
• Traditional color assay sensitivity is not adequate, however, using the chemiluminescent test method of the invention, up to 12 log reduction of bacteria can be guaranteed.
• the chemiluminescent test method may be used for the monitoring of the pasteurization of serums, and, for example, acid phosphatase could be demonstrated as a marker for proper pasteurization, with 12 log reductions of various microorganisms.
• the chemiluminescent test method may also be profitably employed in pasteurization and sterilization of liposomes, and liposome containing products, including a receptor antibody or enzyme which has the chemiluminescent activity heat treatment and can thus be monitored in short time periods such as seconds or minutes. More particularly, heat processed food products, such as meats, vegetables and fruit (canned or juices) , can be monitored by monitoring reduction and peroxidase activity, for instance, for example, horseradish peroxidase, glucoronidase phosphatase (for example potato acid phosphatase) or epirase, at very low levels, down to IO "15 mols or lower.
• the chemiluminescent method enhances greatly the sensitivity and provides for rapid, accurate real time monitoring of bacterial reduction in the heat- treated sample being tested.
• the chemiluminescent test method is carried out by obtaining a sample of the product to be tested, either directly from the surface or interior of the product, or from any surface contaminated by the product, and typically in one case may be employed by using a swab on a stick, with the swab dry or premoistened with water or a sterilizable saline or buffer solution, to retain the sample of the product.
• the sample thus obtained is admixed, either before or after the incubation step with a chemiluminescent compound which is susceptible to the enzyme or receptor present in the sample, or inherently or integrally which is added to the sample as an enhancer.
• the chemiluminescent compound may be selected through a wide variety of compounds, but more particularly, for example, may be a 1,2-dioxetane compound by Tropix, Inc. of Bedford, Massachusetts. Generally, chemiluminescent compounds are also employed in combination with a sufficient amount of enhancers to provide for increased amplification of sensitivity in connection with the enzymatic cleavage, which enhancers are usually selected based upon the particular enzyme, such as phosphatase, which is to be cleaved.
• the sample which contains a receptor or more particularly a cleavable enzyme, which is either integral with the sample or which is added, for example, may be phosphatase.
• Chemiluminescent compounds and the enhancers may be either added before or after incubation; however, incubation is carried out with the sample for a selected time period and temperature, usually with a chemiluminescent compound present. Incubation is terminated employing a stopper solution, and immediately thereafter, the sample, typically in a translucent or transparent glass or container, is placed within a luminometer such as portable luminometer well, and the luminescence of the sample, in relative light units (RLU) are determined relative to time in order to provide for a determination of the amount of receptor or enzyme remaining in the heat-treated sample.
• RLU relative light units
• the chemiluminescent test method thus permits the determination as to whether the treated product was subject to a proper heat-treating process, for example, as required by the U.S. Code of Federal Regulations which specifies cooking temperatures in connection with food products, with the acid phosphatase as an indicator for the cooking time and temperature.
• the chemiluminescent substrate reagents used in the test may vary; however, some substituted 1, 2-dioxetanes and enhancers suitable for use therewith are described in U.S. Patent 5,330,900, issued July 19, 1994, and U.S. Patent 5,338,847, issued July 23, 1996, both hereby incorporated by reference.
• the reagents may be employed in solution or in tablet form.
• the reagent may comprise a compressed tablet of a bulking agent (like microcellulose or other inert filler material) , and optionally with other additives like stabilizers, anti ⁇ oxidants, binder agents, dyes, etc.
• a 30 milligram cellulose tablet having about 1-20 micrograms of the reagent and enhancer may be used to detect alkaline phosphatase or alkaline phosphatase conjugates in the test sample.
• test method will be described for the purposes of illustration only in connection with certain preferred illustrated embodiments, however, it is recognized that various changes, modifications, additions and improvements may be made to the illustrated test method embodiment by those persons skilled in the art, all falling within the spirit and scope of the invention.
• Fig. 1 shows a flow diagram of the meat patty test procedure
• Fig. 2 is a graph depicting the 4 log reduction of phosphatase activity from ground chicken meat when heated at cooking temperatures ranging from 630C to 830C;
• Fig. 3 is a graph illustrating the detection limit of residual uncooked meat (raw chicken meat) portion in a sample
• Figs. 4-7 are graphs illustrating the reduction of phosphatase activity under treatments at four different temperatures and cooking times. Description of the Embodiments
• the Food Safety and Inspection Service the USDA's agency responsible for safety of meat and poultry product, has established new requirements for cooked uncured meat products designed to "protect the public health by reducing the risk of foodborne illness from certain types of meat products prepared in USDA inspected plants” .
• the rules as set forth in the "Requirements for Cooked Uncured Meat Products", published in the F_S £ Back g rounder. August 1993, pages 1-4, and the "Code of Federal Regulations", published in the Federal PCH RI-P-T-. August 2, 1993, 9 CFR Parts 318 and 320, set heating, cooking, handling and storage requirements for meat processing facilities (including supermarkets) , plants, restaurants, hotels and other institutional kitchens.
• the program's goal is to reduce pathogens throughout the food chain "from the farm to the consumer's fork”.
• Requirements include:
• the phosphatase assay is a rapid tool for quality assessment of these requirements.
• the use of phosphatase as an indicator for heating process has been recognized by the milk industry worldwide for more than two decades. Phosphatase activity has been previously reported as a possible indicator for the presence of uncooked meat in a cooked or pre-cooked poultry product.
• the use of acid phosphatase measurement as a monitor of end-point temperature in poultry tissue has been presented at the 1993 IAMFES (International Assoication of Milk, Food and Environmental Sanitarians) meeting by a group headed by Carl E. Davis from USDA's Russell Research Center in Georgia. They found a similar time/temperature dependent decrease in mean chicken breast acid phosphatase activity.
• the test method is a valuable tool in quality control, as it is rapid, simple and easy to interpret.
• the simplicity and minimal preparation of samples make the test applicable to on-line monitoring, post cooking sampling and on-site testing using a portable unit. Multiple samples can be prepared simultaneously, which makes the procedure even more desirable.
• Such meat samples can be extracted, buffered (Buffer Table provided) and tested in under 15 minutes.
• the kit uses a stable, ready-made chemiluminescent reagent substrate (e.g., AP reagent) and a stopping solution to stabilize the results.
• the precooked meat patty rule also establishes requirements for prevention of cross-contamination in common areas used for handling raw meat as well as fully cooked patties. Efficiency of cleaning and sanitizing of the handling areas can be monitored by swabbing areas and testing. Any residual raw product can be easily detected and results can be recorded for HACCP documentation. So far, test performance has not been affected by the presence of sanitizers routinely used by the processing industry (e.g., chlorine, detergents) .
• Fig. 1 of the Drawings A flow diagram of the meat patty test procedure is shown in Fig. 1 of the Drawings.
• the test results, obtained from a luminometer, are expressed in relative light units (RLU) .
• Luminometers are available in portable or bench top laboratory models.
• the AP reagent comprises
• Enzyme activity was measured versus the regulatory requirement of temperature and time of cooking. The results indicate that response curves of phosphatase activity are correlated with cooking time of 0-3 minutes, at temperature ranges of 63-690C.
• Fig. 2 of the drawings shows the 4 log reduction of phosphatase activity from ground chicken meat when heated at temperatures ranging from 630 C to 830C. The sensitivity level of the assay was established in regard to the detection of residual uncooked portions of the sample.
• Fig. 3 shows the detection limit determined to be as little as 0.005% of residual raw meat in a cooked chicken meat sample.
• Beef phosphatase activity was measured at four different temperatures and compared to log E. coli reduction over three minutes.
• the temperature of beef muscle extract was raised (measured by thermocouple probe) to 63, 67, 67.8 and 68.90C and maintained at these temperatures.
• Maximum of 4 log reduction in phosphatase activity could be measured by the test method up to three minutes.
• At least 6 log reduction of E. coli was measured at 2 log reduction of phosphatase activity under any of these treatments, as shown in Figs. 4-7 of the drawings.
• Table 3 summarizes the predicted log reduction when
• the reduction in phosphatase activity is 2 logs or more.
• Table 3 show ⁇ examples of test method results from uncooked, raw, partially cooked (60OC) , and fully cooked (69.40C) beef.
• a t 60OC (140OF) there is a 50% or greater drop in RLU values, at 69.40C (1570F) , the difference is greater than 99%.
• a screening level can be set at 99% reduction (2 logs) which give a 95% confidence level for fully cooked beef burger. Any food processing plant contains a potentially hazardous situation in terms of microbial contamination from raw meat or milk contaminating the finished product .
• This test measures the activity of phosphatase as indicative of raw tissue, milk or serum in cooked product ⁇ (e.g., pasteurized milk, cheese, cooked meat, salami, cold cuts, smoked fish) . It also can be used to detect cross- contamination from raw material on processing surfaces, equipment or packaging intended for finishing products.
• raw tissue, milk or serum in cooked product ⁇ e.g., pasteurized milk, cheese, cooked meat, salami, cold cuts, smoked fish
• a sample is obtained using a probe with a sample- collecting swab at one end.
• the swab can be dry for sampling wet surfaces, or is moistened with a water/buffer solution, for example, for meat products and solid dairy products, like cheese.
• test sample is added in a test container, such as an elongated plastic tube having a transparent well at one end, to a buffer solution such as saline buffer, pH 6-10, with preservatives (e.g. benzoic acid, sorbate) , and a pH indicator such as phenol red at 0.001%.
• a buffer solution such as saline buffer, pH 6-10, with preservatives (e.g. benzoic acid, sorbate) , and a pH indicator such as phenol red at 0.001%.
• a chemiluminescent compound which is cleaved by phosphatase is employed, such as a tablet with a 1,2- dioxetane phosphatase substrate, (comprised of, e.g., CPD-
• a stopper solution for example, a stopping solution 0.0025-0.025 ethylene diamine tetroacetic acid (EDTA) , 0.05-0.2M Tris base or other biological buffers, and optionally a pH indicator, 0.1-0.3 NaCl, pH 8-11) .
• EDTA ethylene diamine tetroacetic acid
• Table 5A Test results in Relative Light Units (RLU) of various ground beef samples held at various temperatures and times
• the chemiluminescent test uses the presence of phosphatase activity to determine whether cooked meats have met CFR specified cooking temperatures.
• the test method uses a chemiluminescent substrate for rapid determination of phosphatase activity.
• the procedure includes the sampling step, which includes using a premoistened wet swab to sample the core of the meat
• the swab is brought into contact with the chemiluminescent substrate, for one to ten minutes at a temperature range from room temperature to 650C, for example, 550C for one minute.
• the reaction is terminated by raising the pH, and stabilized by adding a stopping solution and immediately counting relative light units (RLU) using a photometric luminometer and placing the well of the test container with the sample, buffer and chemiluminescent compound, in the luminator.
• RLU relative light units
• the average test for raw beef is in the range of 15,000 to 20,000 RLU, while fully cooked beef gives results in the range of 0-300 RLU (see Table 2) .
• the chemiluminescent test method accurately detects raw meat and also can distinguish fully cooked meats from incompletely cooked meats. Meat processed at a temperature 20C below CFR specifications and for thirty seconds too short a time (Sample 4) , was identified as positive in this study. Samples properly processed, and hamburgers purchased from a local restaurant, were negative for residual raw meat.
• the test method has also been employed for ultra high temperature (UHT) processed milk (e.g., milk treated in a microwave ultra high temperature short time process) by testing for the enzyme lactoperoxidase in the milk, as well as for or an alkaline phosphatase test for milk.
• UHT ultra high temperature
• the UHT milk test includes dispensing 1ml of a buffered lactoperoxidase to lOO ⁇ l milk sample, incubate at 350C for one minute, add lOO ⁇ l of reagent LPO (tablet or liquid) and count in analyzer.
• the LPO reagent comprises a luminol (2,3 aminophthalhydrazide) and perborate as chemiluminescent substrate for lactoperoxidase. Sodium carbonate pH 11 is added as an enhancer at the end of one minute of incubation) .
• the alkaline phosphatase test for milk includes: mixing a reagent AP (alkaline tablet with CDP-StarTM chemiluminescent substrate and enhancer from Tropix, Inc.), and milk in a test tube; incubating for three minutes at 350C, adding a stopping solution to the incubated sample and analyzing or counting by a luminometer the emitted light.
• the alkaline phosphatase test covers the pasteurization range from 60°C to 75°C, whil ethe lactoperoxidase test covers UHT treatments up to 95°C.
• Example 4 The test method may further be employed as a validation test for microwave heat treatment of blood, blood derivatives, body fluids, or fermentation processes of therapeutics to eliminate bacteria, viruses and mycoplasme.
• This ultra-high temperature, short-time (UHT) process is described in U.S. Patent 5,000,000, incorporated herein by reference. Enzymes like acid and alkaline phosphatase, galactosidase, glucoronidase, and peroxidases can be used to determine is sufficient heat was applied in the UHT process.

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