WO1997005287A1 - Chemiluminescent method of monitoring products after heat treatment - Google Patents

Abstract

A method of monitoring heat processes in pharmaceutical, clinical, food or non-biological products, which method includes the steps of combining a test sample or sample extract from the product with chemiluminescent compounds or compounds specifically susceptible to enzymes, while triggering the decomposition of the chemiluminescent compound to emit light. The enzyme can be indegenous or added before the heat process as an indicator. The sample is incubated and integration of chemiluminescent light emission over time is measured as indicative of the past temperature and time profile of the sample.

Description

Chemiluminescen Method of Monitoring Products After

Heat Treatment

Reference to Prior Applications This application claims the benefit of the priority date of U.S. Provisional Patent Application S.N.

60/001,706 filed July 31, 1995, titled "Chemiluminescent

Heat Monitoring Process and System for Biological Activity" .

Background of the Invention

It is desirable to validate rapidly and effectively the heat treatment of products in the preparation of pharmaceutical biologicals and food products with a rapid, sensitive and accurate method to verify that the product haε been heat-treated to an adequate or certified temperature within the time in which the product has been so heat-treated. The determination of acid phosphatase (AP) in products as an indicator for cooking to a temperature has been reported in associative literature. Enzymatic tests have been used for monitoring milk pasteurization, such as the Scharer Test (the "Standard Method for Examination of Dairy Products", 16th Ed., 1992, R. T. Marshall, Ed., ch. 11, p.418) , which is used worldwide to monitor standard pasteurization, however, it takes about thirty minutes to detect one unit of AP in the Scharer Test. New pasteurization and ultrapasteurization procedures are at a level of 0.01U, and thus cannot be monitored with the Scharer Test. A test known as the Fluorophos acid test (the "Standard Method for Examination of Dairy Products", 16th Ed., 1992, R. T. Marshall, Ed., ch. 11, p. 18) using a fluorescent substrate is also used; however, this test has a limit of detection of 0.05U, and takes about three minutes to perform. It is desirable to provide for a new and improved, effective, rapid, chemiluminescent method and test with low limits of detection for the monitoring of heat processes in various products. Summary of the Invention

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. In the preferred embodiment, 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. 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.

In the past, 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. Thus, 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. 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. For example, 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. For example, 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.

The 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.

Brief Description of the Embodiments 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^grounder. 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:

1. Cookinσ Temperature vs. Time:

Operators are instructed to measure the temperature, every hour, of at least one product, in the center using a device accurate within lOF, to ensure control of heat processing (see Table l, for permitted heat processing time and temperature, from the Code of Federal Regulations 9 CFR Parts 318 and 320.

Table 1

Source: Code of Federal Regulations (9 CFR Parts 318 and 320) 2. Prevention of Cross-contamination: Prevent bacteria from raw products from cross- contamination of fully cooked products. Handling areas for raw and cooked meat must be either physically separated or products should be processed at different times after thorough cleaning and sanitizing of the area. Detailed sanitation instructions include:

-Surfaces, equipment - germicidal sanitizer equivalent to 50 ppm chlorine -Employee's hands - similar to above

-Garments, aprons and gloves identified and designated only for either fully cooked or uncooked processing

-Fully cooked product to be stored with other product shall first be packaged or covered.

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. This research was published in an article titled "Rapid Fluorometric Analysis of Acid Phosphatase Activity in Cooked Poultry Meat", by Carl E. Davis and William E. Townsend, in the Journal of Food Protection. Vol. 57, No. 12, pp. 1094-1097. The Applicants have found that beef phosphatase thermal stability coincides with the time/temperature requirement established by regulatory agencies and accepted by the Industry (see Table 1) . Phosphatase thermal stability correlates well with log reduction of bacteria. The sensitivity of the assay (linear over 4 logs of activity) makes the chemiluminescent test method useful for determining if temperature requirements for either partially or fully cooked meat products have been met at the processing plant or other locations.

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) .

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

1,2-dioxetane, a chemiluminescent substrate CSPD™

(Disodium 3- (4-methoxyspiro{l,2-dioxetane-3,2 ' - (5 ' chloro) tricyclo [3.3.l.i.^1,2] decan}- -yl)phenyl phosphate - of Tropix, Inc., of Bedford, Massachusetts) and a luminescent enhancer material, LAMS, Luminescence Amplifying Materials, e.g., TMQ, poly(vinylbenzyltrimethylammonium chloride) , and others as listed in U.S. Patent 5,330,900. Phosphatase activity was examined in raw chicken, beef, pork and turkey. Samples were obtained from local supermarkets and tested up to the expiration day. All samples exhibit high phosphatase activity with results ranging from 1 to 3 x 10⁶ RLU (see Table 2) When samples were fully cooked to 750C and tested again, phosphatase activity dropped 4 logs. Results from the fully cooked meats establish the range for negative phosphatase.

Table 2

Table 2: Charm Phosphatase Test- Typical results for partially cooked and fully cooked ground beef

Matrix Temperature Results Reduction

°F °C RLU %

Raw NA NA 1 ,250,000 0 partially cooked 60 140 500,000 >50 fully cooked 69.4 157 200 >99

To evaluate further the applicability of the test to the marketplace, hamburgers from various fast food restaurants were tested. In all but one, the phosphatase activity was net zero, indicating over 4 log reduction in activity (over 12 log reduction of E. coli ) . In one sample, bought during rush hour as medium rare, low phosphatase activity was detected (3 log reduction) . This indicates the potential of undercooking when rare to medium meat patties are served at peak hours.

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

USDA time and temperature requirements are met. Hold time requirements will increase the log reduction of E. coli by an additional 6 logs. The total log reduction of E. coli at 630C is 12.1, 670C is 18.5, 67.80C is 19.1, and 68.90C is 18.8.

Table 3

Table 3: Correlation of 99% Reduction in Phosphatase Activity and Log Reduction of E.coli

Figure Cooking E.coIi Reduction

Temp. At 0 seconds During Dwell Time Total

# °C (log) (sec) (log) dog)

4 63.0 6.1 180 6 12.1

5 67.0 8.5 60 6 18.5

6 67.8 1 1 .1 16 6 19.1

7 68.9 10.8 10 6 18.8

The reduction in phosphatase activity, as measured by the test method, is 2 logs or more. Table 3 showε examples of test method results from uncooked, raw, partially cooked (60OC) , and fully cooked (69.40C) beef. _At 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 . Similarly, in distribution centers, supermarkets and restaurants, cross-contamination from raw meat to finished product can occur when same surfaces or equipment is used. Phosphatase activity has been tested as a marker for contamination. By modifying the standard swab procedure for microbial quality, a rapid test method has been developed which identifies contamination by raw meat, blood or serum at extremely low levels. 0.1 mg tissue or O.lμl serum spread over 10 x 10 cm area can be detected using a sterile swab and testing for phosphatase activity ⁽see Table 4) . After cleaning with 50 ppm chlorine solution, phosphatase activity was removed. Chlorine at 50 ppm did not interfere with phosphatase activity.

Table 4

Table 4: Sanitation testing using CPT on 4 by 4 Inch square areas

Square Initial Cleaning Treatment Re-contamination RLU* SOppm chlorine then... RLU Source (mg) RLU

A 49956 rinsed with water 508 bovine serum (0.2) 3441

B 36636 rinsed with water 380 bovine serum (0.2) 3261

C 11580 no rinse, air dry 348 ground beef (10) 147969

D 2492 no rinse, air dry 396 ground beef ( 10) 297621

*raw meat processing surface

The following examples further illustrate the effectiveness of Applicants' invention.

Example 1

Testing residual raw milk/meat/fish or other food products.

This test measures the activity of phosphatase as indicative of raw tissue, milk or serum in cooked productε (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.

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.

The 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 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-

Star™, a product of Tropix, Inc.) , freeze dried and made into a tablet for use in the test method.

After incubating, the cleaving reaction is stopped by adding 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) .

Results in testing cooked ground beef hamburgers is shown in Table 5A.

Table 5A: Test results in Relative Light Units (RLU) of various ground beef samples held at various temperatures and times

Sample # 1 Sample 82 Sample #3 Sample UA Sample #5 Sample «6

Temp °C (°F) 53 ( 128) 57 ( 135) 59 ( 138) 63 ( 145) 65 ( 149) 69 ( 156) Hold Time •> 60 sec 60 sec 60 sec. 60 sec. 60 sec 16 sec

Replicate tt RLU RLU RLU RLU RLU RLU

« 1 10536 12490 1 1622 2795 10 123 n 17784 22940 5481 3903 0 0

#3 14325 841 1 5040 21 13 0 0

UA 1 1979 6309 17881 2060 0 0

#5 21310 12832 10475 4969 186 0

«6 21426 6264 1 1022 5766 188 227

I Average 16227 1 15 1 10254 3601 64 58 S -'• Range 4676 6285 4704 1542 95 96

)

( % activity 95 68 60 21 3.8 3 4 Table 5B: Test of a dozen hamburgers purchased at a local food chain restaurant

Hamburger # CHEF (RLU) Hamburger U CHEF (RLU)

1 0 7 37

2 0 8 0

3 0 9 0

4 0 10 0

5 0 1 1 0

6 0 12 o

Example 2

The purpose of this study is to demonstrate the test method's performance, precision and accuracy in predicting completeness of cooking level of cooked ground beef . Inadequate cooking has been the major cause of stomach poisoning from pathogenic bacteria like E. coli and salmonella.

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

(after splitting the meat sample to expose the inner core) . Also, it can be used to swab an equipment surface

(e.g., a slicing machine, counter top surface) , or other surfaces to test for residual raw meat/milk. In the incubation step, 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. After incubation and at the reading step, 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.

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) .

Results for ground beef heated to various temperatures and hold times are listed in Table 5A.

Using the results for fully cooked meat, a cut off for determining incompletely cooked meat can be set cat the upper range (e.g. 300 RLU) . In the Applicants' field samples (Table 5B) , all the hamburgers were properly cooked (all results below 300 RLU) . In the Applicants' own cooking experiment, (Table 5A) , the Applicants effectively screened low temperature cooked products

(Samples 1-4) from adequately processed and cooked products (Samples 5 and 6) .

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.

Example 3

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.

The UHT milk test includes dispensing 1ml of a buffered lactoperoxidase to lOOμl milk sample, incubate at 350C for one minute, add lOOμl 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-Star™ 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.

Claims

What is claimed is:

Claims Claim 1. A chemiluminescent test method for monitoring and validating the heat processing of a product subject to heat-treatment, which method comprises: a) obtaining a sample of the heat-treated product which heat-treated product contains therein a receptor or enzyme; b) incubating the sample for a selected incubation time period and incubation temperature; c) combining said sample, either before or after the incubating step, with a stable, chemiluminescent compound, susceptible to the enzyme or receptor in said sample, which triggers a decomposition of said chemiluminescent compound, to provide for emitted chemiluminescence; d) terminating the incubating step; and e) determining the chemiluminescent light units relative to time to obtain a profile of the amount of receptor or enzyme remaining in the treated sample.

Claim 2. The method of claim 1 which includes incubating said sample for a time period of from about one to fifteen minutes at a temperature of about 25-65°C.

Claim 3. The method of claim 1 wherein the chemiluminescent compound comprises a 1,2-dioxetane compound.

Claim 4. The method of claim 1 wherein the enzyme for the cleavage of the chemiluminescent compound, and to be determined in the sample, comprises phosphatase.

Claim 5. The method of claim 1 which includes obtaining said sample by contacting the heat-treated product or a surface containing the heat-treated product with a premoistened swab.

Claim 6. The method of claim 1 which includes buffering the sample at a pH of about 3-10 in a buffer solution.

Claim 7. The method of claim 1 which includes stopping the incubating step by adding a stopper solution having a pH of about 8-11.

Claim 8. The method of claim 1 which includes adding an effective amount of an enhancer to enhance the sensitivity of said chemiluminescent compound.

Claim 9. The method of claim 1 which includes carrying out the test method in a transparent test container and inserting a lower portion of the test container in a luminometer to determine the relative light units relative to time.

Claim 10. The method of claim 1 which includes immediately after the incubating step or immediately after adding the chemiluminescent compound and after incubation, determining the relative chemiluminescent light units relative to time.

Claim 11. The method of claim 1 wherein the sample is derived from a previously heated meat or dairy product.

Claim 12. The method of claim 1 wherein the enzyme to be determined comprises lactoperoxidase.

Claim 13. The method of claim 12 wherein the sample comprises ultra high temperature treated milk.

Claim 14. The method of claim 1 wherein the chemiluminescent compound is in compound tablet form.

Claim 15. The method of claim 1 wherein the product comprises cooked meat, and which includes accepting the cooked meat product when the relative light units are 300RLU or below.

Claim 16. A chemiluminescent test method for monitoring and validating the heat processing of a product subject to heat-treatment, which method comprises: a) obtaining a sample of said heat-treated product by contacting said heat-treated product or a surface containing said heat-treated product with a premoistened swab, which heat-treated product contains therein an enzyme; b) buffering and incubating said sample for a selected incubation time period of from about one to fifteen minutes and incubation temperature of about 25- 65°C; c) combining said sample, either before or after the incubating step, with a stable, chemiluminescent compound, which chemiluminescent compound comprises a 1,2-dioxetane compound, susceptible to the enzyme in said sample, wherein the enzyme for the cleavage of said chemiluminescent compound and to be determined in the sample comprises phosphatase, which triggers a decomposition of said chemiluminescent compound, to provide for emitted chemiluminescence; d) terminating the incubating step by adding a stopper solution; and e) determining the chemiluminescent light units relative to time to obtain a profile of the amount of enzyme remaining in said treated sample.