Carl E. Davis

Rapid fluorometric analysis of acid phosphatase activity in cooked poultry meat

A new quantitative f1uorometric assay was used to determine poultry-muscle acid phosphatase (ACP) activity at five end-point temperatures (EPTs). Nonfrozen or frozen ground broiler and turkey breast and turkey dark meat (16 9) were packed in glass tubes (25 ×150mm) and heated to 62.8, 65.6, 68.3, 71.1 and 73.9°C in a water bath set 1.5°C above target EPT. After heating, samples were removed and immediately chilled (2-3°C). A 75 μl portion of an aqueous meat extract (1 meat/2 H2O wt/wt) was added to 2.0 ml ACP substrate and the kinetic increase in fluorescence monitored at 38°C. The experiment was replicated three times. A curvilinear decrease in mean (N = 12) ACP activity occurred within each muscle type. Although freezing before cooking lowered ACP activity, it was not different (P>.05) from that of nonfrozen meat. Acid phosphatase activity (mU/kg) means (N = 12) with standard errors for EPTs between 68.3 and 71.1°C for broiler and turkey breast and turkey dark meat were as follows: 9270 ± 873 and 5548 ± 562; 9313 ± 665 and 6808 ± 521; and 4821 ± 398 and 3370 ± 281, respectively. This procedure provides a rapid (3 min instrument time), sensitive analytical method for quality-assurance process-control technicians or regulatory analysts to monitor EPT in cooked poultry.

Pyruvate Kinase Activity as an Indicator of Temperature Attained During Cooking of Cured Pork

Muscle pyruvate kinase (PYK) activity was established as a biochemical indicator of temperature attained during cooking in both a model system and a commercial-type pullman-style canned cured pork product. Water extract (model system) or pressed-out meat juice (commercial-type pullman-style canned cured pork) was incubated (37°C up to 40 min) with reagent containing adenosine diphosphate, phosphoenolpyruvic acid and NADH. Lactate dehydrogenase (LDH) necessary for the reaction is provided by the muscle extract or meat juice. When muscle PYK activity is present, NADH is oxidized resulting in a loss of fluorescence (reaction mixture spots on filter paper viewed under long-wave ultraviolet light). Model system results showed high PYK activity (no fluorescence) remained in samples heated to 67.7°C. PYK activity progressively declined in samples heated to 68.3°C and 68.9°C. No PYK activity was detected in samples heated to 69.5°C or 70°C. Canned product attaining a core temperature of 62.9°C had high PYK activity. PYK activity progressively declined in product heated to 65.6°C and 68.6°C. Essentially no PYK activity was detected from product processed to 69.9°C.

Effect of Heat on Biuret-Positive Water-Extractable Porcine Muscle Proteins

Ground pork longissimus was heated in glass tubes in a controlled temperature bath at 2 (control), 20, 40, 45, 50, 55, 60, 62.5, 65, 67.5, 70, 75, or 80°C for 15 min after the sample reached the desired temperature, removed and chilled (2°C) immediately. Treated samples were homogenized with deionized water at a ratio of 1:3.3 (w/v) muscle to water. The resulting water-extractable proteins were determined by the biuret method. Eight ml of clear extract from each treatment was reheated for 15 min at 70°C, removed, and chilled (2°C) immediately. Coagulated proteins were removed by filtration (0.45 μm). Soluble protein was used as an index of heat denaturation. Water-extractable biuret-positive protein losses were 5.7% from 2 to 45°C, 69.7% from 50 to 67.5°C and 4.3% from 70 to 80°C. Reheating each treatment extract to 70°C yielded 20.2% baseline biuret-positive soluble materials. The ratios of soluble proteins at each treatment temperature with the baseline critical value of 70°C were 5.1, 5.1, and 4.9 from 2 to 45°C; 4.5, 3.9, 2.7, 2.2, 1.5, and 1.2 from 50 to 67.5°C and 1.1, 1.0, and 1.0 from 70 to 80°C. This indicates that coagulation of water-extractable biuret-positive compounds is nearly constant at about 70°C. These results suggest that a ratio of water-extractable biuret-positive proteins from heat treated porcine muscle may be useful in determining the temperature to which pork has been heat processed.

Effect of Heating on Water Soluble Biuret-Positive Compounds of Canned Cured Pork Picnic Shoulder

Protein solubility loss as a result of heat denaturation/coaguiation was followed by a ratio of extractable biuret positive compounds (EBPR). Extracts of water-soluble proteins were evaluated by isoelectric focusing (IEF) on polyacrylamide gels. Four heat treatments (60°C, 62.8°C, 65.6°C and 68.8°C) were employed in processing canned (No. 300×407) cured pork. Center cores from canned samples were ground for water soluble protein extraction utilizing a 1:3.3 meat-to-water ratio by high-speed blending (Sorvall Omni-mixer) for 1 min at 0-2°C, centrifuging 10 min at 27,000 ×g at 0-2°C and filtering (0.45-μa.m) with vacuum assist. Eight ml of the clear extract was re-heated in a glass tube for 15 min at 70°C, removed, and chilled (0-2°C) immediately. Coagulum was removed by filtration. EBPR was calculated from mg of protein/ml of initial muscle extract divided by mg of protein/ml of reheated extract for each temperature treatment. EBPR values were 1.75, 1.24, 1.13, and 1.10, respectively. Using 70°C as the critical temperature, an upper 95% confidence limit EBPR value of 1.12 was calculated. Portions of protein extract were isoelectrofocused on thin layer (0.8 mm) low concentration (5% monomer) polyacrylamide gels (pH gradient 3-10). IEF gels generally showed resolution of 12 to 23 protein bands in the muscle extracts, depending upon temperature treatment. Certain bands with apparent isoelectric points (pis) ranging from 7.4 to 8.5 decreased in staining intensity (silver stain) as temperature increased. The general protein separation profiles correlated with decreasing EBPR values as temperature increased.

Time Temperature Heating Effect on Biuret-Positive Water-Extractable Porcine and Bovine Muscle Proteins

Ground pork longissimus or beef semimembranosus muscle was heated in stoppered glass tubes in a controlled temperature bath at 60, 65, 67.5, 70, or 75°C and held for 0, 7.5, 15 or 30 min after the sample reached the desired internal temperature, removed, and cooled (0-2°C) immediately. Heated samples were homogenized with deionized water at a ratio of 1:3.3 (w/v) muscle to water. The amount of water-extractable proteins was determined by the biuret method. Eight ml of clear extract from each treatment was reheated for 15 min at 70°C, removed, and cooled (0-2°C) immediately. Coagulum was removed by filtration (0.45 μm), and a biuret measurement made on the clear extract. These two values were used to calculate a water-extractable biuret-positive ratio (EBPR) value for a specific time/temperature treatment. The base value of 70°C was selected for the ratio because it represents a temperature slightly above that necessary for thermal inactivation of certain animal viruses required by USDA-APHIS/FSIS for certain imported canned meat products. Heat denaturation/coagulation of water-extractable, biuret-positive bovine and porcine compounds with subsequent solubility loss was a time/temperature-dependent process through 70°C. EBPR values for bovine and porcine muscles heated up to 60, 65, 67.5, 70, or 75°C with no holding time were 2.11, 1.23, 1.08, 1.07, 1.02 and 2.88, 1.65, 1.13, 1.04, and 1.02, respectively. Using 70°C as the critical denaturation/coagulation temperature, EBPR values for beef and pork were 1.07 ± .024 and 1.04 ± .066, respectively. Upper 95% confidence limits were 1.30 for beef and 1.12 for pork.

Acid Phosphatase Activity and Color Changes in Consumer-Style Griddle-Cooked Ground Beef Patties

The U.S. Department of Agriculture and the Food and Drug Administration have issued temperature requirements to help consumers cook beef patty products that are free of pathogens. Verification of end-point temperature (EPT) is needed in cooked meat products due to concerns over outbreaks of Escherichia coli O157:H7. Acid phosphatase (ACP) activity was studied as a potential method for determination of EPT in ground beef patties cooked nonfrozen, patties frozen 7 days and thawed at room temperature 4 h in a refrigerator or by microwave, and patties made from ground beef frozen in store packages, then thawed in a refrigerator overnight. Pressed-out meat juices were analyzed from patties (n = 314) cooked to 57.2 degrees C (135 degrees F). 65.6 degrees C (150 degrees F), 71.1 degrees C (160 degrees F), and 79.4 degrees C (175 degrees F) target EPTs. Expressed meat juice and internal meat patty color decreased in redness as EPT increased. Freezing whole packs with slow refrigerator or room temperature thawing caused significantly greater loss of redness in expressed cooked meat juice than did other handling methods. Log10 ACP had a significant linear (R2 = 0.99) response to EPT. Results show that the 3- to 5-min ACP test could be used to verify EPT in griddle-cooked hamburger patties.

Evaluation of a rapid method for measurement of catalase activity in cooked beef and sausage

Catalase (CAT) activity in ground beef and pork was determined on samples cooked from 60 to 71.1 degrees C. One-gram samples of ground round (4% fat), hamburger (24% fat), and commercial pork sausage (38%fat) were cooked in a controlled-temperature waterbath at 65, 68.3 and 71 degrees C. Chilled samples were immersed in direct contact with the cooking water; the test samples were removed every 15 s and immediately immersed in an ice-water bath (O to 1 degrees C) to quick-chill the samples to prevent temperature over-run. Samples retained high (HMB value 20+, over range) CAT activity through 90, 60, and 45 s at 65, 68.3, and 71 degrees C, respectively, before showing rapid activity decreases. Four USDA-FSIS approved meat patty heating processes (66.1 degrees C, 41 s; 67.2 degrees C, 26 s; 68.3 degrees C, 16 s; and 69.4 degrees C, 10 s) were analyzed for CAT activity in meat frozen prior to cooking was slightly lower (P < 0.05) than in degrees C meat. CAT activity decreased (P < 0.05) among meat treated at 66.1 degrees C for 41 s, at 67.2 degrees C for 26 s, and at 68.3 degrees C for 16 s, but the treatment at 68.3 degrees C for 16 s was not different (P < 0.05) from that at 69.4 degrees C for 10 s. These results show this rapid (20 to 25 min) CAT activity test could be used to establish activity values at specific end-point temperatures for model heat-processed ground beef or sausage products and may be useful to USDA FSIS process inspectors and food processors in quality assurance and HACCP (hazard analysis critical control points) programs for thermal input verification.

Evaluation of Creatine Phosphokinase Activity as a Means of Determining Cooking End-Point Temperature

The influence of cooking end-point temperatures (EPTs) of 62.8, 66.7, 67.8, 68.9, 70.0, 71.1, 73.9, and 76.7°C on residual creatine phosphokinase (CPK) activity in laboratory prepared model systems of ground chicken and turkey breast meat was determined. CPK activity was also assayed in commercially prepared chicken, turkey, and meat products using a Sigma #661 CPK test kit. Three tenths milliliter of 0.9% saline extracts obtained from the chicken, turkey, and meat products was substituted for 0.3 ml serum specified in the test kit procedure. For the model samples, there was a marked decrease in CPK activity as EPT increased from 66.7 to 76.7°C; however, model samples heated to 76.7°C did retain low amounts of CPK activity. In general, very low levels of CPK were found in commercially prepared chicken and turkey products (0 to 10.6 Sigma units/ml). Results of CPK activity in commercially prepared meat products would indicate that the test is product dependent, with values ranging from zero for beef franks to 258 Sigma units/ml for hard salami. Thus, while CPK activity may be useful for detecting cooking EPT for quality control purposes, it should not be used as a regulatory procedure where experience with the specific product is not available.

Evaluation of a Sound Velocity Analyzer for Estimating Maximum Internal Temperature to Which Meat Products Have Been Heat Processed

The sound velocity (SV) values of a variety of solutions (distilled water, saline, silicon dioxide, bovine serum albumin, coagulated beef protein) were determined as the temperature of the solution was increased from 10 or 20° to 71°C. Few differences were found in the SV values for the various types of distilled water; however, there was an increase in SV values as the concentration of saline increased. Level of silicon dioxide or bovine serum albumin had no significant effect on SV values; however, there was an increase in SV values as percentage of precoagulated beef protein solids in solution increased. The sound velocity analyzer used in this study was found not to be sensitive enough to detect the onset of coagulation of saline soluble proteins and, therefore, would not lend itself as an objective method which could be related to end-point temperature of meat products.