Determination of the moisture and fat content of meat

Transcription

1 802 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 FOOD COMPOSITION AND ADDITIVES Determination of Moisture and Fat in Meats by Microwave and Nuclear Magnetic Resonance Analysis: Collaborative Study TIMOTHY P. LEFFLER,CINDY R. MOSER,BOBBIE J. MCMANUS,andJOHN J. URH 1 CEM Corp., 3100 Smith Farm Rd, Matthews, NC JIMMY T. KEETON and AMY CLAFLIN Texas A&M University, Department of Animal Science, 2471 TAMU, College Station, TX Collaborators: K. Adkins; A. Claflin; C. Davis; J. Elliot; P. Goin; C. Horn; J. Humphries; K. Ketteler; P. Perez; G. Steiner Ten laboratories participated in a collaborative study to determine the total moisture and fat in raw and processed meat products by microwave drying and nuclear magnetic resonance (NMR) spectroscopy. Meat products were prepared following the AOAC Method and analyzed using CEM Corp. s SMART Trac Moisture and Fat Analysis system. SMART Trac provides moisture results by measuring the weight loss on drying by microwave energy. The dried sample is then analyzed by NMR spectrometry for fat content. Moisture and fat results are displayed and reported by the SMART Trac as a percentage (g/100 g). Microwave drying is an AOAC-approved reference method (Method ), Moisture in Meat and Poultry Products. NMR spectrometry is a secondary technique used to determine the concentration of various constituents in biological, organic, or chemical samples. The study design was based on Youden s matched pair principle for collaborative tests. For the purposes of this study, 10 laboratories each tested 10 Youden matched pairs, for a total of 20 samples. The study samples represented a range of products processed daily in plant operations. Included were raw meat samples (beef, pork, chicken, and turkey) as well as processed meats (beef hot dog, pork sausage, and ham). The total moisture content of the undiluted samples, as received for the purposes of this study, was determined by AOAC Method and ranged from to 74.99%. The total fat content of the undiluted samples was determined by AOAC Method and ranged from 1.00 to Submitted for publication June The recommendation was approved by the Methods Committee on Commodity Foods and Commodity Products as First Action. See Official Methods Program Actions, (2008) Inside Laboratory Management, May/June issue. 1 Author to whom correspondence should be addressed; john.urh@cem.com 29.79%. Statistical analysis of study results for total moisture yielded a relative standard deviation for repeatability (RSD r ) range of 0.14 to 0.95% and a relative standard deviation for reproducibility (RSD R ) range of 0.26 to 0.95%. Statistical analysis for total fat yielded similar RSD r and RSD R range of 0.74 to 4.08%. Results for turkey had higher RSD r and RSD R values, both at 12.6%, due to low fat content and possibly to the separation of the samples observed by some of the collaborators. Results demonstrate that microwave drying with NMR is a rapid, practical method providing results equivalent to AOAC Methods (Forced Air Oven Drying) and (Soxhlet Ether Extraction) in raw and processed meat products. Determination of the moisture and fat content of meat products is commonly performed for raw material acceptance, in-process control, pricing, quality assurance, and regulatory compliance. The purpose of the study was to quantify the performance of CEM s SMART Trac system for the rapid determination of moisture and fat for raw and processed meat products. This is of particular interest as the SMART Trac is already in widespread use in this industry. Traditional methods for determining the moisture and fat content of foodstuffs include loss on drying and solvent-based chemical extractions. The time lag inherent in the widely used traditional methods prevents production processes from operating at optimal efficiency. Furthermore, traditional methods require solvents that are expensive, often hazardous, and pose disposal problems. Accordingly, producers have sought alternative means for determining moisture and fat content in samples. Currently, an AOAC Official Method exists for rapid moisture analysis by microwave drying (1) and for fat analysis, which uses methylene chloride (2). The proposed rapid method also uses microwave drying for moisture analysis but includes nuclear magnetic resonance (NMR) for

2 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, Table 1. Youden pair identifiers were assigned to the following samples Pair No. Pair Sample No. I Ham, low-fat 17, 18 II Ham, high-fat 19, 20 III Pork, low-fat 6, 8 IV Pork, high-fat 5, 7 V Beef, low-fat 2, 4 VI Beef, high-fat 1, 3 VII Chicken, low-fat 9, 10 VIII Beef hot dog, high-fat 13, 14 IX Turkey, low-fat 11, 12 X Pork sausage, high-fat 15, 16 fat analysis. This method of analysis was previously described in AOAC Peer-Verified Method PVM 1:2003 (3). Collaborative Study Ten laboratories participated in the collaborative study. The collaborators received 20 samples [10 Youden (4) matched pairs; see Table 1] comprising the primary meat categories: beef, chicken, pork, ham, and turkey in various concentration ranges for moisture and fat in both raw and processed meat. Prior to the beginning of the study, a trial run was conducted between the Department of Animal Science at Texas A&M University (TAMU) and CEM using practice beef samples. Because all of the collaborators were experienced in the meat and food processing industry and proficient in sample analysis, they were not included in the trial run. The primary purpose of the trial run was to confirm sample preparation, packaging, delivery, and reporting. TAMU prepared the samples included in this study as follows: To minimize water loss during preparation and subsequent handling, samples weighing kg (10 15 lbs) were used. Ground material was kept in containers with air- and watertight covers. Samples were prepared for analysis as follows: (1) Approximately 6.8 kg (15 lbs) of fresh product required for samples to be split (e.g., beef, high-fat, approximately 30%) and beef high-fat diluted to 5% of the initial fat value (e.g., % fat) were obtained fresh from national meat processing plants and stored at 4 Cfor 3 days. Additional samples necessary for the method setup (i.e., to establish a standard curve for a particular product) required only 2.3 kg (5 lbs) of material. Table 2. Results of AOAC Methods for moisture (950.46) and fat (960.39) Moisture, % Fat, % Sample No. Sample name Mean a SD RSD Mean a SD RSD 1 Beef, high-fat Beef, low-fat Beef, high-fat, diluted Beef, low-fat, diluted Pork, high-fat Pork, low-fat Pork, high-fat, diluted Pork, low-fat, diluted Chicken Chicken, diluted Turkey Turkey, diluted Beef hot dog Beef hot dog, diluted Pork sausage Pork sausage, diluted Ham, low-fat Ham, low-fat, diluted Ham, high-fat Ham, high-fat, diluted a Ten replicate determinations.

3 804 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 (2) Whole-muscle meat product samples were diced into approximately 5.08 cm (2 in.) cubes and passed through a Hobart grinder (Model 4612; Troy, OH) held at 4 C and equipped with a 1.27 cm (1/2 in.) plate. Coarse ground samples then were reground using a fine cm (1/8 in.) plate. All ground meat products then were homogenized in a chilled (4 C) Robot Coupe bowl chopper (Model R10; Jackson, MS) to a paste or pâté consistency. Ground material was placed in the chilled bowl chopper and chopped for 30 s. Then the inner side wall and bottom of the bowl were wiped down with a spatula [plastic or rubber spatula with cm (2 4 in.) straight-edge blade], and the gathered material was transferred from the spatula to the bulk of the test sample. The process was repeated for an additional 30 s. Once the fat content was determined, Youden pairs were prepared with the balance of the meat homogenate. This was done by weighing a portion of the sample to the nearest gram and adding sufficient chilled, distilled, deionized water to dilute the fat content of the sample by approximately 0.4%. The samples were then rehomogenized as above to thoroughly incorporate the water, collected, and frozen. These prepared samples and their Youden pairs were analyzed following AOAC standard methods for moisture analysis, (5), and fat analysis, (6). All tests were performed 10 times each on 20 samples (10 Youden matched pairs) at TAMU. The results are reported in Table 2. After preparation of the Youden matched pairs of samples, the moisture levels varied from to 75.91% and fat levels had a range of 0.74 to 29.79%. The homogeneity of the samples was evaluated for acceptability by determining the relative standard deviation (RSD) of the 10 replicate analyses for the samples using the criteria described by Thiex (7), where the resulting value should be <2.0. All results were below the critical value with the exception of fat results for turkey and low-fat ham. The relatively high RSD for these samples was attributed to the low levels of fat present. Additional samples that were required to establish a standard curve for each product were divided and frozen as above. All samples then were divided into 56.7 g (2 oz) aliquots and stored in plastic containers with screw-cap lids allowing a minimum amount of air space. They were then packaged in cardboard-covered insulated freezer boxes and frozen at 40 C. It was necessary to freeze all samples before analyses for consistency of evaluation, because the collaborator laboratories received frozen samples from TAMU. Two 56.7 g (2 oz) containers of each homogenized meat product were provided to each laboratory for evaluations in this study. Only one sample was needed for the study. The second aliquot was provided as a backup sample in case of a problem with the first. Data were gathered on 5 Youden pairs during September 2006 and a second set of 5 Youden pairs was analyzed during November Samples were shipped with ice packs by overnight delivery, 2 shipments per laboratory, for a total of 20 samples (10 Youden matched pairs) per laboratory. (3) All samples were thawed at 4 C at least overnight, but not more than 72 h. It was imperative for all laboratories to thoroughly stir each sample before analysis. In addition, samples were prepared as quickly and consistently as possible to minimize moisture loss and subsequent skewing of results. Collaborators were instructed to analyze each study sample once as described in the method, and make no modifications of any kind. They were then instructed to submit the sample results to the Study Director. AOAC Official Method Moisture and Fat in Meats Microwave and Nuclear Magnetic Resonance Analysis First Action 2008 [Applicable for the determination of moisture and fat in meat and meat products (fresh, finished products and emulsions of beef, pork, and poultry).] See Tables A and B for the results of the interlaboratory study supporting acceptance of the method. A. Principle The method includes a microwave-drying step followed by nuclear magnetic resonance (NMR) analysis. Moisture is evaporated from the sample by using microwave energy. Weight loss is determined by electronic balance readings before and after drying and is converted to moisture content by the system s microprocessor. Fat is determined by pulsed radio frequency (RF) energy while within a static 0.47 Tesla magnetic field. The resulting NMR signal is recorded and analyzed for the total proton activity of fat present in the sample. The loss-on-drying by microwave is a reference method (985.14), and the NMR method is a secondary technique to predict the concentration of fat in biological or organic samples. NMR was discovered in the middle of the last century and is based on the observation that certain nuclei will absorb and re-emit RF energy over a narrow band of frequencies when placed in a static magnetic field. The frequency at which the NMR effect occurs for a given nuclear isotope is dependent on the magnetic field strength of the magnet, and the phenomenon is caused by the interaction between the nuclear magnetic dipole of a nucleus and the magnetic field it experiences. (The latter is the reason the word nuclear is included in the description of the phenomenon. NMR does not involve the emission of ionizing radiation.) Although many nuclei can be made to generate an NMR signal, the overwhelming majority of NMR experiments involve the excitation and detection of signals from the 1 H nucleus, and this branch of the science is commonly known as proton NMR. NMR has been widely used as the basis of an analytical spectroscopy technique (NMR spectroscopy) for several decades and is the basis of magnetic resonance imaging (MRI), which has been used as a clinical diagnostic tool for nearly 20 years. The NMR technique incorporated into the SMART Trac system is based on low-resolution time domain NMR (often called LR-NMR). This is a small offshoot of NMR

4 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, Table A. products Interlaboratory study results for the determination of percent moisture in raw and processed meat Repeatability Reproducibility Sample Youden pair No. of labs Mean s r RSD r s R RSD R HorRat Beef, high-fat VI Beef, low-fat V Pork, high IV Pork, low III Chicken VII Turkey IX Beef hot dog VIII Pork sausage X Ham, low-fat I Ham, high-fat II spectroscopy that has also been used for over 20 years for industrial quality control. The vast majority of LR-NMR is proton NMR. The main difference between LR-NMR and NMR spectroscopy is in the effects used for discriminating between different hydrogen-containing constituents of a sample. In NMR spectroscopy, these constituents are distinguished by small variations in the magnetic field that 1 H nuclei experience in different molecules and different parts of the same molecule. These variations are caused by differences in the electronic structure of molecules and lead to small differences in the NMR frequencies of 1 H nuclei in different molecules that can be used to discriminate between the different constituents within the sample. This phenomenon is known as the chemical shift effect. In LR-NMR, it is not possible to detect chemical shift effects in samples containing 1 H nuclei because of the low field strength and homogeneity of the magnet used to generate the static magnetic field. Instead, differences in the rate of decay of the signal from different constituents (commonly known as transverse relaxation or T 2 decay) are used to distinguish between NMR signals from different constituents within the sample. Transverse relaxation can generally be approximated as an exponential decay with time constant T2. In food that has undergone microwave drying, the main constituents that contain significant amounts of protons are fat, protein, and carbohydrate. There are significant differences between the proton transverse relaxation times (T 2 ) of these constituents. In particular, both protein and carbohydrate in dried foods exhibit solid-like behavior and have transverse relaxation times that are very short (typically of the order of 10 S or less) and the signal from these substances decays very rapidly. However, for fat the Table B. products Interlaboratory study results for the determination of percent fat in raw and processed meat Repeatability Reproducibility Sample Youden pair No. of labs Mean s r RSD r s R RSD R HorRat Beef, high-fat VI Beef, low-fat V Pork, high IV Pork, low III Chicken VII Turkey IX Beef hot dog VIII Pork sausage X Ham, low-fat I Ham, high-fat II

5 806 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 Figure Illustration of sample handling techniques. transverse relaxation times are considerably longer (typically of the order of 10 ms or greater) and thus the signal decays relatively slowly. In addition, any very small amounts of residual moisture that remain following microwave drying of the sample are associated with the nonlipid molecules within it (i.e., protein and carbohydrate) and also exhibit solid-like behavior. Thus, it is possible to discriminate between the fat and the other principal constituents of a dried food by exciting the system, waiting for the solid-like signals to decay, and then acquiring the remaining signal, which, in the absence of moisture, is predominantly from protons contained in fat within the sample. An NMR signal acquired from a dried food sample using the methodology described above will be directly proportional to the number of protons within the fat contained in the sample and, for many samples, is directly proportional to the fat content of the sample. B. Apparatus (a) Microwave moisture analyzer. 0.2 mg H 2 O sensitivity; moisture range of % in liquids, solids, and slurries; 0.01% resolution. Includes automatic electronic balance (0.1 mg readability), microwave-drying system with

6 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, Table 3. Interlaboratory study results (Youden pairs) of moisture Pair I Pair II Pair III Pair IV Pair V Pair VI Pair VII Pair VIII Pair IX Pair X Lab A B a C D E F G b H I J a Pair rejected by Cochran test. Pair rejected by Single Grubbs test. b Table 4. Interlaboratory study results (Youden pairs) of fat Pair I Pair II Pair III Pair IV Pair V Pair VI Pair VII Pair VIII Pair IX Pair X Lab A a B C D E F G b H b I J Pair rejected by Single Grubbs test. Pair rejected by Double Grubbs test. a b

7 808 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 temperature control, and microprocessor computer control (CEM Corp., Matthews, NC), or equivalent. (b) NMR RF pulse generator. Pulse power 250 W nominal; pulse times variable in 100 ns increments; transmit and receive phases selectable 0, 90, 180, and 270 ; nominal 90 pulse times 4 s (18 mm probe). Magnet: permanent, thermally stabilized, 0.47 T (20 MHz), homogeneity >10 ppm. Signal detection: dual channel (quadrature) detection with programmable low-pass filtering, programmable data acquisition rate up to 4 MHz per pair of points (CEM Corp.), or equivalent. (c) Glass fiber sample pads. CEM Corp., or equivalent. (d) Trac film. Used to contain a sample while it is in the NMR; made of a proprietary material that does not interfere with the NMR determination (CEM Corp.). (e) Plastic sleeves. CEM Corp. (f) Compression tool. CEM Corp. (g) Teflon-coated spatula. (h) Grinding apparatus. As described in AOAC C. Safety It is recommended that persons with heart pacemakers or other magnetically sensitive devices do not approach within 0.3 m (11 in.) of the SMART Trac magnet component. Certain heart pacemakers or other magnetically sensitive prosthetic devices may be affected by magnetic fields as low as 0.5 mt. D. Sample Preparation Follow method as described in AOAC E. Determination (1) On the SMART Trac main menu screen, select Load Method, and then select the appropriate preprogrammed item to be analyzed, e.g., ground beef. Note: Different types of sample matrixes and fat will exhibit different responses on the NMR system. In order to obtain accurate fat readings, 2 or more samples of the specific sample type must be analyzed by AOAC Method , Soxhlet Ether Extraction. Samples should cover the entire fat range to be analyzed. Preferably, one high-fat reference sample and one low-fat reference sample should be analyzed. The reference values are stored in the SMART Trac system and replicate runs of each sample are then performed to determine the appropriate NMR signal values for that specific sample type. After completing the reference scans, the SMART Trac system will establish a linear relationship for fat determination for that type of sample. (2) Press the Ready key to initiate the analysis. Place 2 square glass fiber sample pads onto the balance pan in the SMART Trac microwave chamber and press Tare on the key pad. Tare weight is automatically recorded. (3) Transfer approximately 4 g of sample with a spatula to the center of one of the tared sample pads. Spread the meat sample evenly across the square pad (see Figure , illustrations I and II). Note: Sample size should be 3 5 g. (4) Cover the sample with the other tared square pad, similar to making a sandwich, and place the pads back onto the SMART Trac s balance pan. (5) Press Start on the key pad to begin the drying process. A temperature control system allows rapid temperature measurement of the sample during drying to adjust the microwave power delivery. Percent solids (g/100 g) results are displayed on the screen (±0.01%) after the sample has dried to a constant weight. Note: Five short beeps are heard when drying is complete. (6) Remove the sample pads with the dried meat sample and rollboth in Trac film(see Figure , illustrations III and IV). (7) Compress the rolled sample in the plastic sleeve using the compression tool, and insert sample into the NMR chamber for analysis. (8) Press Ready on the SMART Trac to continue the fat analysis, and then press Start to analyze for fat. Percent fat (g/100 g) is displayed on the screen (±0.01%). Table 5. Comparison of mean moisture percent values to reference values Study Reference Sample Youden pair Mean s R Mean SD Recovery, % Beef, high-fat VI Beef, low-fat V Pork, high-fat IV Pork, low-fat III Chicken VII Turkey IX Beef hot dog VIII Pork sausage X Ham, low-fat I Ham, high-fat II

8 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, Table 6. Comparison of mean fat percent values to reference values Study Reference Sample Youden pair Mean s R Mean SD Recovery, % Beef, high-fat VI Beef, low-fat V Pork, high-fat IV Pork, low-fat III Chicken VII Turkey IX Beef hot dog VIII Pork Sausage X Ham, low-fat I Ham, high-fat II Reference: J. AOAC Int. 91, 802(2008). Results and Discussion Collaborative study results for each material are presented in Tables 3 and 4. Data analysis was conducted using AOAC s Interlaboratory Study Workbook Version 2.0 for Blind (Unpaired) Replicates (8). In order to apply the Youden pair data set shown in Tables 3 and 4 to this workbook, the data had to be modified. This modification determined the difference between the reference values reported in Table 2 for each Youden pair of samples. This difference was then applied to one of the Youden pair results. The resulting data set was essentially made up of blind duplicate results from each laboratory. Analysis of the collaborative study data found 5 result pairs that were identified as outliers by either the Cochran or Grubbs detection methods. The Cochran test showed that moisture in high-fat ham from Laboratory B should be rejected. The Single Grubbs test statistic showed that the moisture result for high-fat ham from Laboratory G and the fat result for low-fat ham from Laboratory A were to be rejected. The Double Grubbs test showed that the percent fat data from Laboratories G and H were to be rejected for turkey. The statistical analysis of the study is summarized in Tables A and B. Moisture yielded a relative standard deviation for repeatability (RSD r ) range of % and a relative standard deviation for reproducibility (RSD R ) range of %. Statistical analysis for total fat yielded an RSD r and RSD R range of %. Collaborators' comments mentioned that turkey and chicken samples showed some separation of moisture from the solid components of the sample. This, in combination with the low fat concentration, may have contributed to the relatively high RSD reported for the turkey. It is believed that the relatively higher amount of fat present in the chicken helped to reduce the RSD r and the RSD R to 2.28 and 2.35%, respectively. HorRat values were found to be acceptable for samples, rangingfrom0.12to0.44formoistureand0.31to1.18forfat, except for turkey. Turkey produced a HorRat for fat of 3.16, which exceeded the critical value of 2.00 for this statistic. The high result was due to the low amount of fat in the sample. Percent recovery reported in Tables 5 and 6 was determined by comparing the overall mean of the collaborator values from Tables A and B to the reference values reported in Table 2. The recovery is excellent for all constituents. Recovery for moisture ranged from to 99.97%, while fat ranged from to %. The data indicate that the moisture and fat results compare favorably with the AOAC results (Table 2) and are suitable for determining total moisture and fat in raw and processed meat products. Collaborators' Comments A number of collaborators reported that moisture had separated to the top of the container for Sample 10, diluted chicken, and Sample 12, diluted turkey. Collaborators followed protocol instructions to rehomogenize the sample. Recommendations Based on the results of this study, it is recommended that the method for determination of moisture and fat in meats by microwave and NMR analysis be adopted First Action. Acknowledgments We wish to acknowledge Jimmy Keeton of TAMU, Department of Animal Science, College Station, TX, and his staff for assistance with sample collection, preparation, and distribution to the collaborative laboratories. In addition, a special thanks to Brandy Broglin of CEM Corp. for assistance with the study.

9 810 LEFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 We thank the following collaborators for their time and effort involved in the study: Gina Steiner, Jones Dairy Farm, Ft. Atkinson, WI Patricia Perez, Quality Sausage, Dallas, TX Kay Ketteler, 5 Star Custom Foods, Ft. Worth, TX Paula Goin, Tyson Prepared Foods, N. Richland Hills, TX Amy Claflin, Texas A&M University, College Station, TX Chaunda Davis, CEM Corp., Matthews, NC Kevin Adkins, Wayne Farms, Dobson, NC Chris Horn, Wayne Farms, Douglas, GA Janet Humphries, U.S. Department of Agriculture, Blakely, GA Jill Elliot, Deibel Laboratories, Madison, WI References (1) Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method (2) Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method (3) Keeton, J.T., Hafley, B.S., Eddy, S.M., Moser, C.R., McManus, B.J., & Leffler, T.P. (2003) J. AOAC Int. 86, (4) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of the AOAC, AOAC INTERNATIONAL, Gaithersburg, MD (5) Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method (6) Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method (7) Thiex, N.J., & Van Erem, T. (2002) J. AOAC Int. 85, (9) AOAC Interlaboratory Study Workbook for Blind (Unpaired) Replicates (2006) AOAC INTERNATIONAL, Gaithersburg, MD, Version 2.0