INDUXXRIAL A N D ENGINEERING CHEMISTRY
598
Vol. 16, No. 6
Some Factors Concerned in the Autoxidation’ of Fatsa With Especial Reference to Butter Fat By George R. Greenbank and George E. Holm RClI LABORATORIES, DAIRYDIVISION, DEPARTMENT OF AGRICULTURE, WASHINGTOIN, D.c. RESEA
MOITG the many
The presence of water or water vapor seems to retard the autoxiEXPERIMENTAL factors that have dation of fats. The formation of those compounds that are responsibeen mentioned as A pure, dry butter fat ble for the tallowy odor is also partially prevented or retarded by the accelerators in the producwas exposed to oxygen a t presence of water vapor. Fatty acids act, probably indirecfly, as tion of rancidity of oils and 95’ C. with rapid stirring, catalysts for aufoxidation,through the liberation of unsaturated acids fats, or tallowiness in butter and the absorption of oxywhich are strongly catalyfic. gen was m e a ~ u r e d . ~The fats, are heat, light, and The resistance of a butter fat to autoxidation may be increased by absorption was of an automoisture. Authors generthorough washing of the fat or by passing steam through the fat, but ally agree that light and catalytic nature, as shown prolonged heating or steam treatment will decrease its resistance to heat accelerate the reaction, in Fig. 1. autoxidation. but there seem to be conSince t h e experiments flicting views as to the exact were carried out a t 95” C . role that water plays when present. Smith3and Schestakoff4 the periods of induction are comparatively short (2 to 3 hours); ascribe the best keeping quality of fats to a low moisture con- a t lower temperatures this period would be considerably tent. Ryan and Marshall5maintain that water and light have longer. If samples are taken a t various intervals in the little to do with the production of rancidity. Winckele in period of induction, it will be found that there is little or no earlier publications considers moisture as a catalyst for the loss of color, little tallowy odor, and but a faint Kreis test. production of rancidity, but in later work discounts it as a At the moment that the reaction enters into the logarithmic factor. It is interesting to note that with butter he was not phase, where the absorption of oxygen is rapid, three pheable to obtain rancidity tests as with other fats, when butter nomena are noticeable-vis., (1) the bleaching or loss of and,the other fats were similarly treated. Acidity and enzyme color of the butter fat, (2) tallowy odor, (3) pronounced Kreis test. action have also been mentioned as catalyzing factors. Much work has been done by Rogers and his associates’ on the effect of acidity upon the keeping quality of butter. The general conclusions were that acidity has a decided effect upon the keeping quality and that sweet-cream butter always possesses a better keeping quality than does butter made from ripened cream. They also concluded from their studies that enzyme action plays a minor role in the changes in the flavors of butter, since “the destruction of the enzymes by pasteurization has no great effect upon the nature of the flavor changes in the butter.” In summary, Rogers8 concludes “that the principal changes in the flavor of butter are probably due to spontaneous chemical changes in which oxidation plays an important part and that the oxidation is accelerated by the presence of catalyzers in the form of metal salts,” and that “the action of microorganisms is indirect and the major change is brought about by spontaneous chemical action.” Numerous studies have been made of the action of metaIs / 2 3 4 5 6 7 8 9 upon butter fats, and it has been shown that they have a T h e -hrx powerful catalytic influence in oxidation. FIG. RELATIVE EFFECT OF WATER UPON THE RATEOF AUTOXIDATION The nature of the autoxidation of butter fat under various OF BUTTERFATAT 9 5 O C. conditions has been studied in order to find the conditions that are necessary for an improved keeping quality. InasThe changes that occur when butter fat is autoxidized may much as many of the oils and fats contain oleic acid glycerides be tabulated as in Table I. (mixed or otherwise) and in view of the fact that oxidation OF AUTOXIDIZED BUTTERFAT IN THE VARIOUS of other acid radicals (linoleic) is similar in nature, the re- TABLEI-PROPERTIES ABSORPTION PHASESOR OXYGEN sults obtained may be applied to other fats and oils. PERIOD O F LOGARITHMIC
A
L The term “autoxidation,” as commonly used in this connection, refers t o what is correctly called “autocatalytic oxidation.” 2 Presented befors the Division of Agricultural and Food Chemistry at the 66th Meeting of the American Chemical Society, Milwaukee, Wis., September 10 to 14, 1923. 8 Pharm. J., 95, 4 (1915). 4 Chrm. Rm. Felt- Have-Ind., 9, 180 (1902). 6 A m . J . Pharm., 79, 308 (1907). 6 Apoth. Zfg., 69, 690 (1905). 7 Rogers and Gray, Bur. of Anima2 Ind., Bull. 114 (1909); Rogers, Thompson, and Keithley, Ibid., 148 (1912). 8 Third International Congress of Refrigeration, Washington-Chicago, September 16 t o October 1, 1903.
Expt. PERIODOF INDUCTION 1 No oxygen absorption 2 Very slight or no change in color of butter fat 3 Negative or faint Kreis test 4 Sweet odor
ABSORPTION Oxygen absorbed rapidly Bleaching (loss of color of butter fat) Positive Kreis test Tallowy odor
The experimenk shown in Fig. 1 and Table I indicate that loss of color, tallowiness, and an intense Kreis test appear simultaneously. It has been proved in the authors’ previous work9 that the intensity of the Kreis test is directly proportional to the oxygen absorbed. No attempt will be 9
Holm and Greenbank, THISJOURNAL, 15, 1051 (1923).
INDUSTRIAL A N D ENGINEERING CHEMISTRY
June, 1924
made here to explain the ultimate cause of any of these phenomena, but to show how tallowiness occurs and how its production is influenced by various factors. From time to time it has been noted that the presence of small amounts of water in butter fat or lard when autoxidized at 95" C. always increased the length of the period of induction for those fats. Fig. 1 shows also the retarding effect of the presence of water (15 cc. in 400 grams of butter fat) upon the autoxidation a t 95" C. The length of the induction period is materially increased, but the same phenomena are observed in the various stages that are observed in the case of dry butter fat. The effect of the presence of water' upon the rate of autoxidation a t room temperature was determined, and similar results were obtained. A variation in the vapor pressure of the atmosphere in which a fat is stored has been shown to affect the reactions involved in the autoxidation process when judged by the olfactory sense. T o determine the effect of humidity upon the actual rate of oxidation, samples were stored over sulfuric acid and glycerol and over water. I n atmospheres of low vapor pressure the rate of oxidation seemed to be most rapid, while in atmospheres of high humidities the oxidation was checked somewhat. The interesting observation was made, however, that in separate cases, where the rate of oxidation was the same when judged by the Kreis test, it was always found that the one stored in a high humidity produced little rancid odor (not hydrolytic changes) until oxidation was well advanced. These experiments indicate that the presence of water vapor has a retarding influence upon the rate of formation of those compounds that cause tallowiness. HarrislO reports that one of the objections to the wet method of rendering fats is the high free fatty acid content produced. He also reports observations upon lard of very low free-acid content that had been kept in storage for a long time and still possessed a sweet odor and taste. The observation of Rogers and Gray7 that butter made from swcet cream seemed to keep better than butter made from ripened cream is significant from the standpoint of the quality of pure butter fat, since the latter always contains a small quantity of fatty acids. The variation in the keeping quality of various butter fats supposed to be sweet also makes it probable that some constituent of the fat is largelyresponsible for the differences in the variation in keeping quality. A sample of pure butter fat was prepared from freshly churned butter. Autoxidation curves were obtained upon this sample and upon similar samples containing small amounts of butyric and oleic acids. The period of induction was found to be greatest in the check sample and the shortest where oloic acid was added. To verify these experiments, which were carried out a t 95" C., various amounts of different acids were added to 10 cc. of fresh butter fat and the samples exposed to air in the absence of light. As soon as one of the samples had lost its color the samples were all tested with the phloroglucin hydrochloric acid reagent to determine the extent of the oxidation in the various samples. Table I1 shows the progress of the oxidation in terms of color intensity with the Kreis reagent. T A B L E II-CATALYTIC
SAMPLE Check Check butyric acid Check valeric acid Check cirproic acid Check oleic acid Check oleic acid
+ + + + +
EFFECTS OF ACIDSI N
COLOR Natural Very slightly bleached Very slightly bleached Bleached Completely bleached Completely bleached
OXIDATION
Kreis Test 0 1
1 1.2 12
60
Table [I indicates that fatty acids are catalysts for this autoxidation and tends to show that the fatty acids of 10
Chem. Met. Eng., 29, 93 (1923).
599
greater molecular weight %lahiegreater catdyti acid evidently owes its strong catalytic power not only to its acidic properties, but also to the ease of oxidation of the double bond and the catalytic action of the by-products of the oxidation. That the natural constituents of a butter fat are what determine the rate of oxidation or the susceptibility of a fat toward oxidation may be shown in a very striking manner, as follows: Connect in series three small vessels containing equal amounts of fresh, dry butter fat in such a manner that air may be blown through the three samples. To the last vessel connect an upright tube to act as a condenser. Immerse the three vessels in water kept a t 100" C. and pass air through the series. It will be noted that No:3 of the series will be the first to oxidize, and No. 1will resist oxidation the longest. This can be explained by the fact that the volatile acids and perhaps other volatile products of a catalytic nature found in Nos. 1 and 2 will be carried over into No. 3 and consequently this sample will be catalyzed the strongest. A series carried out in this manner showed oxidation to occur in the various times noted: No. 1, 16 hours; No. 2, 13 hours; No. 3, 6 hours.
I
I FIG.%-RELATIVE
2
3
4
5
T h e - hrs.
6
7
8
9
EFFECTS OF ACIDSUPON THE AUTOXIDATION OF BUTTER FATAT 95O C.
Aged fats respond to oxidation more readily than do fresh fats. This is sometimes due to a higher acidity, but in most cases it will be found that aged butter fats having the same acidity as fresh butter fats will yield to oxidation much more readily. The same observations have been made in the case of lard. Though lacking in experimental proof, it is probably safe to assume that during storage slight hydrolysis has taken place due to the presence of stronger acids of lower molecular weight, thereby liberating acids from the glycerides (especially oleic, which, as the writers have shown, has a catalytic action), with a probable formation of slight amounts of oxidizing substances. NEUTRALIZATION-In view of the fact that the fatty acids are not highly soluble and that the acids of higher molecular weight, or those which seem to possess the greatest catalytic action, are practically insoluble in water and consequently are difficult to wash out, an attempt was made first to neutralize the butter fat with dilute sodium hydroxide, using phenolphthalein as indicator, and then wash out the soluble acid salts. When the fat had been thoroughly washed it still showed an acid reaction. This was undoubtedly due to hydrolysis of the glycerides. The quality of this fat was much inferior to the check, and resisted oxidation by a current of air a t 100" C. for only 1 hour, whereas the check remained unchanged for over 4 hours. The prepared sample also oxidized readily a t room temperature when exposed to air.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
600
It is apparent from the data presented that no factors control the keeping quality to a greater extent than do the acidity of the fat and the temperature a t which it is stored. The question of temperature can as a rule be eliminated when we are dealing with cold storage, but in the presence of a high acid content it is doubtful if even low temperatures will prevent the slow changes that occur. I n view of the fact that a fresh butter fat of low acid content will strongly resist oxidation, even at room temperatures, with free access to air, there can be no doubt that acidity is one of the crucial factors to be considered in preparing fat of good keeping quality. STEAMTREATMENT-steam distillationll suggested itself as a possible method of removing the acids and perhaps other compounds of a catalytic nature. Accordingly, 400 cc. of butter fat were steam-distilled and a t intervals of 15 minutes samples were taken and the acidity was determined upon each sample. The remainder of the sample was then placed in a beaker and stored a t room temperature without access to light. At intervals of 2 weeks the samples were tested, and odor, color, taste, and intensity of color with the Kreis reagent were recorded. TABLE 111-KEEPING
Time - - of -.
QUALITY OF
Distil- ---Aciditylation Cc. 0.05N NaOH per 10 cc. Minutes 0 2 4 8 Check 0 . 3 0.3 0.3 0.3 15 0.2 0.2 0.2 0.2 30 0.15 0.15 0.15 0.15 45 0.15 0.15 0.18 0.18 60 0.25 0.25 0.25 0.30 Tal. Tallowy. SB = Slightly bleached. B = Bleached.
-
As in the previous experiment, the samples of lowest acidity possessed the best keeping quality, and the samples that were steam-distilled the longest possessed a poor keeping quality. The acidity of the distillates parallels that of the fat samples and consequently can be used as a control, thus eliminating fat sampling in the distillation process. As the distillation is prolonged there is evidence of hydrolysis and liberation of volatile and nonvolatile acids, with deterioration in the keeping quality of the fat. The experiments recorded in Table IV as well as other experiments that were made indicate that the loss of acidity through distillation, when calculated from the total acidity of the fat and distillate, is in no case equal to the total acidity of the original sample. This loss of acidity may be due to condensation of the free acid,s with some constituents. Other methods of freeing fats from the acids have been tried. Vacuum distillation a t 40" C. improved the product materially, but did not accomplish such good results as did the steam distillation. Steam distillation at reduced pressures with temperatures of 30', 50", 70', and 100' C. showed that the best results were obtained with the 100" C. distilla-
BUTTERFATWITH RRDUCED ACIDITY, AS SHOWN --Color--0 2
4
. . . . . .
. . . .
. . . .
--Odor----0 2
8 SB
. . . .
. . . .
Table I11 indicates that the sample where conditions produced the lowest acidity possessed the best keeping quality, and the sample distilled the longest was of a poor keeping quality, though its acidity was less than that of the check. There is no doubt that here the higher acidity is due to hydrolytic action with the result that oleic acid is set free, which, as has been shown, has a strong catalytic action in the oxidation of butter fats.
+++ ++ -
++
++ +++
Inasmuch as the sampling and titration of the fat were not extremely satisfactory and in view of the probability that hydrolytic action goes on from the beginning of the distillation, it was decided to follow the loss of acidity of the butter fat through the distillate. Samples were taken a t each interval and tested a t the end of 8 weeks' storage, as before. (Table IV) 11 "Steam distillation" as used throughout this paper refers to the process of the passage of steam through a fat.
TABLEV-EFAECT
OF
Acidity 7 0.05N NaOH per 10 Cc. 0 2 4 8 0.3 0.3 0.3 0.3 0.25 0.25 0.25 0.25 0.15 0.20 0.20 0.25 0.20 0.25 0.25 0.25 0.25 0.25 0.30 0.30
TESTSMADEIMMEDIATELY AND
8 Tal. Tal.
:
Tal.
Taste-----0 2 4
8
. . . . . .
off
. . . .
Off
. . . . . . off . . . . . . . . . . . . . . Off
AFTER
2, 4,AND 8 WEEXS
---Kreis 2
--0 -
--
Test-
--4
8
++ - ++- + ++ +++
tion. This again indicates the probable validity of the foregoing theory, that some acidity is lost through condensation; it also shows the role of heat in improving the keeping quality of fat. The contention is sometimes made that enzyme action has much to do with the keeping quality of a fat; hence pasteurization at ordinary and high temperatures is resorted to in order to improve keeping quality. I n order to destroy any natural enzymes and to show the general effect of heat upon a sample of butter fat with regard to its keeping quality, a sample was placed in a beaker, the beaker was placed in a glycerol bath kept at 100" C., and the contents were stirred to obtain uniform heating. At regular intervals samples were removed, the acidity was determined, and the keeping quality was tested by exposing samples to air in Petri dishes without access tolight. The results are shown in Table V. These experiments indicate that enzyme action has little to do with the keeping quality of butter fat, with tespect to oxidation. It is shown, on the contrary, that continued heating, especially a t the temperature used here, is detrimental to the keeping quality. The reason for this is undoubtedly the hydrolytic action or displacement of oleic acid by the stronger acids. WASHING-Attempts were made to prepare butter fat of good keeping quality by another method-namely, by thoroughly washing the fat. A sample of butter fat was washed for 6 hours by stirring in a streamof water held at 35" to 40' C.
TABLEIV-VARIATIONSIN ACIDITY orr FATAND DISTILLATE WITH CONTINUED DISTILLATION Acidity of Acidity of Distillate Distillate. Fat (10 Cc.) Kreis Test SamDle Cc. Cc. 0.05 N NaOH Cc. 0.05 N NaOH after 8 Weeks 0.30 0:45 0.25 0.20 0.20 0.20 0.15 0.10 0.12 0.10 0.10 0.20 0.10 0.20 0.20 0.30 0.25 0.30 0.30
Time of Heating at looo C. Minutes Check 15 30 45 60
4
BY
. . . . . . . . . . . . . . . . . . . .
. . . . SB . . . . . . . . . . SB 'B
Vol. 16, No. 6
HEATUPON THE KEEPING QUALITY OF BUTTER FAT,TESTEDIMMSDIATELY A N D AFTER 2,4, AND 8 WEEKS
7 -
----Color0 2
4
.. .. ..
SB
8 SB
SB SB B B
-0
. . . . .
. . . . .
. . . . .
2
. . . . .
Odor---4 ,,
.. ..
Tal. Tal.
8 Tal. Tal. Tal. Tal. Tal.
---Taste-0 2
. . . . .
. . . . .
. . . . .
. .
4
. . . . . . . . . Off
8
off
off off off Off
---Kreis 0 2
-
-
Test-4
8
++ ++ + ++ + ++ + ++ +++ -
+-
INDUSTRIAL AND ENGINEERING CHEMISTRY
June, 1924
The acidity of the fat had decreased from 0.35 cc. to 0.10 CC. 0.05 N sodium hydroxide per 10 cc. of butter fat. The butter fat was then dried and stored at room temperature open to the air, and in vacuum with access to sunlight. The samples stored a t room temperature and exposed to air showed no signs of oxidation after 2 months, nor did the sample stored in vacuum. The check in each case oxidized readily. TABLEV I-EFFECT Sample 1 2 3 4 5
SUNLIGHT U P O N STEAM-TREATED AND WASHED BUTTER FAT DESCRIPTION TIMEOP STORAGECOLOR Kreis Test Treated (moist) 2 months Bleached -IUntreated (moist) 2 months Bleached -!Treated (dry) 2 months Natural Bleached Untreated (dry) 6 weeks Washed (dry) 2 months Natural OF
+ ++ ++ -
The tffect of sunlight upon the steam-distilled sample in vacuum was also tested, with the results shown in Table VI.
DISCUSSION Fig. 1 indicates the nature of the reaction in the autoxidation of butter fat a t 95" C . The same type of curve is obtained upon autoxidation of any unsaturated fat Containing oleic acid. At the temperature used the rate of reaction is comparatively rapid, but the curve expresses the nature of the reaction. At lower temperatures, however, the length of the induction period would be greatly increased. Similarly, the difference between the length of the induction period of dry and moist butter fats would be increased. Inasmuch as oxidation goes on a t an accelerating rate when once begun, it becomes of practical importance in the storage of fats to keep them within the period of induction if oxidation is to be prevented. Evidence seems to indicate that the presence of water somewhat retards the rate of oxidation at ordinary temperatures. The effect of water upon the rate of oxidation seems to be twofold. The action where water is mixed with the butter fat is in all probability due to extraction of catalyzing substances from the fat. The explanation for the greater tallowiness a t lower humidities, or lower moisture contents, lies undoubtedly in the fact that in the absence of moisture the autoxidation proceeds to the aldehyde stage, while when moisture is present it proceeds directly to the acid stage, thus giving little of the tallowy odor produced by the aldehydes and the other by-products. In the procesd of oxidatian of two butter fats, one dry and one containing water, it has often been noted that the two q a y proceed with equal rapidity when judged by the Kreis test, but when judged by the olfactory sense the one containing water seldom shows traces of tallowiness until highly oxidized, while the dry sample has a very tallowy odor and taste even before the color is lost. This indicates that water prevents the formation of those compounds that give rise to the tallowy in a former paragraph, and again proves the contention made'by the authors in a previous paper,9 that tallowiness judged by the olfactory sense and by the Kreis test are not synonymous and that the Kreiatest is not in all cases an absolute measure of the amount of the substances giving rise to what are termed rancid odors (not hydrolytic changes). Fatty acids have been shown to produce a catalytic action in the oxidation of butter fat, and the acids of higher molecular weight seem t o produce the strongest catalytic effect. Old fats are very susceptible to oxidation, even though their acidity may be low. This is in all probability due to the liberation of some of the weaker acids of higher molecular weight (probably oleic) from the glycerides by the stronger acids of low molecular weight, and a probable formation of oxidizing wubstances.
601
The importance of the removal of acids to improve the storage quality of butter fat is twofold. First, it removes direct catalyzing acids and perhaps other compounds; and second, the removal of these acids excludes the subsequent formation of the higher acids of greater catalyzing power. It is difficult to say just what is the nature of the catalytic action by acids. They undoubtedly aid hydrolysis of the glycerides and may serve as oxygen carriers through formation of peracids. They perhaps aid the formation of intermediate compounds; and if the production of tallowiness involves a rearrangement, they may serve in some manner as catalysts for the rearrangement. The results indicate that either steam distillation or thorough washing of a butter fat will materially increase its ability to withstand oxidation changes. The danger in the method of steam treatment lies in the possibility of continuing too long and thus producing changes, probably hydrolytic, which increase the possibility and probability of oxidation. While the writers have no proof that oleic acid is set free in the process thay believe this to be the case where steam distillation is prolonged or where heating is continued. There may be similar changes if washing is prolonged, but undoubtedly to a very slight extent. Heat seems to be an important factor in the preparation of a good quality butter fat by the steam distillation process. The fact that the keeping quality of butter fat can be so materially increased by subjecting it to a process which removes the fatty acids is of great practical importance, not only from the standpoint of the butter-oil industry, but also from the point of view of butter manufacturers and manufacturers of oils and fats. The foregoing experiments indicate the importance of the use of sweet cream in the manufacture of butter for storage, and indicate also that a thorough washing of any fat that is to be held in storage is not only advisable but necessary when excellent keeping quality is desired. Though all the experiments reported, in which washing was used to improve the keeping quality of the fat, were carried out at 40" C., it is reasonable to assume that washing a t higher temperatures would be desirable and would yield better results in shorter time. The question of neutralization has been but briefly mentioned, but the probable effect of overneutralization and its dangers have been indicated. This question is being given further consideration.
APPLICATION TO OTHERFATSAND OILS In former publicationsQJ2the writers have shown that the oleic acid radical is one constituent involved in of fats, and in the production of rancidity (n changes). Most fats contain olein or a mixed glyceride of oleic acid, and are therefore subject to the same changes of oxidation that are found in butter fat. The factors that influence the oxidation of butter fat are, therefore, to be considered in the storage of other fats and oils. Some preliminary experiments with lard indicated that a steam-distilled sample was less subject to oxidation than was the untreated sample. Though the writers have not tried other fats and oils, the data given in this paper are suggestive of what conditions are to be carefully regulated to preclude the probability of oxidation of fats and oils manufactured for the market. I n the light of the experiments recorded, the main proMem to be considered in $torage products is freshness. To be able to resist oxidation such a product must still lie within the period of induction from the standpoint of oxidation, for any product that has passed this stage contains autocatalysts and will rapidly become unfit for use. 11
Proc. SOC.Enpfl. Biol. M i d . , 20, 176 (1922).
