comparison of fat extraction methods for analysis of meat · introduction historically, covance has...

IntroductionHistorically, Covance has seen a discrepancy between Soxhlet and acid hydrolysis fat results, which prompted an investigation into the possible impact of methodology on total fat results. In addition, the long reflux time for Soxhlet extractions makes the recent use of microwave accelerated technology and other methods advantageous when quick results are needed. Determining total fat within meat is important as consumers have become more health conscious and obesity has grown to be a major health problem in many countries. Food from animal sources may contain high amounts of fats, which are highly associated with calories. There are a wide variety of methods available for the analysis of total fat in meat that may provide varying results.

Presented here is a comparison of four commonly used methods for the determination of total fat in meat: acid hydrolysis, Soxhlet, chloroform-methanol extraction (CHCl3-MeOH) and microwave accelerated extraction (MAE). The methods each rely on different solvents and extraction techniques to determine the amount of crude fat. Three different types of meats (ground beef, skinless chicken breast and bratwurst) were analyzed in a raw and cooked state. Five replicates of each meat type were analyzed on one day. In order to demonstrate the day-to-day variance of the methods, an additional day of five replicates was analyzed for the ground beef.

MethodsFat by Soxhlet Extraction (Soxhlet)The samples were weighed into a cellulose thimble containing sea sand and dried to remove excess moisture. The fat was extracted for 5 hours using pentane as the extraction solvent. The extract was then evaporated, dried and weighed.

Reference: Official Methods of Analysis of AOAC INTERNATIONAL, Method 960.39 and 948.22, 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD (2005).

Fat by Acid Hydrolysis The samples were hydrolyzed with 8.3 N hydrochloric acid and transferred to a Mojonnier flask. The fat was extracted using ethyl ether and hexane. The extracts were evaporated over steam, re-dissolved in hexane and filtered through a column of sodium sulfate. The filtered extracts were then evaporated, dried and weighed.

Reference: Official Methods of Analysis of AOAC INTERNATIONAL, Methods 922.06 and 954.02, 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD (2005).

Fat by Chloroform-Methanol (CHCl3-MeOH)The samples were enzymatically hydrolyzed with taka-diastase and extracted using a mixture of chloroform and methanol. A portion of the chloroform layer was filtered through a column of sodium sulfate, evaporated, dried and weighed.

Reference: Official Methods of Analysis of AOAC INTERNATIONAL, Method 982.23, 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD (2000).

Fat by Microwave Accelerated Extraction (MAE)The samples were weighed into a CEM Green Plus Vessel containing a small amount of sea sand and dried for 6 minutes using 75% power, 1600 watts. The fat was extracted using petroleum ether as the solvent. The extraction temperature was ramped over 10 minutes to 100°C and held for 20 minutes. The extract was filtered through a column of sodium sulfate, evaporated, dried and weighed.

Reference: Microwave Accelerated Extraction of Fat from Food Samples, Greg LeBlanc, Arve Hubbard, and Chaunda Goldson, CEM Corporation, Matthews, NC.

Materials Analyzedn Ground beef (93% lean/7% fat) n Chicken breasts, skinless n Bratwurst

Experimental DesignCookingEach sample type was analyzed raw and after cooking. The ground beef was cooked by preparing beef patties. To achieve uniform sizing for the broiled patties, 112 grams of raw ground beef were pressed into each patty. All ground beef patties were cooked to a final internal temperature of 70°C in a preheated conventional oven. No fat was added during cooking. Chicken breasts were cooked by roasting in a preheated conventional oven to a final internal temperature of 60°C. The chicken breasts were positioned in the center of the rack in an uncovered roasting pan and no oil or water was added. The bratwurst samples were cooked to a final internal temperature of 80°C in a preheated George Foreman Grilling Machine. The bratwurst samples were evenly spaced in the center of the cooking grate and the grill lid was closed.

Analysis of Fatty AcidsAfter extraction, a portion of the fat was saponified with 0.5 N methanolic sodium hydroxide and methylated with 14% BF3-methanol. The resulting methyl esters were extracted with heptane containing an internal standard.

Reference: Official Methods and Recommended Practices of the AOCS, Ce 2-66, 6th Ed., AOCS, Champaign, IL (2009).

Statistical AnalysisResults from the analysis of the fat and fatty acids in the various sample types were statistically compared using Analysis of Variance (ANOVA). The mean results from each method were compared separately for the raw and cooked samples. In addition, a test for equal variances was performed to compare the variance in the results from each method. All statistical analyses were performed at a 95% confidence level.

Comparison of Fat Extraction Methods for Analysis of MeatSharon Habeck, Barb Mitchell and Darryl SullivanCovance Laboratories Inc., Madison, Wisconsin

ResultsGround Beef (93% lean/7% fat)In the raw ground beef, the Soxhlet method identified 7.6% fat, which was less than the other methods (Table 1). After cooking, the different methods provided similar results in that the Soxhlet method yielded slightly less fat than the other methods (Table 2). All methods provided acceptable within- and between-day precision.

ANOVA analysis indicated the mean results by Soxhlet were statistically lower than the other methods (Tables 3 and 4). The acid hydrolysis method yielded higher values for the cooked sample than the other methods.

Replicate Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 21 8.1 7.8 7.5 7.8 8.3 8.6 8.2 8.02 8.2 8.0 7.7 7.5 8.2 8.3 8.4 8.03 8.2 8.0 7.7 7.6 8.2 9.3 8.1 7.94 8.1 7.8 7.6 7.7 8.1 8.2 8.3 7.95 8.2 7.9 7.6 7.6 8.3 8.4 8.0 7.6

Mean 8.2 7.9 7.6 7.6 8.2 8.6 8.2 7.9SD 0.05 0.10 0.08 0.11 0.08 0.44 0.16 0.16

RSD (%) 0.6 1.3 1.1 1.4 1.0 5.1 2.0 2.0

Overall Mean 8.0 7.6 8.4 8.0Overall SD 0.16 0.09 0.35 0.23

Overall RSD (%) 2.0 1.2 4.2 2.9

MAECHCl3-MeOHAcid Hydrolysis Soxhlet

Table 1. Fat Results for Raw Ground Beef (g/100g)

Replicate Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 21 13.9 14.0 13.4 12.9 13.8 13.6 13.1 13.12 13.9 14.1 12.8 12.9 12.5 13.5 13.1 12.83 13.4 13.7 12.4 13.2 13.5 13.3 12.8 13.04 13.3 13.7 12.8 12.7 12.9 13.3 13.1 13.15 13.0 13.8 12.6 12.5 14.1 13.3 13.1 13.0

Mean 13.5 13.9 12.8 12.8 13.4 13.4 13.0 13.0SD 0.39 0.18 0.37 0.26 0.65 0.14 0.13 0.12

RSD (%) 2.9 1.3 2.9 2.0 4.9 1.0 1.0 0.9

Overall Mean 13.7 12.8 13.4 13.0Overall SD 0.35 0.30 0.45 0.12

Overall RSD (%) 2.6 2.3 3.4 0.9

MAEAcid Hydrolysis Soxhlet CHCl3-MeOH

Table 2. Fat Results for Broiled Ground Beef (g/100g)

Method N Mean GroupingCHCl3-MeOH 10 8.39 A

MAE 10 8.04 BAcid Hydrolysis 10 8.03 B

Soxhlet 10 7.63 CMeans that do not share a letter are significantly different

Table 3. Tukey Method Grouping Raw Ground Beef (g/100g)

Method N Mean GroupingAcid Hydrolysis 10 13.68 ACHCl3-MeOH 10 13.38 AB

MAE 10 13.02 BCSoxhlet 10 12.82 C

Means that do not share a letter are significantly different

Table 4. Tukey Method Grouping Broiled Ground Beef (g/100g)

The total amount of fatty acids as a ratio of the amount of fat (% total fatty acids/fat) was similar across all methods. The lower ratio for the chloroform-methanol method was expected due to the higher polarity of the solvents used. Higher polarity solvents will extract more polar material, such as phospholipids, which contain a lower percentage of fatty acids by weight (Tables 5 and 6).

Acid Hydrolysis Soxhlet CHCl3-MeOH MAETotal Fat (Gravimetric) 8.0 7.6 8.4 8.0

Total Fatty Acids 7.15 6.87 7.18 7.37% Total Fatty Acids/Fat 89.4 90.4 85.5 92.1

Monounsaturated Fat 2.96 2.90 2.97 3.07Polyunsaturated Fat 0.350 0.204 0.336 0.270

Saturated Fat 3.13 3.05 3.16 3.26Trans Fat 0.395 0.389 0.400 0.429

Table 5. Fatty Acid Results for Raw Ground Beef (g/100g)





Table 6. Fatty Acid Results for Broiled Ground Beef (g/100g)

Chicken BreastsIn the raw chicken breasts, the Soxhlet method again provided statistically lower results than the other methods, and the chloroform-methanol method provided statistically higher results (Table 7). The acid hydrolysis and chloroform-methanol extractions yielded the same result for the roasted chicken breasts (Table 8). Evaluation of the fatty acid analysis showed higher recovery of fatty acids for the microwave accelerated extraction in the raw chicken breasts when compared to the other methods. However, for the roasted chicken breasts, the % total fatty acids/fat was similar for three of the methods, with the chloroform-methanol method being lower, as was the case with the ground beef.




Saturated Fat 0.521 0.364 0.616 0.510Trans Fat <0.020 <0.020 <0.020 <0.020

Table 7. Fatty Acid Results for Raw Chicken Breasts (g/100g)




Saturated Fat 0.986 0.849 0.927 0.811Trans Fat <0.020 <0.020 <0.020 <0.020

Table 8. Fatty Acid Results for Roasted Chicken Breasts (g/100g)

Statistical analysis indicated again that extraction by Soxhlet was lower than the other methods (Tables 9 and 10). For roasted chicken breasts, the chloroform-methanol and acid hydrolysis methods were statistically the same, but both statistically greater than the microwave accelerated extraction and Soxhlet methods.


MAE 5 2.04 BAcid Hydrolysis 5 1.96 B


Table 9. Tukey Method Grouping Raw Chicken Breasts (g/100g)


MAE5 3.46 AAcid Hydrolysis5 3.04 B


Table 10. Tukey Method Grouping Roasted Chicken Breasts (g/100g)

BratwurstThe higher fat bratwurst samples displayed less variation between methods than the previous samples. In the raw and cooked samples, the acid hydrolysis and microwave accelerated extraction methods provided the highest results (Tables 11 and 12). The ratio of fatty acids to total extracted fat was consistent between all four methods.





Table 11. Fatty Acid Results for Raw Bratwurst (g/100g)





Table 12. Fatty Acid Results for Grilled Bratwurst (g/100g)

Results from the statistical analysis indicated that for the raw bratwurst, the mean of the chloroform-methanol results was significantly lower than the other three methods (Table 13). For the grilled bratwurst, the mean results for all four methods were statistically equivalent (Table 14).

Method N Mean GroupingAcid Hydrolysis 5 21.64 A

MAE 5 21.52 ASoxhlet 5 21.16 A

CHCl3-MeOH 5 18.88 B


Table 13. Tukey Method Grouping Raw Bratwurst (g/100g)

Method N Mean GroupingAcid Hydrolysis 5 25.28 A

MAE 5 24.66 ASoxhlet 5 24.36 A

CHCl3-MeOH 5 24.28 A


Table 14. Tukey Method Grouping Grilled Bratwurst (g/100g)

DiscussionIn general, the data generated by the Soxhlet method produced the lowest fat content. The Soxhlet method utilized a non-polar solvent (pentane) whereas the acid hydrolysis and chloroform-methanol methods used polar solvents (chloroform and ethyl ether). As the solvents increase in polarity, so does the ability of the solvent to extract polar lipids. When the research requires minimization of polar lipids, the Soxhlet method may be the method of choice.

The acid hydrolysis and chloroform-methanol methods tended to extract higher concentrations of fat in all samples. This difference between methods was more pronounced in the lower fat samples (raw and roasted chicken) with the methods not being significantly different on higher fat samples. The ratio of fatty acids to extracted fat was greater in higher fat samples than lower fat samples indicating possible concentration dependence. Excluding the chloroform-methanol method, this ratio was similar across the different methods within each sample type, indicating that the majority of the extracted material was fat.

In cooked products, the acid hydrolysis method yielded higher results. A more rigorous method may be required to extract the fat due to the chemical changes that occur during cooking. As the acid hydrolysis method yielded higher fat recoveries, while maintaining high percentages of fatty acids per extracted fat, it gave a better representation of the total fat content. Although the chloroform-methanol method also yielded higher results, it was not as safe or “environmentally friendly” as the other methods. The increased fat recovery that was gained was outweighed by the disadvantages of using chloroform. The results demonstrated that microwave accelerated extraction can be used as an alternative to traditional methods. The advantages of the microwave accelerated extraction method were a reduction in the amount of solvent used and shorter extraction times.

ConclusionsThe differences between the four commonly used methods for determination of total fat in meat appear to be dependent on the level of fat in the meat and the state of the meat – raw or cooked. While the results demonstrated statistical differences between the methods, the final results for each method yielded values within 1 gram of fat per 100 grams of sample.

In conclusion, the results indicated that acid hydrolysis is a better choice for cooked meats and performed as well as chloroform-methanol for uncooked meats. Ultimately, the selection of a crude fat method for meat should be based on the regulatory guidelines or intended scientific use of the data, as different methods will produce slightly different results.

AcknowledgementsWe would like to thank J. Polywacz for the statistical analysis and the following Lipid staff for their research help: Y. Herr, A. Dyer, H. Baganz, E. Kenseth, C. Richard, D. Fletcher, M. Borde, R. Connelly, S. Trimble, R. Stangl, S. O’Kroley, R. Gotz, D. Saylor, T. Yang, P. Linzmeier, E. Kennedy, A. Gates, K. Wincapaw, T. Rindfleisch, M. Shafer, S. Kanable, J. Day and J. Maly.

Presented at the AOAC 127th Annual Meeting & ExpositionChicago, Illinois25–28 August 2013

Comparison of Fat Extraction Methods for Analysis of Meat

Sharon Habeck, Barb Mitchell and Darryl SullivanCovance Laboratories Inc., Madison, Wisconsin

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Web Site: www.covance.com

Covance is an independent, publicly held company with headquarters in Princeton, New Jersey, USA. Covance is the marketing name for Covance Inc. and its subsidiaries around the world.

© COPYRIGHT 2013, COVANCE INC.

comparison of fat extraction methods for analysis of meat · introduction historically, covance has...

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