Preparation of Animal Tissue Fats for Determination of Peroxides and Free Fatty Acids B. N. ROCKWOOD, J. M. RAIMSBOTTOhI, AND V. C. MEHLENBACHER Swift & Company, Research Laboratories, Chicago, 111.
A method is presented for the complete extraction of fat from meat tissues by the use of a high-speed mixing and cutting apparatus which comminutes the mass rapidly in the presence of the solvent. The fat is not altered in its free fatty acid or peroxide content during extraction, so that the procedure is applicable to studies of the fatty tissue of meat.
T
HE fatty tissues of fresh and cured meats may deteriorate in quality during prolonged storage as a result of oxidation and hydrolysis of the fat. The extent of this deterioration is commonly measured organolept'ically as well as by the extent of peroxide development and free fatty acid formation. Organoleptic methods are always subject t o the variations of human judgment. An increase in free fatty acids is associated with the hydrolysis of fats. Similarly, an increase in peroxide values is associated with oxidation of fats. Determinations of free fatty acids and peroxides have been used by various investigators in studies of meat storage. Pennington and IIepburn ( 5 ) determined the free fatty acid content of chicken tissue fat. They ground the sample in a food chopper, heated it with neutral 95% alcohol t o boiling, and titrated it immediately with sodium hydroxide, using phenolphthalein as indicator. Lea ( 4 ) studied the free fatty acid content and the peroxide value of mutton and lamb fat. He chilled small strips of fatty t,issue t o -20" U., wit,h a scalpel cut the st,rips in slices 0.5 mm. thick, and further reduced the small slices in size by cutting wit,h scissors. A chloroform-acetic acid mixture was at1dt.d t o 1 gram of the crushed fatt'y tissue and the peroxide viilue determined in the presence of the tissue. Lea (3) also invwtigat,ed the susceptibility of pork fat to oxidation, but had difficulty in obtaining uniform samples. Because strips from f:tii,ly zadjscnnt regions in the back fat of the sanie animal did not al\\-ays oxidize a t the same rate, he combined several thin sections to malie one sample. Cook and White ( 1 ) investigated tho formution of peroxides and free fatty acids in poultry and rnndc: preliminary studies on methods of preparing the fat for awilysis. The skin and fatty tissues from frozen birds viere choppc,d at freezing tempcrat,ures. They obtnin'cd best results by drying the fatty tissue under vacuum a t 50" C., follon-ed by Soshlct extraction in a darkened room at, 4.5' CJ. Sclireiber and associates ( 6 ) , who extracted t,issue fat with et,iiylene chloride and filtered it while hot, reported that, the oxidation induction period of extracted fat did not give a reliable pi,etiiction of the stability of poultry fat i n situ during frozen storage. They suggested that part of the fat peroxides may have bwri decomposed in removing the solvent. Khceler ( 7 ) used a mixture containing 60Ci, glacial acetic acid and 40 yo chloroform as :Lsolvent for glyceride fats. He measured the peroxide eontent by adding powdered pot.assium iodide and titrating the 1ibcr:tted iodine with sodium thiosulfate, using starch as indicator, anti expressed the results as millimoles of peroxide per 1000 grams of fnt. Lat'er King, Roschen, and Irwin (21,modified the Wheeler procedure and expressed the results in milliequivalents per 1000 gr:tnis of fat'. They also correlated the peroxide value n-ith the induction period. Free f a t t y acids and peroxides are preferably Measured by extracting the fat and making the determinations on the extracted material. Otherwise there is no assurance that water, protein, or other foreign substances may not complicate the reaction and lead t o erroneous results. However, if the analyses are t o be made on the extracted fat, the method of preparation must not change the character of the fat. Anything but the mildest extraction procedure Kill influence the results, especially the peroxides. Furthermore, unless the extraction is practically complete, the extracted f a t may not bc representative. There-
fore, work was undertaken to find a simple and rapid procedure for removing the fat without altering the peroxide and fatty acid levels. METHOD
Special Apparatus. A Waring Blendor or equivalent highspeed mixing and cutting apparatus, available at almost any chemical supply house. Reagents. Sodium thiosulfate, 0.01 N , and sodium hydroxide,
Preparation of Sample. Cut about 80 to 100 grams of the tissue fat into small cubes (about 10 mm.) on a clean surface with a stainless steel knife, placc, diced fat in a ]Taring Blendor with 250 ml. of chloroform, and jtir for 30 seconds. Filter the mixture of comminut,ed fat and chloroform immediately through filter paper into a clean 400-ml. bcaker, and withdran- three 25-ml. portions with a pipet at'oncc for analyses. Place one 25-1111. portion in a previously tared 15O-ml. beaker. Place each of the other 25-ml. portions in a 125-1111. Erlenmeyer flask. Evaporate the chloroform in the 150-ml. beaker on a water bath under a st,reani of nitrogen, and dry for a few minutes (to constant might'i i n an oven at 101' * 1O C. Use this weight of fat as t,lie sample w i g h t for calculation of peroxides and free fatty acids. -Analyze onc of the remaining 25-1111. portions for peroxides and t,he last for frce acids. Test,smust be completed as promptly as possible. Procedure for Peroxide Value. To one 26-m1. portion of t h e chloroform filtrate, add 10 ml. of glacial acetic acid and 1 ml. of a saturated solution of potassium iodide. Let stand x i t h occasional swirling for exactly 1 minute. Add 30 ml. of distilled water and titrate with 0.01 S sodium thiosulfate, using * - starch as the indicator. C.iLcurA4rIox. Rlilliequiralmts of pcroxide per 1000 grams ml of Na?S?O?X S of fat = x 1000 weight of fat ivhcrc -1-= nor;alit,y of sodium thiosulfate. Procedure for Free Fatty Acids. To one 25-ml. portion of t h e chloroform extract add 25 ml. of previously neutralized alcohol and 1 ml. of phenolphthalein indicator. Titrate with 0.05 N sodium hydroxide to the first permanent pink color. CALC~L.\TIOS. c; of free fatty acids as oleic = ml. of alkali X :V X 28.2 iveight of fat where *Y = normality of sodium hydroxide. NOTE. It is essential that after the preparation is started t h e complete procedure be finished as promptly and quickly a s possible. EXPERIIZENTA L
Elperinient I was designed to determine the degree of disintegration of the sample necessary for fat extraction (Table I). Eight 100-gram samples were taken from a fresh pork loin which had been chilled to 35 C. following daughter, requiring about 23 hours, and then treated a s described bebm. Each determination was carrird out in duplicate,
854
V O L U M E 19, NO. 1 1
Table I.
Effect of iMethod of Disintegration on Meat
Method of Comminution
Period of F a t Extraction in Chloroform
1
Blendor
See. 30
2
Knife
Sample
3 -i
Blendor
30
F a t in
Chloroform Removal
Evaporated
Not evaporated
Knife
25 M I .
of Solution
Table 111. Time
Free Fatty Acids
Peroxide Values
8.550 8.965 0.830 0.445
0.26 0.28 0.10 0.10
11.0 18.0 34.0 17.5
8.550 8.965 0.830 0.445
0.26 0 25 0.10
0.0 0.0 0.0 0.0
8.265 7.615 2.260 3.695
0.25 0.25 0.28 0.25
8.0 7..0 2.0 5.0
8.265 7.615
0.27 0.29
2.260
0.12'
0.0 0.0 0.0 0.0
0.10
5
Blendor
6
Knife
i
Blendor
8
1
1
Evaporated
Not evaporated
Knife
3.696
0.07
Effect of Stirring Time in Waring Blendor
Period of Mixing In Blendor
25 M1. of Solution
F a t in
Free F a t t y Acids
Min.
Grams
70
Peroxide Values
75 75 75 75
1
2
7.5
0.22 8.Ql 11.84 0.06
36 36 36 .. - 20 - 20
0.21 0.58 0.06 0.06 0.11
Table IV.
NO
2 3 4 5 6 7
0.0 0.0 0.0 1.3 17.9 0.0 0.0
0.08
0.10
'
2 9 0
1
Table 11.
..
0 1
Peroxide Values M1./ 1000 g. fat 0.0 0.0
% 0.05 0.05 0.10
F.
O
0 0 1 1 2 4 9
Sample
Free F a t t y Acid
Temperature
Days
Hour
Effect of Storage Temperature
0.0 0.0 0.0 0.0 0.0
Effect of Mixing on Fat
Free F a t t y Acids (as Oleic) On rendered On rendered fat mixed fat in Blendor
70
%
0.28 0.37 0.48 2 50 3.30 8.50 9,60
0.28 0.36 0.42 2.40 3.10 8.10 9.10
Peroxide Values On rendered fat mixed in Blendor
O n rendered fat
MI./
10.4 4.0 62.8 4.4 9.0 16.0 15.0
8.7 3.8 60.0 4.3 9,7 16.6 14.6
1000 8.jar
fatty acids (Table 11). These results indicate that variation in the mixing time between 30 and 240 seconds made no significant difference in the results. I n experiment I11 the progress of tissue fat deterioration with time and temperature was determined, to see if the method would show the differences normally expected under storage eonditions. Sample 1 was cut into 0.5-inch cubes, comminuted for 30 seconds in a Waring Blendor with 250 ml. of peroxide-free chloroform, and filtered immediately. The chloroform was evaporated, the fat residue was weighed, and suitable quantities were used for determinations of free fatty acids and peroxides. The portion used for the peroxide determination was dissolved in chloroform-acetic acid, and the procedure carried out by the King-Roschen-Irwin ( 2 ) modification of the Wheeler ( 7 ) method. The free fatty acid content was obtained by adding 50 ml. of neutralized 95% alcohol and titrating with 0.05 N sodium hydroxide, using phenolphthalein indicator. Sample 2 was handled in a similar manner, except that it was chopped to a fine pulp with a stainless steel knife, covered immediately with peroxide-free chloroform, and filtered after 30 seconds. I t is evident from the data that the extraction from the samples cut with a knife was incomplete and that the peroxide values were appreciable. Samples 3 and 4 were a repetition of 1 and 2, respectively, except that the chloroform was not evaporated. Ten milliliters of acetic acid were added to 25 ml. of the extract and the peroxides were determined as usual; 50 ml. of neutralized 95% alcohol were added to 25 ml. of the extract and the,free fatty acids were titrated. The presence of the chloroform in the free fatty acid determination did not appear to influence results appreciably, as is indicated by Tables I and IV. Comparison of the data obtained on the first four samples suggests that even as mild a treatment as evaporation of the chloroform tends to increase the peroxide value. I t is assumed that the actual peroxide value of fats as fresh as these should be a t or near 0. Samples 5, 6, 7, and 8 were repetitions of samples 1, 2, 3, and 4 except that the extraction in chloroform after mixing was continued a t 32" F. for 1 hour instead of 30 seconds. I t is obvious that 30 seconds' comminution in the Blendor with the solvent provided sufficiently fine chopping to permit rapid and complete extraction. In no case was complete extraction obtained on the samples chopped with a knife. Free fatty acids obtained on three of the portions cut with a knife were low. I t is the authors' opinion that this was because the amount of fat extracted was not aufficient to be representative of the entire meat sample. Experiment I1 was made for the purpose of determining the effect of duration of mixing of the sample in the Waring Blendor. The samples were filtered immediately after stirring and the fat-chloroform mixtures werr anaIyeed for peroxides and free
Tissue fat was obtained from a pork loin which was removed from the carcass 2 hours after slaughter. The fatty tissue was diced in 0.5-inch cubes and thoroughly mixed. Representative portions were tested immediately and the remainder of each sample was stored in Mason fruit jars with loose-fitting covers a t temperatures of -20°, +36", and $75" F. Portions were withdrawn from storage and tested a t intervals over a 9day period. Each sample (100 grams) was stirred in a Waring Blendor for 30 seconds in the presence of 250 ml. of chloroform. The free fatty acid content and peroxide value were determined on 25-1111. portions of the fat-chloroform mixture. The results, indicating the tendency for the free fatty acid and peroxide value to increase TTith increasing storage time and temperature, are shown in Table' 111. Experiment IV was designed to determine if the process of stirrring the sample in the Waring Blendor with chloroform had any tendency to increase the free fatty acids and peroxide values. Samples of ordinary rendered fat of varying quality were selected. The free fatty acids and peroxide values were determined in the regular manner and portions of the same samples were then mixed in the Waring Blendor with 250 ml. of chloroform and the acids and peroxide values again measured as before. Comparison of these results (Table IV) indicates no significant difference. The method here,in described has been in use for several months and has given very satisfactory performance. LITERATURE CITED
(1) Cook, IT. H., and White, W. H., Food Research, 4, 43340 (1939). (2) Klng, -4.E., Roschen, H. L., and Irwin, W. H., Oil and Soup, 10, 105-9 (1933). (3) Lea, C. H., Dept. Sci. Ind. Research (Brit.), Food Invest. Rept., 1938,60-4 (1939). (4) Lea, C. H., J . SOC.Chem Ind., 50, 207-13T (1931). (5) Pennington, D. E., and Hepburn, J. S., J . Am. Chem. SOC., 32, 568-72 (1910). ( 6 ) Schrieber, M. L., vsii, G. E., Conrad, R. M., and Payne, L. F., Poultrv Sci., 26, 14-19 (1947). (7) Wheeler, D. H., Oil and Soup, 9, 89-97 (1932). RECEIVED March 14, 1947.
