George L. Long Pomona College Claremont, California 9171 1
I
I
Analysis of Lipid Content and Composition of Ground Beef
(
An undergraduate biochemistry experiment
T h e isolation and analysis of the major lipid fraction from animal d e p o t f a t presents itself a s a simple a n d relev a n t hiochemistry teaching laboratory exercise. Quantification of lipid c o n t e n t in ground heef i s also a timely exercise i n t h i s age of renewed consumer awareness, a n d can h e performed with simplified methods a n d equipment. O n e of t h e most valuable a n d straightforward techniques developed for the characterization of lipid components is gas chromatography. Probably greater t h a n 99% of t h e lipids isolated from ground heef a r e triglycerides b y virtue of t h e ground d e p o t f a t (1). Furthermore, only t h r e e f a t t y acids account for about 90% of t h e t o t a l lipid fraction (2). T h e simplicity of t h e lipid composition permits one t o analyze t h e various components a n d their relative a m o u n t s b y gas chromatogr a p h y without further purification. This p a p e r outlines methods for t h e isolation, quantitation, a n d gas chromatographic analysis of lipid from ground beef; a n d presents t h e results obtained in a n undergraduate hiochemistry lahoratory.
Experimental Lipid was extracted from ground heef obtained from local supermarkets by the method of Bligh and Dyer (3). Seven grams of tissue was disrupted in 33 ml distilled water: trichloromethane: methanol (3:20:10 by volume) with the aid of a small mortar and pestle and 0.5 g clean sand. The solvent mixture was filtered through glass wool contained in a glass filter into a 250-ml separatory funnel. The macerated tissue was reextraded with 38 ml H20: CHC13:CHzOH (8:20:10) and the solvent filtered as above. To the combined solvent fractions was added 40 ml H20:CHC13(1:l). The contents were gently mixed and allowed to stand until the solvent phases separated.' The CHC13 phase was collected in a 125-ml Erlenmeyer flask and the solvent was removed by gentle swirling on a steam pot contained in a well-vented hood. When the solution stopped boiling vigorously it was transferred to a tared 5-in. watchglass with a Pasteur pipet. The flask and pipet were rinsed with 2 ml CHCh and the rinse was added to the residue. The residue was dried to a cake by gentle swirling and blowing near an air vent and allowed to stand overnight on the lsb bench. The watchglass was then weighed and the grams lipid per gram of ground heef was calculated. Methyl ester derivatives were made from the above isolated lipid fraction by a modification of the procedure of Allen and Good (4). About 1 mg of the lipid fraction was transferred to a clean tared 4-in. screw cap vial. Ten milliliters of 5% H&04 in methanol was added and the lipid was dissolved. The vial was placed in a
Gas chromatographic elution profile fafatty acid melhyl esters. Elution was on a 5% LAC column on Chromasorb G. 60-80 mesh. 1% in. X 6 Ill at 150°C, flow rate of 32 ml He per minute. The anow denotes the paint of sample (1 @I) inhduction. A: NIH Standard "D" containing the methyl esters of Cleo (I). Cleo (Ii), C m (111). C l e : ~(IV). C,=I (Vl fatty acids. 6: Ground beet lipid sample containing compounds l-V plus the C,.., (VI) and C,a:;. (VII) methyl esters.
70°C oven for 3-5 min and the cap tightened.z The mixture was allowed to incubate for 1-2 hr a t 70°C and then cooled. Ten milliliters water was added and the mixture shaken. The aqueousmethanolie solution was extracted twice with 4 ml hexane and the pooled heaane phases were washed in a test tube with 5 ml water. A "pinch" of NaHCOz was added to the heaane, the solution was mixed, and the NaHCOs allowed to settle. The hexane phase was removed and evaporated to dryness in a 2-in. screw cap vial over a steam pot. The remaining residue was dissolved in ahout 10 drops of carbon disulfide for gas chromatography. A sample volume of 1-10 ul was run on a 5% LAC column (a linear alternating polymer of diethylene glycol and succinic acid) in a Varian Aerograph series 2700, gas chromatograph which was attached to a l-mV pen re-
'
Emulsification has not been a problem using the above procedure. However, the rate and degree of phase separation could he enhanced by centrifugation a t this point. V n cases where the use of an oven might result in ignition of the limited amounts of CH30H vapors, a constant temperature metal heating block or water bath can be used.
Tabla 1. Summary of Lipid Content in Ground Beef %
Pricelib
Source
Market A Market A Market A Market B Market B Market B. Market C a Market C Markst D Market D Pomona College Dining Hail
0.88 1.09 1.38 0.92 1.09 1.29 0.87 1.17 0.88 1.19 5700/Sem.
$
Grade Regular Lean
X-lean Regular Lean
X-lean Regular X-lean Re4ular Lean Bulk
Lipid lindividuall Iexfracfion6)
31.7. 33.3. 32.8 29.6, 28.3, 29.2, 30.7, 28.5 20.1,19.1, 20.1.19.5 27.0. 27.4. 27.7, 27.0 23.1, 23.5. 22.4 16.4. 18.1. 16.3. 18.6 26.5. 26.3. 24.8 16.9. 15.1,15.1. 17.0 23.8. 22.7.24.5. 23.5 14.6. 15.8; 17.3. 16.1. 16.6 16.5, 16.7, 16.6, 15.5
Average
Pricellb nonlipid material
32.6 29.6 19.9 27.5 23.0
$1.30 1.55 1.72 1.27 1.42
16.1 16.3
1.42
. ..
'Market c Publicly claimed that their regular and extra lean ground beef contained approximately 25 and 16% fat, respectively. Volume 52,Number L?, December 1975 / 813
Table 2.
Percent Composition of Fatty Acids From Ground Beef
Fatty Acid
a Values are
%
observeda
% Reported (Ref.
(I))
based on the relative areas of the peaks depicted in the
figure.
corder. (Many other polar columna could be used as well.) A small ~amplcof the NIH standard mix D containing myristate, palmitate, palmitoleate, stearate, and oleate methyl esters was run as a reference. The structural assignment for each of the major unknown peaks was made on the~basisof the standard's elution profile and the straight line obtained by plotting the log (retention time of the unknownlretention time of the standard (mvristate)) versus the molecular weixht of the saturated fatty arid methyl erterr. A similar plot for the unsaturated fatty acid methyl esterswill give a straight h e whose slope is parallel to that of the saturated compounds. The relative amount of each of the major components was determined by cutting out the peaks and weighing them. Resuits and Dlscusslon Table 1lists a summary of the results obtained by a biochemistry student laboratory regarding the percent lipid extracted by the above procedure. The individual values in
814 / Journal of Chemical Education
each line represent results from replicate extractions by a single team of two students. The reproducibility of values for each student team is remarkably good for such a simple extraction nrocedure. The averaee ~ e r c e n lt i ~ i dvalues reveal a great deal of variation for'different'markets and mades of meat. Thev eenerallv confirm other studies which i;ave suggested tha
