WO1999043847A1 - Method for detection of microbial contamination on food products - Google Patents
Method for detection of microbial contamination on food products Download PDFInfo
- Publication number
- WO1999043847A1 WO1999043847A1 PCT/US1999/003894 US9903894W WO9943847A1 WO 1999043847 A1 WO1999043847 A1 WO 1999043847A1 US 9903894 W US9903894 W US 9903894W WO 9943847 A1 WO9943847 A1 WO 9943847A1
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- WO
- WIPO (PCT)
- Prior art keywords
- test
- phosphatase
- microbial
- swab
- test sample
- Prior art date
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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
- C12Q1/34—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
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
Definitions
- ATP-bioluminescent (Adenosine Triphosphate) based methods with beef and poultry carcasses, for the detection of bacteria on surfaces have been described for differentiating between bacteria ATP and somatic ATP (see "Sanitation Technology", October 1997, pp. 53-54).
- the described method of detection has not been found to be satisfactory in practice, due to high levels of false positive and false negative results.
- test swab is then reinserted in the CHEF TEST ® device and threadably moved to puncture the first chamber and mix the test sample with only the microbial phosphatase with dioxetane solution.
- the CHEF TEST ® device is then incubated, e.g. , 35oc for 5 minutes to accelerate the phosphatase-dioxetane reaction and then the test swab is threadably moved into the second chamber to stop the reaction.
- the transparent test vial at the end of the body unit is then inserted into the wall of the photometer for RLU to be determined.
Abstract
A test method for the detection of microbial phosphatase like fecal matter or contaminants on the surface of an animal carcass or tissue by a luminescent test, which includes heating a product sample to be tested to a temperature and for a time sufficient to destroy residual bovine phosphatase and other heat-sensitive phosphates, and to provide a test sample for luminescent testing, only with microbial phosphatase as a measure of microbial contamination.
Description
METHOD FOR DETECTION OF MICROBIAL CONTAMINATION
ON FOOD PRODUCTS Reference to Prior Applications
This application claims the benefit and filing date of U.S. Provisional Application Serial No. 60/075,836, filed February 24, 1998.
Background of the Invention
PCT Patent Application Serial No. US96/12482, filed
July 30, 1996, based on U.S. Provisional Patent Application Serial No. 60/001,706, filed July 31, 1995, hereby incorporated by reference; discloses a luminescent test method for validating the prior heat processing or treatment of a product, such as detecting fully cooked meat from incompletely cooked meat, or processed milk from unprocessed milk, for example, by measurement of phosphatase activity.
This method, known as the CHEF TEST® (a trademark of Charm Sciences Inc. of Maiden, Massachusetts), comprises a luminescent test method for monitoring and validating the heat processing of a product subject to heat-treatment. This method comprises obtaining a sample of the heat-treated product, which heat-treated product contains therein a receptor or enzyme; then incubating the sample for a selected incubation time period and incubation temperature; combining said sample, either before or after the incubating step, with a stable, luminescent compound susceptible to the enzyme or receptor in said sample, which triggers a decomposition of said luminescent compound, to provide for emitted luminescence; terminating the incubating step; and determining the emitted luminescent light units, relative to time, to obtain a profile of the amount of receptor or enzyme remaining in the treated sample. siimmary of the Invention The invention comprises a method for the detection of microbial contaminants, such as fecal matter, on or in a product or product surface, such as an animal tissue or
carcass by measuring the microbial phosphatase level or concentration from a product sample.
The CHEF TEST* (cooking heating efficiency) , as described in the PCT application in one embodiment, is directed to the measurement by luminescence of the total phosphatase level of the product sample. The prior described test method on a positive result requires confirmation that the positive result is not from microbial phosphatase, but only from residual bovine phosphatase. Confirmation is accomplished by heating the sample to pasteurization and then repeating the phosphatase test. The pasteurization heating of the sample kills the residual bovine phosphatase, but not the microbial phosphatase. Then, if the sample remains positive after heating, the positive test result will be due to the presence of only the microbial phosphatase. In the detection of milk samples; milk; or milk products, the heated sample would be reported as a negative sample. In the method of the invention, particularly with fresh-killed animal carcasses; or parts; or tissues, the detection of fecal matter, like coliforms or E.coli ; or other microbial contaminates provides for the detection of microbial phosphatase and not residual bovine phosphatase or total phosphatase.
The method of the invention comprises heating a product sample derived, for example, from meat carcass or tissue, to a sufficient temperature, e.g., pasteurization temperature and time, e.g., 5 to 15 minutes, to kill or destroy substantially all the residual or interfering phosphatase and to leave the product sample with substantially only the microbial phosphatase to be detected, as described in the PCT application, or other phosphatase detection methods.
ATP-bioluminescent (Adenosine Triphosphate) based methods, with beef and poultry carcasses, for the detection of bacteria on surfaces have been described for differentiating between bacteria ATP and somatic ATP (see "Sanitation Technology", October 1997, pp. 53-54). However, the described
method of detection has not been found to be satisfactory in practice, due to high levels of false positive and false negative results.
The luminescent CHEF TEST® measures residual bovine phosphatase. The method to detect microbial phosphatase as an indicator for fecal contamination provides very few false negatives or false positive. The method comprises heating, e.g. , by pasteurizing the product sample, such as swabbed from the carcass surface to be tested. All non-microbial phosphatase in the product sample should be destroyed, leaving only microbial phosphatase for detection. The majority of intestinal microbes that produce phosphatase are fecal coliforms. There potentially could be a positive result from a phosphatase producing non-coliform bacteria, but that is unlikely, and in any event, would still indicate unacceptable contamination. The method provides for the detection of at least about 10 ppm of fecal contamination.
The outside of the fresh-killed carcass should either not be contaminated with fecal matter in the first place or should have all fecal matter washed off prior to processing. There is a large market and need for a rapid and simple luminescent test to determine if a carcass, carcass parts, or tissue ( e .g. , meat, chicken or fish) or other food, is free of fecal or spoilage microbial matter. There is an old AOAC (Association of Analytical Chemists) approved method using phosphatase as an indicator for fecal matter; however, this AOAC test is a color test which takes a few hours to run. The luminescence method of the invention takes less than five minutes to complete and is at least 100 times more sensitive. Background of the Invention
Microbial phosphatase is more heat-stable than animal- derived phosphatase, which has variable heat-time destruction properties, depending on the species tested. For example, chicken phosphatase takes a greater time, e.g. , about 13 to
15 minutes at 75oc to be destroyed than beef phosphatase, e.g., about 7 to 8 minutes at 75°C to be destroyed.
The invention will be described for the purpose of illustration only in connection with certain illustrated embodiments known as the FCI TEST™ (Fast Contamination Indicator, a trademark of Charm Sciences, Inc. of Maiden, Massachusetts, USA); however, it is recognized that various changes, modifications, additions and improvements may be made in the illustrative embodiments without departing from the spirit or scope of the invention.
Brief Description of the Drawings Fig. 1 is a bar chart-graph summary of test method detection results of fecal vs. ingesta contamination on a carcass. Fig. 2 is a bar chart-graph summary of test method detection results of fecal vs. ingesta contamination on various portions of a carcass.
Description of ha Kn odiments
The test results of Figs. 1 and 2 were obtained by employing a test tube vial with a sterile water buffer, a CHEF
TEST® test device, an incubator, and a photometer to measure emitted light. The CHEF TEST® device is a threadable
POCKETSWAB® (a trademark of Charm Sciences, Inc. of Maiden,
Massachusetts, USA) test apparatus with a top cover with a threadably moveable test swab (optional with an end sheath), an intermediate body unit, and a transparent, removable or fixed, test vial at one end thereof to insert in the photometer.
The CHEF TEST® device includes two membrane-separated swab-puncturable chambers. The first chamber contains an AP substrate, such as a dioxetane derivative solution designed to
react with the microbial phosphatase from the test sample and to provide bioluminescence emitted light to be measured by the photometer. The second chamber includes a stopping solution, such as a chelating agent like an EDTA (ethylenedia inetetraacetic acid) solution to stop the dioxetane-phosphatase reaction. The test swab is used to swab an area of a carcass or tissue, then washed in the sterile buffer solution incubated to destroy bovine residual phosphatase. The test swab is then reinserted in the CHEF TEST® device and threadably moved to puncture the first chamber and mix the test sample with only the microbial phosphatase with dioxetane solution. The CHEF TEST® device is then incubated, e.g. , 35oc for 5 minutes to accelerate the phosphatase-dioxetane reaction and then the test swab is threadably moved into the second chamber to stop the reaction. The transparent test vial at the end of the body unit is then inserted into the wall of the photometer for RLU to be determined.
The FCI TEST™ is a self-contained, single service device designed to quickly detect microbial phosphatase on meat and food products. The test measures heat-stable phosphatase as an indicator of the presence of microbial contamination. On the surface of freshly slaughtered animals, this contamination is primarily due to fecal matter. The test may be used in food industry safety programs as a rapid detector of surface contamination and/or as a rapid assessment of HACCP (Hazard Analysis Critical Control Point) samples for unacceptable levels of fecal matter. This test enhances food safety programs with intermediate feedback for corrective action. A Charm Sciences, Inc. LUMINATOR™ displays test results in relative light units (RLU) .
The control point is the cutoff value (RLU) between acceptably clean carcass (negative) and suspect for fecal contamination (positive) carcass. Establish a control point
To Determine Control Point for Negative Carcass:
Run three swabs of negative sample (clean carcass or skin) .
Average the results. Multiply by 2. This is the control point.
The control point should be entered as the Limit of the LUMINATOR™ (AP Assay) or customized channel. Any sample result greater than the limit (control point) is suspect positive. Positive Control
Uncap a Preheat Vial and add 1 Phosphatase Positive Control Tablet.
Insert swab, swirl 20 times to dissolve tablet. Remove swab and start assay in step THREE (incubation in 45oc for 2 minutes).
Complete test procedure.
EXAMPLE
ONE Swab
Uncap Preheat Vial and place in 75 °C incubator.
Withdraw swab from the test device body by gently twisting and pulling.
Remove red sleeve exposing swab.
Swab the carcass, suspicious spots, or swirl in
HACCP rinse or sponge sample* allowing the swab to absorb liquid (approximately 1 minute).
If testing later, replace sleeve over swab and reinsert swab assembly into body. Test within
15 minutes .
For assessment of low level contamination, filter
10 ml HACCP sample through 0.45 μm filter and swab filter. (See Filter Addendum for FCI Test™ ) .
TWO 75 ° C Preheat If swab with sleeve was reassembled for later use, withdraw swab from sleeve.
Place lower part of device (swab housing and attached vial) in 45°C incubator.
Insert swab into Preheat Vial (75°C) and twirl initially at least 5 times to mix. Incubate for time listed in Table 1:
Table 1 : Incubation time (75°C) to incubate sample - Step TWO
PRODUCT SWAB TEMP. TIME
Pork/Beef room/cold* 7 minutes
Turkey room 8 minutes
Turkey cold* 9 minutes
Chicken room 12 minutes
Chicken cold* 13 minutes
* Swab is still cold from swabbing cold carcass.
THREE Incubate 45 C
Remove swab from the Preheat Vial and reinsert in test device. While holding up to light to view.
SU.BSTITUTESHEET(RULE26)
engage threads. Screw the handle down slowly until the swab tip punctures the first seal only and is in the first yellow reagent, as shown.
Mix by screwing the swab tip out and then back into the first reagent once. Swab should remain in first reagent for incubation.
Incubate for 2 minutes at 45°C. FOUR Stop
Screw the swab fully down through the remaining seals.
Screw the handle back to the starting position so the swab tip is out of the microtube.
Shake back-and-forth with tapping to get liquid to bottom of microtube. FIVE Read
Immediately insert the FCI Test™ into the chamber of the LUM-T™ or Luminator K. For LUM-T™, insert entire test device into chamber. Depress gently until fully seated. For Luminator K, break off the microtube and close chamber.
Press ENTER to read on [AP Assay]. After the beep, note RLU reading.
Remove test device from analyzer and discard.
I N T ERP R E TAT I O N O F R E S UL T S
Sample results less than the control point are negative.
Sample results greater than or equal to the control point are suspect positive for microbial phosphatase.
Positives should be retested along with controls.
Positives indicate phosphatase of fecal or microbial origin.
Consult Luminator operating instructions for printing or downloading to spreadsheets.
The Pathogen Risk Reduction Rule for HACCP requires monitoring carcasses for the fecal indicator organism E.coli , and if necessary. Salmonella .
Fecal contamination is the major source for food pathogens. In food animals and fish, these pathogens are part of the normal flora in the intestine. The FCI TEST™ is a single use swab device that monitors bacteria phosphatase associated with fecal contamination. Thus, the FCI TEST™ gives the slaughter establishment a tool to assess rapidly pathogen risk and predict in 10 to 15 minutes whether E.coli monitoring samples will be within HACCP specifications.
The FCI TEST™ is based on phosphatase enzyme production by intestinal microflora. This microbial phosphatase is easily distinguished form animal tissue phosphatase by its heat resistance. Bovine, porcine, fish and poultry phosphatases are inactivated by exposure to temperatures of 69 to 7loc, while intestinal microbial phosphatase is heat- stable. The FCI TEST™ uses a small portable incubator to destroy animal phosphatase. Then, using a luminescence substrate, the FCI TEST™ detects low levels of microbial phosphatase activity, which is measured by a hand-held photometer. The entire test takes 10 to 15 minutes (depending on tissue type) and can be performed on or off site.
The FCI TEST™ device has numerous advantages over the current microbial and visual inspection methods:
Flexibility: The singe service device allows for random testing of carcasses whenever there is suspect visual contamination. It can also be used to predict HACCP sample E. coli levels.
Confirmation of
Visible Matter: Particulate contamination can come from feed, crop (poultry), stomach or intestinal/fecal matter. Only intestinal/fecal matter presents risk of
pathogens. High microbial phosphatase is only present in intestinal/fecal material. The FCI TEST™ can distinguish between phosphatase from intestinal/fecal matter and stomach ingesta.
Real Time .Assessment of HACCP Compliance: Real time process improvement becomes a possibility in about 10 to 15 minutes.
Interpretation of Results The photometer produces a digital result, expressed in relative light units (RLU) , and PASS/FAIL. The FCI TEST™ correlates well with E.coli counts as noted in Table 1. The cut-off or control point is easily adjusted to the level of concern for poultry, beef, fish and swine. Sensitivity can be enhanced with filtration of carcass rinses.
TABLE 1. FCI TEST" Correlation to E.coli on Turkey Carcasses Spiked with Ingesta/Fecal Matter
SAMPLE BLIΪ E.coli Count (2,500 Cut-Off)
Turkey skin rinse 1,580 Pass <100 CFU/ml (25g/99ml)
Turkey skin rinse spiked 4,530 Fail «4,000 CFU/ml with 1:10,000 dilution of fecal matter (0.01g/99ml)
Turkey skin rinse spiked 26,000 Fail «40,000 CFU/ml with 1:1,000 dilution of fecal matter (0.1g/99ml)
Turkey skin rinse spiked 800 Pass <100 CFU/ml with Ingesta (lg/99ml)
10
SUBSTITUTE SHECT (RULE 26)
The LUM-T™ (is a trademark of Charm Sciences, Inc. of
Maiden, Massachusetts, USA) photometer stores and tracks data on carcass type, sampling site, date, time result, interpretation, and plant ID# as illustrated in Table 2. Data may be downloaded to any spreadsheet for trend analysis.
TABLE 2. Typical Data Download at Hog Slaughter
Establi shment
Assay Date Time Plant ID# Loc#: RLU Limit Result
FCI 04/19/1998 07:05:53 412 JOWL 32,091 <5000 Fail
FCI 04/19/1998 07:06:44 412 BELLY 550 ^ 5000 Pass
FCI 04/19/1998 07:07: 24 412 BELLY 234 < 5000 Pass
FCI 04/20/1998 07:00: 17 412 BELLY 672 5000 Pass
FCI 04/21/1998 07: 00: 51 412 HAM 1 , 663 ^ 5000 Pass
FCI 04/22/1998 07:08: 12 412 JOWL 2,056 <5000 Pass
FCI 04/23/1998 07:00: 20 412 FAT 445 5000 Pass
FCI 04/24/1998 07:01:38 412 BELLY 25,435 <5000 Fail
TABLE 3. LUM-T™ Microbiological Risk Assessment Tools
HACCP Criteria Test Name Description Time
Sanitation/ 1. FCI Test Fast Contamination 9-15 minutes* Hygiene Indicator Test
2. Micro-Q Microbial Quality 3 minutes of Water
3. PocketSwab ATP Hygiene Swab 30 seconds
4. CHEF Swab Raw Meat Cross 3 minutes
Contamination
Thermal Saftey 5. CHEF Test Endpoint 'Doneness ' 5 minutes
Indicator en Oxtød l^feat
* 9 minutes for beef or swine. 12 minutes for turkey, T 5 minutes for chicken
11
The FCI TEST™ distinguishes animal ingesta from fecal matter, and can assess level of E. coli sensitivity required by HACCP regulations. It permits on-line prediction of pathogen contamination, and allows for continuous improvement over time, which is in keeping with HACCP principles. The FCI TEST™ offsets reliance on subjective visual checks by increasing the emphasis on objective scientific standards. The FCI TEST™ is one of may HACCP friendly tools available on the LUM-T™ photometer food safety test menu. The Examples describe the steps of the test method of the invention wherein the surface of the product carcass is swabbed with a swab, placed in a buffer solution, such as a low molarity solution of either water or saline and then the buffered product sample is incubated for 8 minutes at 75°C to destroy any residual bovine phosphatase on the beef carcass product sample and to retain only the more heat-stable microbial phosphatase which is detected by a bioluminescence test. The test method was used to determine the test results as set forth on the drawings of Fig. 1 and Fig. 2. Fig. 1 shows test results in bar graph form of phosphatase activity (mU/ml) versus fecal contamination level in parts per million.
Fig. 2 shows test results based on fecal phosphatase (microbial) surviving 72©c for 1 minute while tissue phosphatase is destroyed as shown in the Graph of CHEF results on clean hog carcass contaminated with barely visible amounts of intestinal (fecal) or stomach contents on fat side and skin side.
The CHEF TEST® test apparatus is described in U.S. Patent No. 5,827,675, issued October 27, 1998. LUM-T™ and LUMINATOR® are trademarks of Charm Sciences, Inc. of Maiden, Massachusetts, for a luminescent light measuring photometer to measure emitted light from the transparent test vial of the POCKETSWAB® device.
12
Claims
Claim 1. A test method for the detection of surface microbial phosphatase on an animal carcass or tissue, which method comprises: a) obtaining a test sample from the surface of an animal carcass or tissue to be tested; b) heating the test sample to a temperature and for a time to destroy substantially phosphatase other than microbial phosphatase; and c) testing the heated test sample for the concentration of microbial phosphatase therein.
Claim 2. The method of claim 1 which includes obtaining a test sample from the surface or tissues of a fresh-killed animal carcass.
Claim 3. The method of claim 1 which includes detecting the microbial phosphatase by a luminescence test.
Claim 4. The method of claim 1 which includes heating the test sample to a temperature of about 69 to 75┬░C or greater and for a time of about 5 to 15 minutes or greater.
Claim 5. The method of claim 1 which includes: a) swabbing the product surface with a test swab; b) placing the test swab with the test sample in a buffer solution; and c) incubating the buffer solution with the product sample to destroy residual phosphatase.
Claim 6. The method of claim 1 which includes testing the test sample for fecal contamination of at least about 10 ppm.
Claim 7. The method of claim 1 which includes: a) introducing the heated test sample into a dioxetane substrate derivative solution; b) incubating the solution to accelerate the dioxetane-phosphatase reaction; c) stopping the reaction; and
13 d) measuring the emitted light from the stopped reaction as a measure of microbial phosphatase contamination.
Claim 8. The method of claim 7 which includes carrying out the method steps in an elongated swab-containing threadable test device with separate swab-puncturable membrane chambers for the dioxetane substrate solution and the stopping solution.
14
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU27823/99A AU2782399A (en) | 1998-02-24 | 1999-02-24 | Method for detection of microbial contamination on food products |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7583698P | 1998-02-24 | 1998-02-24 | |
| US60/075,836 | 1998-02-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999043847A1 true WO1999043847A1 (en) | 1999-09-02 |
Family
ID=22128294
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1999/003894 WO1999043847A1 (en) | 1998-02-24 | 1999-02-24 | Method for detection of microbial contamination on food products |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2782399A (en) |
| WO (1) | WO1999043847A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997003209A1 (en) * | 1995-07-12 | 1997-01-30 | Charm Sciences, Inc. | Test apparatus, system and method for the detection of test samples |
| WO1997005287A1 (en) * | 1995-07-31 | 1997-02-13 | Charm Sciences, Inc. | Chemiluminescent method of monitoring products after heat treatment |
-
1999
- 1999-02-24 AU AU27823/99A patent/AU2782399A/en not_active Abandoned
- 1999-02-24 WO PCT/US1999/003894 patent/WO1999043847A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997003209A1 (en) * | 1995-07-12 | 1997-01-30 | Charm Sciences, Inc. | Test apparatus, system and method for the detection of test samples |
| WO1997005287A1 (en) * | 1995-07-31 | 1997-02-13 | Charm Sciences, Inc. | Chemiluminescent method of monitoring products after heat treatment |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2782399A (en) | 1999-09-15 |
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