I
J. R. CHIPAULT, 0.S. PRIVETT, G. R. MIZUNO, E. CHRISTENSE NICKELL, AND W. 0.LUNDBERG The Hormel Institute, University of Minnesota, Austin, Minn.
Effect of Ionizing Radiations on Fatty Acid Esters
Irradiation for preserving fats may be feasible in the future but many knotty problems still need solving
SOON
AFTER X-RAYS were discovered in 1895, it was recognized that celldestroying properties of ionizing radiations could be used to sterilize and preserve foods. Not until recently, however, when powerful electron accelerators and atomic energy were developed, has this become a practical possibility. Successful food preservation by highenergy radiations would presumably eliminate heat, refrigeration, and packaging in rigid, heavy glass and metal containers and consequent disadvantages of such processes. In practice, however, sterilizing doses of such radiations produce undesirable flavors and odors in foods, some of which are associated with lipides. Sheppard and Burton (72) irradiated saturated fatty acids with alpha particles in the absence of oxygen, and found hydrogen, carbon dioxide, carbon q o n oxide, water, and volatile hydrocarbons in the gas phase. The nonvolatile products consisted mainly of saturated hydrocarbons with one less carbon atom
than the fatty acid, and minor amounts of water-soluble short-chain fatty acids. The main reactions appeared to be decarboxylation and dehydrogenation. When irradiated under similar conditions, oleic acid yielded the same types of products and, in addition, stearic acid which indicated that hydrogenation also took place. Burton (2) found, also, that polymers resulted from irradiating oleic acid with deuterons for 2 hours a t an average beam intensity of 9 ma. Mead (8) and Polister and Mead (70) showed that solutions of linoleate soap and emulsions of methyl linoleate, when irradiated in the presence of oxygen, gave conjugated linoleate hydroperoxides similar to those obtained by autoxidation. Hannan and Shepherd ( 4 ) irradiated butterfat and found that its induction period was decreased considerably, presumably because antioxidants were destroyed. They also observed that peroxide formation during irradiation varied with temperature and was maximum at
about -20' C. Further, butterfat irradiated and subsequently stored at -20' C. continued to develop peroxides for several days, to a higher level than samples irradiated and stored a t either higher or lower temperatures. Melting the butter either during or after irradiation prevented these aftereffects, and resulted in low peroxide content. Astrack, Sorbye, Brasch, and Huber (7) irradiated vegetable and fish oils in the presence of oxygen with highenergy electron bursts and reported the formation of peroxides and free fatty acids and, in some cases, an increase in viscosity. I n vacuum, the peroxides were not formed. Antioxidants decreased the formation of peroxides but did not prevent organoleptic changes. More recently, Lang and Proctor (6) measured the monocarbonyl compounds in several refined vegetable oils irradiated with cathode rays. Previous studies have been limited to chemical changes produced by irradiation of fats. This work, however, conVOL. 49;
NO. 10
*
OCTOBER 1957
1713
cerns the relationship between these changes and undesirable flavors and odors that accompany them.
Materials Methyl oleate was prepared by first allowing olive oil to react with absolute methanol in the presence of sodium methoxide. The mixed methyl esters were freed from lower molecular weight compounds by fractional vacuum distillation. The undistilled residue was fractionally crystallized from acetone at -60" C., essentially according to the method of Wheeler and Riemenschneider (73). A final fractional distillation of the crystallized material gave essentially pure methyl oleate having an iodine value of 84.6 (theory, 85.6). Methyl linoleate was prepared by first saponifying safflower seed oil and isolating the fatty acids. The more saturated acids were removed by precipitation as urea-inclusion compounds. The acids recovered from the urea filtrate were esterified with methanol, and the esters fractionally distilled to yield a fraction containing at least 98y0 methyl linoleate. This ester was purified further by low temperature crystallization followed by a final distillation through a short column. The final product had an iodine value of 173.8 (theory, 172.4). Methyl palmitate was prepared by esterifying crude palmitic acid with methanol and removing most of the unsaturated contaminants by making a slurry of the esters in petroleum ether. The solid esters were then dissolved in petroleum ether and treated with concentrated sulfuric acid to remove the remaining unsaturated compounds. The saturated esters were fractionally distilled, yielding a methyl palmitate fraction with no unsaturated impurities. Corn oil methyl esters were prepared by allowing corn oil to react with methanol. The esters were then separated, washed, dried, and distilled through a short Vigreaux column. Their iodine value was 124.0. Corn oil, lard, and butterfat were commercial products purchased locally. T o obtain the butterfat, unsalted butter was melted, centrifuged, and the melted fat layer w-as filtered through a heated filter.
Radiation Sources Two sources of high-energy radiation were used. One was a 2-m.e.v. Van de Graaf accelerator with a 0.25 ma. beam current, operated by the High Voltage Engineering Corp., Cambridge, Mass. With this equipment, the samples were placed on a conveyor belt and passed under a scanning electron beam usually at a rate which gave them a dose of 2 X 106 rep. (roentgen-equivalent-physical) per pass. Electron pene-
1714
tration was approximately 6.6 mm. in matter of unit density. Facilities at the Materials Testing Reactor, Idaho Falls, Idaho, were the second source of radiation. They consisted of a source of gamma rays at the bottom of a water canal. The samples, in sealed No. 2 cans, were lowered into the canal and received a dose of 2 X 106 rep. in 20 to 30 minutes.
Preparing Samples Glass containers prevented contamination by metals and permitted the samples to be sealed in various atmospheres or under vacuum. Special thin-walled tubes of rectangular cross section held samples irradiated by the Van de Graaf accelerator. Dimensions of these tubes were such that complete penetration of a 25-ml. sample of fat ~ 7 a sassured. To minimize autoxidation, all samples were shipped to and from the irradiation facilities under dry ice refrigeration. Special precautions were taken to exclude oxygen from samples to be irradiated under nitrogen or vacuum. They were repeatedly degassed and then regassed with oxygen-free nitrogen to ensure removal of dissolved oxvgen. In the more critical experiments. empty glass containers were flamed while under vacuum to remove adsorbed oxygen from the container walls, prior to introducing the sample. Controls, prepared in exactly the same manner and subjected to the same temperature changes as the samples that were irradiated. were included in all shipments. A master reference control, consisting of the original untreated fatty materials, was kept under vacuum at -20" C. in these laboratories.
Anaiytical Procedures Carbon)-l compounds were determined colorimetrically by the method of Henick, Benca, and Mitchell (5). Higher reproducibility and lower blanks were obtained if the 491, potassium hydroxide solution was added with constant mixing t~ prevent high local concentration of alkali, and the final reading taken 15 minutes after addition. Carbonyl content was calculated according to the following equations based on the molar absorbancies at 430 and 460 mp obtained in these laboratories using pure carbonyl compounds: Unsaturated carbonyl = 4.204 Aiao
- 3.266 Ajqo W
Saturated carbonyl
=
5.971 AAZ0- 4.533 W
These values are expressed as millimoles of carbonyl compounds per kilo-
INDUSTRIAL AND ENGINEERING CHEMISTRY
gram of fat. A460 and A430 are the observed absorbancies of the solution at 460 and 430 mp, and W is the weight of fat in grams, dissolved in the .iml. of benzene solution used for analysis. Peroxide contents were determined iodometrically in the absence of oxygen by a method used routinely in these laboratories (7, 77). Light absorption in the ultraviolet region of conjugated diene and triene absorption was measured on alcohol solutions of the sample with a Beckman DU spectrophotometer. Measurements in the infrared region from 2 to 15 microns were obtained on solutions or films with a double beam Perkin-Elmer Model 21 spectrophotometer equipped with a sodium chloride prism, The apparatus used for molecular microdistillation was designed after the model by Paschke, Kerns, and Wheeler ( 9 ) . I t permits quantitative separation of monomer from higher molecular weight polymers in about 1 gram of material with an accuracy of fIO mg. Stability of lard samples toward autoxidation was sometimes examined before and after irradiation. Oxygen absorption in relation to time was measured in a closed manometric system (Warburg respirometer) at 50" C. Relative stabilities were determined on the basis of the time required to reach an arbitrarily selected level of oxygen absorption per unit weight of sample. Samples were examined by an informal taste panel of seven laboratory staff members with experience in detecting oxidized odors and flavors in fat. The coded irradiated samples and shipped controls were presented to the panel members who were asked to compare them with an identified reference sample and to rate each---Le., unchanged, slightly off-flavor, off-flavor, or strongly off-flavor. These classifications were given the numerical values 0, 1, 2, and 3 respectively, and the scores of all panel members were averaged for each sample. From three to six samples were examined at one time.
Res u It s Effect of Irradiation on Dry Fats, with and without Oxygen. Table I shows the changes observed when methyl palmitate, methyl oleate, methyl linoleate, corn oil methyl esters, and lard were irradiated with 2 X 106 rep. at ambient temperature. In the presence of oxygen, irradiation resulted in formation of peroxides, carbonyl compounds, and undesirable flavors. Saturated carbonyl compounds were formed in all substrates, but unsaturated carbonyls, although detected in small amounts in methyl palmitate, were found in appreciable quantities only in unsaturated fats.
I O N I Z I N G RADIATIONS Similar but smaller changes occurred in the absence of oxygen. Little or no peroxide was formed when methyl palmitate was irradiated under vacuum, but appreciable amounts of saturated carbonyl compounds were produced. All samples also showed decreased palatability. Effect of Water with a n d without Emulsifier. Table I1 shows the effect of water, with and without gum ghatti or dextrin, on changes observed when fats were irradiated with and without oxygen. With oxygen present, the only consistent effect of water was an increase in peroxides when unsaturated compounds were irradiated with beta rays. Carbonyl content sometimes increased also. These changes did not occur in samples irradiated with gamma rays. With saturated compounds, water decreased the formation of peroxides and saturated carbonyl compounds. In the absence of oxygen, similar results were obtained. Water had no consistent effect on flavor changes. The emulsifiers exerted little influence on changes. Effect of a n Antioxidant, Propyl Gallate. Table I11 shows changes observed when methyl palmitate, methyl linoleate, and lard were irradiated under oxygen, with and without added propyl gallate, and water. In general, propyl gallate hindered accumulation of peroxides, and this effect was more marked with the more highly unsaturated substrates. A similar but less marked effect on the formation of carbonyl compounds was observed. Flavor changes were slightly decreased in samples containing the antioxidant. Stability of lard containing propyl gallate was greatly reduced by irradiation under oxygen. In the oxygen absorption test a t 50' C., the induction period of the irradiated sample was 30 hours; the nonirradiated control required 180 hours to reach the same level of oxygen uptake, and the shape of the oxidation curve indicated that the end of the induction period had not been reached. Thus, the antioxidant was largely destroyed during irradiation. Temperature and Storage Effects. Effects of different temperatures during. irradiation, and subsequent storage were investigated to some extent. Table I V shows that 3l/2 hours after irradiation, samples of methyl linoleate irradiated a t -55' C. (solid) had higher peroxide values than duplicate samples irradiated at -45' C. (liquid). This was true whether the samples after irradiation were held a t room temperature or packed in dry ice. Also, peroxide values of samples held a t room temperature were much higher than those of samples held at -78' C. With methyl oleate, no significant difference was observed between the
Table 1.
Irradiation with and without Oxygen (2 X 108 rep., ambient temperature) Peroxide Value, Mmole/Kg.
Substrate
Carbonyl Compounds, Mmole/Kg. Unsatd. Satd.
Under Oxygen (8- or y-rays) 2.2 0.3 2.3 0.6 3.2" 0.5 2.8" 0.7 4.6 1.8 12.8 1.8 6.9 3.1 18.5 5.4 19.4 9.3 11.4 8.7 19.6 9.3 10.1" 3.0 10.6" 2.8
Methyl palmitate
Methyl oleate Methyl linoleate Corn oil methyl esters Lard
Flavor Score
4.9 5.0 4.3 3.6 3.7 2.8 3.6
0.7
1.8 1.8
2.0 1.4 0.9 1.3
5.1
1.7
3.5 3.8 5.8 9.8 10.1
2.2 1.2 1.3 2.f 1.7
2.0 2.1 2.6 1.7 1.5 6.8
0.5 1.0 1.7 1.0 1.7 0.7
1.9
0.5
2.0 2.0 2.3 5.9 6.3
0.5 0.5
Under Nitrogen (p- or y-rays) Methyl palmitate
1.1" 1.8" 0.7 0.7 1.8 1.7
Methyl linoleate Corn oil methyl esters Lard
0.1 0.2 1.2 1.9 6.2 1.6
Under Vacuum (y-rays) 0 0.2 0" 0.1 1.0" 0.2 1O.Sb 1.2 0.5a 1.4 0.5" 1.4
Methyl palmitate Methyl linoleate Lard
a Obtained from duplicate samples irradiated a t the same time. other experiments. Value unusually high and considered in error.
1.6 0.9 1.3
Other values obtained in
Table II. Effect of Water and Emulsifiers on Irradiation with and without Oxygen (2 X lo6 rep., ambient temperature)
Sample
Corn oil methyl esters, &rays 5% water 5% water 1% gum ghatti Corn oil methyl esters, y-rays 5% water 5 % water 1% gum ghatti Methyl linoleate, &rays 4% water 0.16% dextrin Methyl linoleate, y-rays 4% water 4% water 4- 0.16% dextrin Methyl palmitate, 8-rays 4% water 4% water 0.16% dextrin Methyl palmitate, y-rays 4% water 4y0water 0.16y0 dextrin
+ + + + + + + + + + +
+
+ +
+
+
Peroxide Value, Mmole/Kg. Under Oxygen 19.4 29.4 25.4 19.6 13.2 18.2 6.9 23.2 12.8 14.9 6.9 2.3 0.8 0.1 2.2 0.8 0.3
Carbonyl Compounds, Mmole/Xg. Unsatd. Satd. Flavor Score
9.3 12.7 13.8 9.3 8.6 9.4 3.1 2.2 1.8
3.0 0.9 0.6 0.2 0.3 0.3 0.2 0.3
3.5 8.4 10.0
5.8 4.3 3.4 3.6 1.9 2.8 0.1 2.4
2.2 2.2 2.0 1.3 1.3 1.0
1.3 1.6 0.9 1.1
1.4
5.0
1.8
2.2 2.3 4.9 1.7 2.1
1.2 0.8 0.7 1.2 1.5
1.5 4.3 4.1 3.9 6.2 7.3 1.7 2.1 2.6 2.5
1.7 1.2 1.5 0.8 1.7 1.0 1.0 1.6 1.7 1.3
Without Oxygen
Corn oil methyl esters, p r a y s
+ 5 % water + 5 % water + 1% gum ghatti Corn oil methyl esters, y-rays + 5% water + 5% water + 1% gum ghatti Methyl linoleate, &rays + 4% water + 0.16% dextrin Methyl linoleate ,-prays + 4y0 water + 0.16% dextrin
1.8
6.0 4.5 13.3 6.8
12.4 0.7 11.7
0.7 7.3
6.2 6.1 6.1 6.3 7.1 8.5 1.9 1.4 1.2 0.4
VOL. 49, NO. 10
OCTOBER 1957
1715
Table 111.
Effect of Propyl Gallate on Irradiation with Oxygen (7-rays, 2 X l o @rep., ambient temperature)
Sample Methyl palmitate O . O l ~ oP.G. water dextrin water dextrin Methyl linoleate f 0.01% P.G. water dextrin water dextrin Lard
+ +
+ +
+ +
+ +
+
++ 0.01% 0.01%
peroxide Value, hlmole/Kg.
Carbonyl Compounds, Mmole/Kg. Unsatd. Satd.
Flavor Score
0.3 0.2 0.3 0.3
4.9 1.9 2.1 2.1
0.7
+ 0.017, P.G.
2.2 0.1 0.3 0.3
+ 0.017, P.G.
12.8 2.6 6.9 3.0
1.8 0.8 0.9
0.7
2.8 3.0 2.4 2.3
0.9 0.9 1.4 0.9
3.0 2.8 2.7 2.6
9.8 10.1 8.2 8.2
2.1
10.1 10.6
P.G. P.G.
7.1 6.8
Table IV.
0.3
1.5 1.2
1.7 2.0 1.4
Effect of Temperature of Irradiation
@-rays, 2 X 106 rep., oxygen atmosphere) Irradiation Temp.,
c.
Sample
Peroxide Value, Mmole/Xg.
Carbonyl Compounds, Mmole/Kn. Unsatd. Satd.
Flavor Score
After 3.5 Hr. at 25' C.
Methyl linoleate
- 20 - 35 - 45
Methyl oleate
- 20
- 35
16.30 16.10
Methyl linoleate
- 45 - 55
19.00 26.30
Methyl oleate
28 - 20 - 78
4.6 4.3
28 - 20 - 20 - 78
Methyl oleate
- 55
14.60 12.90 42.90 53.40
After 3.5 Hr. at -78O C.
After 3 Days at - 78' C.
Methyl linoleate
Table V.
Butterfat
a
3.7 4.6 5.0
1.4
18.5 15.8 18.0 14.1
5.4 5.2 4.7 3.8
5.1
1.7
4.9 4.9 4.6
1.6 1.7
0.7 0.6
...
Effect of Storage Temperature after Irradiation at Dry Ice Temperature
Sample Methyl oleatea
Corn oil
5.3
1.8 1.6 2.8
(P-rays, 2 X 106 rep., oxygen atmosphere) Storage peroxide Carbonyl Compounds, Temp., Value, Mmole/Kg. Days c. Mmole/Kg. Unsatd. Satd. 6 - 78 5.4 1.2 4.1 3 - 30 4.4 1.4 4.1 - 30 4.1 1.0 3.2 4 - 30 4.6 1.2 4.2 6 6 - 20 8.0 1.2 4.2 6 - 78 34.5 13.0 13.1 6 - 30 33.8 12.1 12.6 6 - 20 22.7 10.0 9.3 6 - 10 32.0 12.0 12.4 1 0 12.0 0.6 1.5 2 0 10.0 0.3 0.8 4 0 12.2 0.2 0.7 1 - 20 22.0 0.7 1.6 2 - 20 13.8 0.4 0.8 4 - 20 20.4 0.3 0.9 6 - 20 23.8 0.2 0.8 1 - 40 16.6 0.4 1.2 2 - 40 15.3 0.4 0.9 4 - 40 22.3 0.3 0.9
Flavor Score 1.2 0.8 0.8 1.2 1.6 2.0 2.2 1.6 1.8 1.6 1.9 1.4 2.0 1.9 1.6 1.8 1.7 1.9 1.4
Irradiated a t - 7 8 O C. in the solid state but allowed to melt between irradiation and storage.
1716
INDUSTRIAL AND ENGINEERING CHEMISTRY
peroxide values of samples irradiated in the solid state at -35' C. or in the liquid state at -20' C., and held at C. or room temperature either -78' after irradiation. In another experiment, methyl oleate and methyl linoleate were irradiated at 28', -20", and -78' C., and then stored for approximately 3 days at -78' C. However, even with methyl linoleate, no significant difference appeared in either the peroxide or carbonyl values after irradiation at the several temperatures. The behavior of the linoleate is perhaps accounted for, in part at least, by certain aftereffects in storage, as indicated by experiments that follow, Flavor deterioration of methyl oleate seemed less at the lower irradiation temperatures, but this was not true for methyl linoleate. Further information on relationships between storage temperature after irradiation and the chemical and flavor changes was obtained. The effects depended on the type of fat used, (Table IV), and also on the treatment received before storage. For methyl oleate irradiated at -78' C., melted and held for a few hours at -20' C.: and then stored at either -78', -30', or -20' C., the smallest changes were obtained in the samples stored at -30' C. for 4 days (Table V). Samples stored for either 3 or 6 days at that temperature had measurably higher peroxide and carbonyl values, and the peroxide values of samples stored for 6 days at -78' or -20" C. were even higher. Generally, flavor of samples C. was also more stored at -30' acceptable. Corn oil was irradiated at -78' C. and kept at low temperature in the solid state during shipment and subsequent storage at -lo', -20', -30': and -78' C. for 6 days. Again: the least change was found in the sample stored at intermediate temperature, in this C. The peroxide value of case -20' this sample was only two thirds that of samples stored at either higher or lower temperatures, and the carbonyl content and flavor score were also lower. Butterfat also was irradiated at dry ice temperature, kept solid during ship'O - Z O O , and ment, and stored at , -40' C. for 1 to 6 days. At all temperatures, the peroxide values were lowest on the second day of storage. This change was particularly marked at -20' C. Carbonyl values dropped rapidly between the first and second days of storage and remained approximately constant thereafter. O n the other hand, flavor changes seemed at. their lowest after a 4-day storage period for all temperatures. Although flavor changes during storage were not consistent, flavor deterioration seemed greatest at -20' C. Interestingly, the per-
I O N I Z I N G RADIATIONS
WAVE LENGTH,
Microns
t W
V
Z
WAVE LENGTH, Microns Figure 1 . A. E.
Infrared spectrum of polymers formed from irradiation
Methyl linoleate in solid state Sodium oleate, linoleate, and methyl linoleate-urea inclusion compound
oxide value of samples stored at -20' C. was higher after 1 day than for those stored a t either 0 ' or -40' C., and also showed the most marked decrease in the second day. A peculiar effect of irradiation temperature on polymer formation occurred for methyl linoleate. Irradiation at room temperature with 2 X 106 rep. gave relatively little polymer, as determined by molecular microdistillation. When methyl linoleate was irradiated in the solid state a t -55' or -78' C., however, visual evidence of polymerization was obtained. The irradiated sam-
ples remained partially gelled, even when warmed to 75' C. The solid material was insoluble in petroleum ether, methanol, and acetone, but could be dissolved with difficulty in benzene, carbon tetrachloride, and chloroform. Molecular distillation of one sample showed that it contained approximately 2% of polymeric material. The nondistilled polymers were dissolved in benzene and deposited as a thin film on a sodium chloride plate by evaporation. The infrared spectrum of this material (Figure 1A), indicates presence of hydroxyl groups by the peak at
2.9 microns, and a normal ester carbonyl absorption a t 5.74 microns. The absence of a small peak or shoulder at 3.33 microns shows that very little cis unsaturation remained and a peak at 10.28 microns probably is caused by isolated trans unsaturation. Some conjugated trans, trans-diene is suggested by the absorption maximum a t 10.14 microns. Similar results were obtained when other samples of methyl linoleate were irradiated a t dry ice temperature in atmospheres of nitrogen or oxygen, and when corn oil methyl esters were irradiated in the solid state, but no visible polymers were formed when methyl oleate or corn oil was irradiated in the solid state at -78' C. Sodium oleate, sodium linoleate, and methyl linoleate-urea inclusion compound were then irradiated with 2,000,000 to 10,000,000 rep. at ambient temperature and the samples analyzed for polymer. The soaps were dissolved in water, acidified, and the free acids extracted with benzene. The methyl linoleate-urea inclusion compound was dissolved in water, acidified with hydrochloric acid, and the methyl ester extracted with petroleum ether. The recovered materials were molecularly distilled (Table VI). No solid material was observed in any of the fatty acids or esters recovered from the irradiated soaps or the methyl linoleate-urea complex, although all contained appreciable quantities of polymer. The polymer, moreover, increased with irradiation dose. Sodium linoleate irradiated with 10,000,000rep. in an atmosphere of oxygen gave fatty acids containing 21% of polymer; this sample, however, was oxidized to such an extent that part of the polymer may have resulted from oxidative polymerization. Inclusion of methyl linoleate in urea retarded but did not completely prevent oxidation during irradiation under oxygen, and appeared to have little or no effect on polymerization. Figure 1B shows the infrared spectra of the polymers separated from the recovered fatty acids and esters. Presence of isolated trans double bonds in the oleic and linoleic acid polymers is indicated by the peak at 10.28 microns. In addition, absorption at 10.12 and 10.53 microns in the linoleate products shows that cis, trans, and possibly trans, trans-diene conjugation is also present. The polymer from irradiated methyl linoleate-urea complex contained isolated trans bonds (0.23 mole per mole of ester) and some conjugated trans, trans, but no conjugated cis, trans unsaturation. Peaks at 3.00, 6.01, and 6.25 microns may be caused by Nsubstituted amide groups which would indicate reaction between methyl linoleate and urea. VOL. 49, NO. 10
OCTOBER 1957
1717
Effect of Dose Rate. Dose rate was conveniently varied by reducing the beam current of the Van de Graaf accelerator and increasing the number of times the sample was passed under the electron beam. The speed at which the sample traveled was kept constant. A sample of methyl linoleate sealed in an atmosphere of oxygen was irradiated at ambient temperature with 10 consecutive doses of 200,000 rep. each, with vigorous shaking preceding each irradiation to ensure that as much dissolved oxygen as possible was available within the substrate. Another sample received this same dosage but at -78’ C. in the solid state and without shaking. Chemical changes were much greater in the sample that received small multiple doses a t ambient temperature and was shaken before each irradiation (Table VII). Peculiarly, this sample had a better flavor than the sample that received a single large dose, but because the experiment was not repeated, this result may not be valid. Irradiation of methyl linoleate in the solid state with multiple small doses increased somewhat the peroxide value over that obtained by irradiation with a single large dose and flavor was adversely affected. Other Factors Affecting Flavor and Odor. Passage of ionizing radiations through oxygen or air produces ozone. To determine if an appreciable amount of ozone was produced in the presence of these substances, small, freshly prepared silver mirrors were sealed in glass containers filled with oxygen or air and irradiated with 2 X 106 rep. of gamma rays. The irradiated bulbs containing
Table VI.
Discussion
Irradiation-Induced Oxidation and Autoxidation. Certain similarities between oxidative changes that occur in irradiation and in ordinary autoxidation of unsaturated fatty acid esters have been described by Mead (8) and Dugan and Landis (3). In both cases, hydroperoxides are among the principal initial products, and their formation is accompanied by shifts in double bonds. It was previously found by Lundberg and Chipault (7) that initially in autoxidation of methyl linoleate, conjugated diene accumulates in direct proportion to hydroperoxide, and that the proportion is virtually the same at all tem-
Analysis of Fatty Acids from irradiated Soaps and Urea Complex
Sample
(“/-rays, anihient temperature) Irradiation Conditions peroxiCie Dose. Value. Mmole/Kg. Atmosphere megarep.
Sodium oleate
Vac.
Sodium linoleate
Vac.
Methyl linoleate Methyl linoleate-urea
Vac.
2 6 10 10 2 6 10 10 0 2 6 10
0 2
0 2
Table VII.
air had an odor characteristic of nitrogen oxides and the mirrors were badly discolored a smoky yellow. The mirror in the bulbs containing oxygen showed sufficient deterioration to indicate formation of ozone during irradiation. In another experiment, odorless and tasteless distilled water was irradiated in an atmosphere of air in a sealed glass bulb. The water acquired a slight bitter taste and gave a positive test for nitrate ions. The role of ozone in flavor and odor changes during irradiation was investigated further by direct ozonization of fatty acid esters and comparing such samples with unozonized samples that had been irradiated under oxygen. Flavors and odors were remarkably similar-more nearly alike than those of irradiated samples and samples rancidified by ordinary autoxidation.
0 2
11.1
8.6
7% 97.2 96.4 92.1 94.0 96.0 94.3 93.2 78.8 99.6 97.1 96.1 93.9
Polymer,
% 2.8 3.6 7.9 6.0 4.0 5.7 6.8 21.2 0.4 2.9 3.9 6.1
Effect of Multiple Beta Ray Doses on Methyl Linoleate under Oxygen
Irradiation Conditions Dose, rep. O c.
Peroxide Value, Mmole/Kg.
One2 X Ten2 X One2 x Ten2 X
lo5 108 lo6
1718
INDUSTRIAL AND ENGINEERING CHEMISTRY
IO6
36.6 35.1 40.1 58.5 26.0 22.6 22.6 208.0 6.3 13.4
Monomer,
28 28 - 78 - 78
18.5 68.0 23.0 32.3
Carbonyl Compounds, Mmole/Kg. Unsatd. Batd. 5.4 14.4 3.7 6.2
5.1 13.2 6.8 5.2
Flavor Score 1.7 0.9 1.0 2.2
peratures between 20’ and 80’ C. Figure 2 shows the changes in spectral absorption at 233 mp (representing conjugated diene) in relation to peroxide content of the linoleate fraction of corn oil methyl esters irradiated at ambient temperatures; these are compared with corresponding values for the autoxidation of methyl linoleate. Evidently a higher proportion of conjugated diene was formed in irradiation. This does not necessarily point to any fundamental difference in the basic mechanism of peroxide formation. With unsaturated compounds, evidence favors the view that free radicals, formed by irradiation, then form hydroperoxides by the same mechanism involved in autoxidation. Regarding chain reactions in production of peroxides during irradiation, such reaction chains must necessarily be much shorter than in autoxidation because concentrations of free radicals are much higher. With a limited supply of oxygen and high concentration of free radicals, a high proportion of nonhyd.roperoxidic products \rould form. Therefore, it is not surprising that the ratio of conjugated diene to peroxide value is different from that observed during the initial stages of autoxidation. That peroxides are formed at least in part by a free radical chain reaction mechanism is further substantiated by the observation that peroxide formation was retarded by propyl gallate. The effect was sufficiently great so that it could not be accounted for simply by competitive reactions of linoleate and propyl gallate, although it was evident from stability measurements that propyl gallate, was also destroyed directly by irradiation in the presence of oxygen. However, ,the fact that propyl gallate was much less effective in retarding peroxide formation during irradiation than in autoxidation was also good evidence that the reaction chains were short. Since the chain termination reactions are polymer-forming reactions, the appreciable formation of polymer discussed later, also supports the view that free radicals are formed in appreciable quantities and that the reaction chains by which they form peroxides are short. Although the autoxidation mechanism is important in radiation-induced oxidation of fats from the standpoint of over-all chemical change, there are other important nonautoxidative changes, particularly in flavor and odor, such as in irradiation without oxygen. The complex variety of reactions involved in irradiation of fats as indicated by a variety of products is well known. Flavor and Odor Components. In autoxidation of fatty acid esters at ordinary temperatures, there is a marked correlation between peroxide values and flavor scores. The peroxides themselves do not contribute appreciably to rancid
o/
-
0.60
o
0500.40
0
//
0
/
/
/
Autoxidized Linoleate
Methyl
/
/
-
/
/ I
030-
o/
00
/
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INCREASE IN PEROXIDE VALUE, m.e /kg Figure 2.
Conjugated diene formation in relation to peroxide value
0 Methyl linoleate
fraction of irradiated corn ail esters methyl linoleate
--- Autoxidation of
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Peroxide Sat. Carbonyl
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1.0 1.5 FLAVOR
2.0 2.5
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SCORE
Figure 3. Flavor score in relation to peroxide and carbonyl content in irradiated methyl linoleate
flavors, but a correlation exists because the rancid flavor components are formed to a large extent by further oxidation and decomposition of the peroxides. There is little if any correlation between peroxide values and flavor scoreb in irradiated fatty acid esters (Tables I to V). Somewhat better correlation exists between flavor score and the presence or absence of oxygen-invariably,
irradiation in the presence of oxygen led to more marked flavor changes, indicating that oxidation products are important components of flavor and odor. Nevertheless, oxidation products are only partly responsible because definite flavor and odor changes occurred where special precautions had been taken to eliminate all free oxygen insofar as possible, This, of course, does not necessarily
mean that the major flavor components are devoid of oxygen-a source is available in the carboxy group of the esters and possibly in the glass walls of the containers. Oxidative flavor changes in fats, such as “reversion” and common oxidative rancidity, have been attributed primarily to carbonyl compounds, and because such compounds form also during irradiation, they have been implicated in flavor changes here also. Lang and Proctor (6) have reported yields of monocarbonyl compounds formed during irradiation of refined olive, corn, and cottonseed oils of about 0.2 micromole per gram per 108 rep. They found, moreover, that these yields were essentially constant for doses u p to 107 rep. and for oxygen pressures from 1 mm. of mercury to 1 atm. The yields were slightly lower a t room temExcept for butterfat, the yield of total carbonyl compounds found in these experiments was much higher than that for monocarbonyl compounds reported by Lang and Proctor (Tables I to V). Carbonyl values here varied considerably with substrate, atmosphere, total dose, dose rate, and irradiation temperature, and did not always follow a regular pattern. The points in Figure 3 represent flavor scores and carbonyl or peroxide values for samples of irradiated methyl linoleate. There is some tendency for the more marked flavor changes to be associated with higher peroxide and carbonyl values, but the correlation is low. Considering other experimental data, the poor correlation of flavor and carbonyl value is to some extent explainable. Thus, it was found that the flavor and odor components have sufficiently low molecular weight for complete removal from irradiated fatty acid esters by scrubbing with inert gas at room temperature. O n the other hand, in autoxidation of fats and probably also during irradiatibn, a large proportion of relatively nonvolatile carbonyl compounds is formed under some conditions; these, although not contributing appreciably to flavor, contribute markedly to total carbonyl value. A fairly good correlation between readily volatile carbonyl compounds and flavor and odor could therefore still be expected. Other readily conceivable flavor and odor components in irradiated fats are low molecular weight hydrocarbons and aliphatic acids. Temperature Effects. Several unusual temperature effects were observed in irradiated samples in these experiments. Some were related primarily to temperature of samples when irradiation took place. Others were dependVOL. 49, NO. 10
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OCTOBER 1957
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ent also on the temperature during subsequent storage. Unfortunately, these experiments are not sufficiently complete to characterize and elucidate the temperature effects fully. The results strongly indicate, however, that more detailed studies would shed light on the mechanisms involved in some radiation-induced changes, and would indicate temperature conditions under which the changes could be minimized. The earliest measurements of peroxide and carbonyl values were made 31/2 hours after irradiation of some samples (Table IV). Undoubtedly, certain postirradiation changes took place during this 3l/z-hour period when the samples were warmed to higher temperature for analysis or storage. Nevertheless, an interesting effect is related to temperature of the sample during irradiation for methyl linoleate-certain changes occurring during irradiation in the solid state led to peroxide values higher than were produced when irradiated at a higher temperature in the liquid state. These differences were accentuated during post-irradiation storage ; thus, the linoleate sample irradiated at -55" C. developed substances that measured as peroxides, to a greater degree during storage, than did samples irradiated at -45' C., particularly during storage at room temperature. Paralleling this, irradiating linoleate at lower temperatures in the solid crystalline state also caused formation of insoluble polymeric material not observed at higher temperatures. Neither of these two types of temperature effects was observed in oleate. Another interesting and perhaps related temperature effect occurred, particularly for butterfat (Table V). These results substantiate the findings of Hannan and Shepherd for butterfat ( 4 ) but they also demonstrate certain additional post-irradiation temperature effects. Although all samples were irradiated under the same conditions at dry ice temperature, those stored for 1 day at -20° C. showed significantly higher peroxide values than those C. Perstored a t either 0" or -40' oxide values of all samples declined on the second day, the decline being great est in the sample at -20' C. O n the fourth day, peroxide values had again risen in all samples. I n the meantime, carbonyl values declined a t first and then remained relatively constant. Various effects of temperature during and after irradiation cannot yet be explained because of limited data. Probably the earlier peroxide and carbonyl determinations on samples both irradiated and stored at low temperature, measure in part certain transitory substances that are neither peroxide nor carbonyl compounds. Most of the effects can be rationalized if it is assumed that during irradiation, relatively high
1 720
concentrations of quite stable free radicals are formed. For linoleate, free radicals with potential conjugated diene centers would be expected to form. These radicals should persist for longer periods at low temperatures and thus have greater opportunity to react with oxygen as the latter diffused through the system. This would account for higher initial peroxide values. At higher temperatures on the other hand, unoxidized free radicals, being in a more mobile state, would have a greater chance to react directly with each other to produce dimers. Formation of insoluble polymers when irradiating solid linoleate probably is related to the orientation of linoleate molecules in the crystalline state. Irradiating the crystalline materials might well lead to a number of reactive conjugated diene-free radicals in close proximity, which would have an opportunity to react readily when the cq-stal structure collapsed in melting the sample, to form polymers beyond the dimer stage. Failure of oleate to parallel the behaviour of linoleate in these temperature effects then would have to be explained mainly by the absence of conjugated diene systems. Summary
Effects of beta and gamma radiations on methyl palmitate, methyl oleate, methyl linoleate, corn oil esters, corn oil, lard, butterfat, oleate and linoleate soaps, and methyl linoleate-urea inclusion compounds have been studied under several conditions. Irradiating fats with high-energy ionizing radiations in the presence of oxygen produces appreciable amounts of peroxides and carbonyl compounds. I t also causes flavor and odor changes which do not correlate well with either peroxide values or formation of saturated or unsaturated carbonyl compounds. That flavor and odor changes may be caused largely by carbonyl compounds, however, is not precluded. The flavor and odor components are volatile materials and the volatile carbonyl compounds represent only a fraction of the total carbonyl compounds formed. Ozone and nitrogen oxides may be formed when fats are irradiated in air and some of the flavor and odor components may result from the action of these compounds on the fats, Organoleptic changes also develop during irradiation under vacuum. No conclusive evidence concerning the chemical nature of flavor and odor components has been obtained in this case but carbonyl compounds are not excluded because they are formed, evidently deriving their oxygen from the ester group of the fat compounds.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Some oxidation products resulting from irradiation in the presence of oxygen form by-mechanisms similar to those involved in ordinary autoxidation. The ionizing radiations produce free radicals which enter into reaction chains of the autoxidative type and, with linoleates, form conjugated diene peroxides. With high radiation dose rates, however, the reaction chains are relatively short because high concentrations of free radicals are formed; antioxidants such as propyl gallate are therefore much less effective in preventing oxidative changes than in autoxidation. The chemical and organoleptic changes are sometimes dependent on temperature both during and after irradiation. These temperature effects have not been thoroughly characterized or elucidated, but some are related to formation of transitory compounds, perhaps free radical in nature, that are moderately stable at low temperatures Acknowledgmenf
The authors acknowledge the technical assistance of Fred Pusch. Literature Cited ( 1 ) Astrack, A,, Sorbve, O., Brasch, A., Huber, I V . , Food Research 17, 571 (1952). ( 2 ) Burton. V. L.. J . Am. Chem. SOC. 71, 4117 (1949).
( 3 ) Dugan, L. R., Jr., Landis, P. W., J . Am. 021 Chemists' SOC.33, 352 (1956).
14'1 Hannan. R. S.. Sheoherd. H. J.. Nature 170, io21 (1952); Trans: Faraday SOL.49, 326 (1953); British J . Radiol. 2 7 , 36 (1954). ( 5 ) Henick, A. S., Benca, M.F., Mitchell, J. H.. Jr.. J . Am. Oil Chemists' Sac. 31, 88 (1954). ( 6 ) Lang, D. A., Proctor, B. E., Ibid., 33, \
I
237 (1956). (7) Luidbe;g, G. 0 . : Chipault, J. R,, J . Am. Chem. Soc. 69, 833 (1947). ( 8 ) Mead, J. F., Science 115, 470 (1952). i9 ) Paschke, R. F., Kerns, J. R., Wheeler, D. H:, J . Am. Obf Chemists' SOC. 31. 5 11954'1. (IO) Polister, B. H.,' Mead, J. F., J . Agr. Food Chem. 2,199 (1954). (11) Privett, 0. S., Lundberg, W'. O., Nickell, Christense, .I. Am. Oil Chemists' Soc. 30. 17 11953). (12) Sheppard: C. Mi:, Burton. V. L., J . Am. Chrm. SOC. 68, 1636 (1946). (13) Wheeley, D. H., Riemenschneider, R. W'., Oil & Soafi 16, 207 (1939).
RECEIVED for review January 14, 1957 ACCEPTED May 6, 1957
Division of Agricultural and Food Chcmistry, 130th Meeting, ACS. Atlantic City, N. J., September 1956. Research undertaken in cooperation with the Quartermaster Food and Container Institutr, No. 723 in series of papers approved for publication. Views or conclusions arc those of the authors and do not necessarily reflect the views or endorsement of the Department of Defense. Hormel Institute publication S o . 150.
