Enzyme Activity in Frozen Vegetables ASPARAGUS 31. -4.JOSLYN -4ND C. L a BEDFORD
factors, It is well recognized undergoes changes in strucUniversity of California, Berkeley, Calif. that the maturity and size of ture and composition after stalk and the time elapsed between harvesting and processing harvesting, with consequent impairment in flavor, appearance, and nutritive value (3, 4). markedly affect the flavor of asparagus. The method and extent of the scalding treatment influence the flavor, strucProgressive deterioration occurs even a t temperatures slightly ture, and appearance of asparagus shoots. Scalding for 2-3 above the freezing point, although the rate a t which ligniminutes in boiling water has been recommended by several fication, reduction in sugar, and increase in crude fiber investigators although steaming is preferred, since it not content occurs is slower at this point than a t higher temperaonly reduces losses in water-leachable vitamins and other tures. Lignification in the pericycle and the vascular bundles soluble nutrients but also results in better flavor retention is usually more pronounced at the base of the tip a t first but (7, 8;10, 16, 18, 20). It has been thought that the scalding progresses towards the tip, and accompanying this there is a should be sufficient to inactivate all enzymes present. The development of an unknown bitter principle (1, 6, 13). The rate and method of freezing rapid deterioration in quality markedly affect the texture and after harvesting is well recogappearance of asparagus (6, 9, nized in the industry, and par18). The storage conditions, ticular care is taken in expeditMaturity, promptness in processing, scaldboth as to uniformity and absoing the prompt processing of ing, and conditions of freezing affect the lute value of temperature, also asparagus after cutting (2, have an effect. The relative 16, 18). The known perishquality of frozen asparagus. Flavor retenimportance of these factors is ability of asparagus makes a tion is closely related to inactivation of not known definitely. study of the factors concerned peroxidase-catalyzing oxidation by hydroAll three factors are involved in its deterioration during freezgen peroxide and gum guaiacum. Catain a t least one phenomenoning storage and their control lase activity decreased sharply with innamely, the loosening and i n t e r e s t i n g , particularly in scaling of the thin epidermis crease in temperature or time of scalding regard to enzyme activity. or outer skin. This condition, Although a c o n s i d e r a b l e but was still present even in overblanched which is particularly objectionamount of work has been done material when tested qualitatively. Acetable, is exaggerated in ason the physical and chemical aldehyde content decreased with decrease paragus stored after harvesting; characteristics of the develop in catalase activity. Peroxidase activity sloughing-off of the skin occurs ing asparagus shoots and of readily after scalding. Scaldwhen measured in terms of pyrogallol the physiological behavior of ing in boiling water, particuthe plant before and after the oxidation did not parallel catalase activity larly when i t is unduly harvest season, particularly in but was more heat resistant at first and prolonged, enhances this scalrelation to its preservation less so later. Peroxidase activity towards ing, and i t occurs to a lesser (1, 3, 47 6, 13, 14, b l ) , Our benzidine was surprisingly heat resistant. extent in steam-scalded than knowledge of the factors inin water-scalded material. The Ascorbic acid oxidase was present in asparav o l v e d is s t i l l r e l a t i v e l y rate and condition of freezing meager. Few data on the gus tissues but was not particularly acalso affect this scaling, slow nature and activity of the tive and did not parallel other enzyme freezing in an air blast resultenzymes present have been resystems in change in activity. Scalding ing in more injury. I n general, corded although measurements in water for 4 minutes at 92" or 3 minutes freshly picked young shoots, of respiration rates have shown steamed and quickly frozen, at 100' C. was adequate for flavor retention. these to be high in comparison exhibit a minimum of scalwith other plant tissues and to Asparagus shoots were thus quite differing. decrease about tenfold for a ent in behavior from the other vegetables 20 O C. decrease in temperature To obtain information on studied; this indicates again that the pro(16). Long green asparagus the types of enzyme systems cedure for processing vegetables varies with respired a t a more rapid involved and the extent of inphysiological activity and anatomical strucactivation necessary for flavor late than did partially white retention, several series of inspears (15). ture. Chemical changes other than enThe quality of frozen asvestigations were made on the zymatic occur to an appreciable extent in paragus is influenced by many effect of scalding on enzyme asparagus tissues. activity and flavor retention 1 Far prevlous pnpeis in this series in asparagus. see literature citation 1 1 .
T
HE asparagus shoot rapidly
702
INDUSTRIAL AND ENGINEERING CHEMISTRY
31.4'1, 1940
AXD TIMEOF BLANCHING O S THE EKZYME ACTIVITY IS T.4BLE I. EFFECTO F TEMPERATURE STORED FOR 4.5 YE.4RS AT -17" C.
-
Temp.,
C.
PO 40 30 60
65
70 75 80 85 90 95 100
100 (steam) 100 (steam) 100 (steam) 100 (steam) 100 (water) 100 (water) 100 (water) 100 (water)
Blanching Conditions-Acetaldehyde, Catalase p. p. m. factor
Time, nun.
e
2 2
e 2 3
2
2
? >
2 2 0.25 0.5 1
2 0.25 0.5 1
2
22.0 29.8 29.0 41.4 26.0 14.1 5.2 3.1 2.2 2.6 1.3 1.7
67.8
53.6 27.2 3.1 37.0 21.1 3.1 1.8
2.63 2.84 2.48 2.24 0.98 0.60 0.07 0.00s 0.009 0 0 0 2.27 0.69 0.11 0.0 0.88 0.41 0.026 0.00
Peroxidase factor 0,0055 0.0056 0.0053 0.0049 0.0016 0.0009% 0.00060 0.00052 0.00021
0.0000 0.0000 0.0000 0,0033 0.0019 0.00092 0,0000 0.0019 0.0011 0,00017 0.0000
Preparation and Storage of Material 1933 SERIES. Partially white asparagus, Martha Washington variety grown in the delta region near Stockton, Calif., was obtained towards the end of the canning season through a local cannery. The asparagus had been cut to 6-inch (15.2-cm.) lengths for canning but was again cut to 4inch (10.2-cm.) lengths (tips) for these tests. Tips n-ere used not only because of the size of tin cans in which they were to be frozen but also because the asparagus was not particularly fresh, some 2 days having elapsed between harvesting and final processing. The asparagus, however, was kept moist and cool in the interim. The stalks averaged 0.5 inch (1.27 em.) in diameter. After being cut to size, the shoots were thoroughly washed and mixed. Aliquots were then blanched in water for 2 minutes at 20", 40", 50", 60 , 6 5 O , 70", 75', SO", 85", go", 95", and 100" C., respectively. The asparagus in 4-pound (1800-gram) lots was immersed in a 45-liter water bath in loosely tied cheesecloth bags. Each successive lot was blanched in thesame water. After blanching, the asparagus was rapidly cooled in running cold water, packed in 1- ound (475-ml.) tin cans, sealed, and stored in air a t -17" C. Hn addition, samples were blanched in water for various lengths of time from 20 seconds to 20 minutes at 55", 65", 75" C., respectively, and in boiling water and in flowing steam for 15 and 30 seconds and 1, 2, and 5 minutes, respectively. In this set of tests the quantity of asparagus needed for the samples was tied in separate cheesecloth bags. All the bags that were to be held a t a constant temperature were then immersed in a 45-liter water bath; a t definite prearranged intervals a bag of asparagus was removed and cooled in running water, packed, sealed, and stored. The water in the bath was changed for each succeeding temperature interval. To test the effect of removal of air from intercellular spaces and of reduction of organic peroxides, samples of asparagus were stored under water for 4, 5 , and 8 hours before freezing, and other samples were impregnated in water, 3 per cent salt solution, 1per cent tartaric acid, and 1 per cent hydrochloric acid solution by a process of vacuumization, followed by release with air, which was repeated for several cycles. The asparagus was under the various solutions mentioned when the vacuum was relieved with air. This treatment forced the liquid into the tissues and waterlogged them. The acid treatment resulted in a marked reddening of certain portions of the shoot, particularly a t the bases of the scales. This was due to the presence of leucoanthocyanins in the asparagus tissues. 1937 in SERIES. Green asparagus, Martha Washington variety, grown the delta region near Clarksburg, Calif., was used. The asparagus was cut to 4.5-inch (11.43-cm.1 lengths and thoroughly washed Aliquots were then blanched in water a t 1000 c. for 1, 2, 3,'and 5 minutes, respectively, and at 87.5" C. for 1, 2, 4, 6, and 10 minutes, respectively. In this set of tests the quantities of asparagus needed for the samples were placed in Monel metal baskets. The baskets were immersed in the water bath, and at definite intervals a basket was removed, cooled in running water, put on trays, and frozen in an air blast a t -30" C. After freezlng they were packed in I-quart (0.95liter) friction-top cans and stored a t -20" C. In addition, the effect of storage before and after blanching and the rate of freezing were studied. Two lots, one in the shade and one in the sun, were allowed to stand for 6 hours before blanching. For the effects of storage after blanching, four lots
703
AND
FLAVOR O F ASPARAGES
-
-Appearance Color after cooking Texture Brownish green Tough, stringy Brownish green Tough, stringy Brownish green Tough, stringy Brownish green Tough, stringy Brownish green Tough, stringy Brownish green Tough, stringy Olive green Tough, stringy Olive green Tough, stringy Oljve green Tough, stringy Olive green Tough, stringy Olive green Stringy Green Stringy Olive green Tough, stringy Olive green Tough, stringy Green Stringy Green Stringy Olive green Tough, stringy Olive green Tough, stringy Green Tough, stringy Green Stringy
Flavor Off-flavor and -odor Off-flavor and -odor Off-flavor and -odor Off-flavor and -odor Off-flavor and -odor Off-flavor and -odor Off-flavor Off-flavor Off-flavor Slight off-flavor Fair flavor Fair flavor Off-flavor and -odor Off-flavor and -odor Slight off-flavor Very slight off-flavor Off-flavor and -odor Off-flavor and -odor Off-flavor and -odor Slight off-flavor
were blanched; one was frozen immediately, the other three were held for 2, 4, and 6 hours, respectively, before freezing. For the rate of freezing three lots were blanched; two were frozen in cans in air a t -17" and -29" C., respectively, and one was frozen on trays a t -17" C. The above lots were all blanched at 100 C. for 3 minutes, and after freezing all were stored a t - 17' C. in 1-quart friction-top cans. 1938 SERIES. Green asparagus, Martha Washington variety, grown in the delta region near Stockton, Calif., was used. Two series were put up. In one the asparagus was cut to Pinch lengths and in the other to 5-inch (12.5-cm.) lengths. Four lots of the &inch length were packed. Three were blanched in water in Chinese bamboo baskets a t 92 C. for 4 minutes and cooled in running water. Two lots were packed in 1-quart friction-top cans; one of these was covered with a 2 per cent salt solution and one was packed in 12-ounce (340-gram) cardboard packages. Another lot was blanched at 100' C. for 2 minutes, cooled, an: packed in 12-ounce cartons. These lots were frozen a t -35 and stored at -17' C. Aliquots of the Mnch lengths were blanched in water in Monel metal baskets at 87.5" C. for 3 minutes: a t 100' C. for 1. 2. and 3 minutes, respectively. After blanching the aspara&' was rapidly cooled in running cold water2 acked in 12-ounce cartons, frozen a t -35", and stored at - 17 O
O
8.
Analytical Methods Samples of asparagus were analyzed for catalase, peroxidase, and ascorbic acid oxidase and aldehyde content as in previously reported investigations of this series (11). The final observations were made on the 1933 series after storage for about 4.5 years, on the 1937 series after storage for 15 months, and on the 1938 series after storage for 3 months. The short storage period sufficed for the latter series since they were adequately blanched and were packed primarily to observe the effect of variation in rate of freezing and freezing storage conditions. Organoleptic observations on these samples were made directly after defrosting and after cooking under constant conditions.
Data and Discussion The catalase and peroxidase activity of asparagus, 1933 series, blanched at various temperatures for 2 minutes is given in Table I together with data on acetaldehyde content. Here as in the case of peas and stringbeans there is a close correlation between acetaldehyde content and catalase activity. The slightly heated sample (40" C.) is likewise more active in catalase. However, the maximum aldehyde content was obtained in the sample scalded at 60" C. As was the case with artichokes, there was a in catalase activity at different sections of the shoot; more catalase, and a correspondingly higher acetaldehyde content were present
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VOL. 32, NO. 5
asparagus tissue was but little affected by heating at 55" C., as was also the case with catalase; heating at 65" C. caused a sharp reduction at first but the peroxidase activity remained practically constant after 8 minutes. At 75" and 100' C. peroxidase activity dropped off a t about the same rate as did catalase. The impregnation with water or salt solution or soaking in water solutions had practically no effect on peroxidase activity. Impregnation with tartaric acid solutions reduced the activity of peroxidase but slightly whereas treatment with hydrochloric acid reduced it markedly. Asparagus packed in 1937 and 1938 had greater peroxidase activity, particularly in the smaller cuts. Steaming for 1 minute markedly reduced this activity, and a t 2 minutes no peroxidase activity was found. Scalding for 1 minute in water a t 87.5" and 100' C. destroyed peroxidase activity when measured in terms of purpurogallin formation. In these samples positive peroxidase activity was observed as measured colorimetrically with both guaiacum and benzidine. Differences of this type-i. e., the marked thermolability of peroxidase when measured with pyrogallol as substrate in comparison with benzidine as substrate-have been reported for other vegetables (11). This behavior leads us t o believe that several peroxidase systems may be involved. Ascorbic acid oxidase in asparagus tissues was not particularly active, and results obtained on the amount of added. ascorbic acid destroyed varied in a rather erratic manner. There was no definite correlation between the activity ofTABLE 11. EFFECT OF DEAERATION IMPREGNATION AND SOAKING ascorbic acid oxidase and scalding conditions, an unexpected ON CATALASE CONTENTAND ACETALDEHYDE CONTENT increase in destruction of added ascorbic acid in extracts (1933 SERIES) prepared from tissues heated a t 55" t o 75" C. being found. Catalase Peroxidase Aoetaldehyde. Impregnating Agent Faotor Factor P. P. M. Fellers et al. (7) showed but little loss in vitamin C content 0.0051 91.1 H a 0 (20' C.) 2.06 of quick-frozen asparagus, and a good retention of vitamin C 0 , 0060 59.8 2.54 HzO (55' C.) in canned asparagus was found by Wassin (19). The rela105.2 0.0055 1.84 NaCI, 3% Tartaric aoid, 1% 1.24 0.0034 75.7 tively greater activity in ascorbic acid oxidase and greater 0.081 0.00063 49.7 0.0054 2.38 Hz0, 1 hr. 22. a nonenzymic loss in vitamin C found in samples which had 2.44 54.0 0.0048 2 hr. undergone longer anaerobic respiration under water is of 2.42 0.0054 5 hr. 117.8 interest in connection with the results obtained by Thornton (17) who found that the vitamin C content of asparagus tissue decreased by 8 t o 25 per cent during storage in an a t m o s The asparagus packed in 1937 and in 1938 was much phere of carbon dioxide, either added as such or formed in fresher and of higher initial quality. Although the raw marespiration. N o recovery of this vitamin C occurred on exterial was of higher catalase activity, it contained in general posure to air. less acetaldehyde. Both catalase and peroxidase were more Qualitative tests for enzymes indicate the presence of a readily and completely inactivated (Table 111). weak oxidase which was readily destroyed by heating. Peroxidase activity, as found previously for other vegetables, depended on the test reagent; the guaiacum reaction was TABLE 111. EFFECT OF TEMPERATURE AND LENGTH OF BLANCHING PERIOD ON CATALASE, PEROXIDASE, AND somewhat more thermolabile than the benzidine. Thus a ACETALDEHYDE CONTENT (1937 AND 1938 SERIES) slight positive benzidine reaction was observed in tissues Aoetaldeheated for 5 minutes (Table IV). Catalase reaction was Tzmp., Time Catalase hyde Peroxidase C. Min.' Year Fsotor P. P. k. Activity reduced in activity on scalding, but evolution of oxygen gas 3.42 1937 15.4 0.0077 .. 1938 (5-in. Unblanched from hydrogen peroxide solutions continued even in definitely cuts) 1 1 . 4 0.0091 3 . 5 8 .. Unblanched overblanched material packed in 1933. The asparagus 0.0142 4.41 outs) 41.7 Unblanched .1. 1938 (4-in. 0.31 5.30 1937 0.0000 87.5 packed in 1937 and 1938 apparently contained a more easily 1.76 0.0074 2 0.0000 1937 87.5 0,0000 1 5.7 0.181 1937 100 destroyed oxidase, an extremely thermolabile peroxidase 0.0000 0.0119 1938 1 ... 100 when tested with guaiacum, and a markedly more resistant 0.0000 0.0126 2 1937 100 ... 2 0.0066 0.0000 1938 . . . 100 one when tested with benzidine, Catalase activity persisted, 0.0007 1938 1 0.071 . . . 100 (steam) 1938 2 0.0066 . . . 0.0000 surprisingly, in commercially overblanched material. The 100 (ateam) difference in the heat resistance of peroxidase when tested with two different reagents indicates the presence of different enzyme systems. The vegetable peroxidases present in the The peroxidase activity showed much less variation with more common garden vegetables are now being carefully reaction time than was found previously for other vegetables, investigated. and the value obtained after 5 minutes is given in Tables The asparagus packed in 1933 was examined periodically I to 111. As with catalase, peroxidase activity was greater in for color and flavor, both before and after cooking. At first the more physiologically active tissue. The peroxidase it was of good flavor even when only slightly scalded although activity did not change appreciably with the temperature of the texture was rather limp and soggy. However as storage scalding in the interval 20" to 60" C., behaved very erratically increased, off-flavors developed in increasing amount until in the region 60" to 80" C., then markedly decreased, and finally after some 4.5 years all samples (Table I) developed reached zero activity at 90" C. The peroxidase activity in in the more physiologically active tissues. Thus the very tips had twice the catalase activity of the next lower section, and this top section had twice the catalase activity of the middle section which was considerably more active than the white butts. The inactivation of catalase at 55" C. was very slow, the sample heated for 20 minutes being approximately as active as the control. At 65" C. the inactivation was more rapid for the first 8 minutes but almost negligible thereafter. At 75" and 100" C. inactivation was more rapid. Inactivation in boiling water was more rapid, uniform, and complete than in steam, largely because of better heat penetration in the former medium. The acetaldehyde content with few exceptions paralleled catalase activity. Impregnation of the asparagus tissues with water (Table 11) had little effect on catalase activity; warm water had less effect. The lower acetaldehyde content in tissues impregnated with warm water may be due in part to greater loss in acetaldehyde. Salt somewhat reduced catalase activity but, surprisingly, increased acetaldehyde content. Tartaric acid solutions appreciably reduced catalase activity and hydrochloric acid reduced it markedly. Forced anaerobic respiration of the tissues did not affect the catalase activity but, as was to be expected, merely increased acetaldehyde accumulation as a result of anaerobic respiration.
fzkA?n
MAY, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
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unpleasant flavors which were a combination TABLE IV. RELATIVE ENZYME ACTIVITYIS ASPARAGUS of bitterness associated with storage after harvestOxidase, -Peroxidaseing and anaerobic-respiration flavors. Scalding Gum G u m guaiacum Benzidine for over 2 minutes and less than 5 minutes in Catalase Guaiacum + Hz01 + HzOp both steam and boiling water was required for Effect of Temperature of Blanching, 1933 Series better flavor retention. Samples scalded for 5 Unblanched ++++ ++++ ZOO C.. 2 min. ++ ++++ ++++ minutes were definitely "cookedJ' in flavor. 40 ++++ ++++ ++++ 50 Steam-blanched asparagus was somewhat superior ++++ ++++ ++ ++++ 60 ++ ++++ ++++ ++++ to water-blanched. 65 ++++ ++++ ++ +++ 70 ++++ ++++ ++ +++ The color of asparagus was influenced by 75 ++++ ++++ + ++f 80 +++ ++++ ++ scalding, the greenness increasing in the uncooked 85 +++ ++++ samples with increase in scalding temperature 90 +++ +++ ++ 95 +$ +++ or time. Here, as previously observed, samples 100 + scalded a t lower temperatures or for shorter times Effect of Length of Blanching Period did not remain green on cooking but changed in 1933 series 5 5 ' C . , 1 min. ++++ ++ ++++ +r++ color owing to pheophytin formation and other 8 min. ++r+ ++++ ++ ++++ changes. However samples scalded for 1 minute 12 min. ++++ ++ ++++ ++++ or over a t 100" C. remained bright green. 65O 1 min. ++++ ++++ ++++ 8 min. +++ ++++ ++++ ++++ Apparently to stabilize the green pigment in 10 min. ++++ ++++ ++++ 20 min. ++++ ++++ + ++++ asparagus a more intensive scalding must be 75' 1 min. ++-+ ++ ++++ ++++ given than is necessary for peas or string beans. 2 min. ++++ ++++ t+ ++++ 4 min. + ++++ ++++ It would appear from comparison of the data +++ 6 min. + ++++ ++++ +++ in Table I with that given in Table IV that the 8 min. ++++ + ++++ 12 min. ++++ +++T + ++ gum guaiac peroxidase reaction is more closely 20 min. +++ +++t correlated with flavor retention than are the 100' C. (steam). 15 sec. ++++ ++++ ++++ 30 aeo. ++T+ ++++ ++++ other criteria of enzyme activity. 1 rnin +++ ++++ ++++ I n the 1937 pack there is again a close rela2 min. ++ +++ +++ 5 min. + tion between inactivation of the peroxidase++++ +++t +t++ catalyzing oxidation of gum guaiac by hydrogen ++++ +++t ++++ ++++ +++ +++ peroxidase and flavor retention. Asparagus ++ ++ scalded for 3 minutes in boiling water retained + + its original flavor and suffered little detectable ++++ ++++ +++++ change in storage. Scalding at 87.5" C. for less ++++ ++++ +than 4 minutes did not inactivate the enzymes +++ responsible for deterioration in flavor, and scald++ looo C., 1 min. ++++ ing over 4 minutes resulted in leaching out of ++++ ++++ 2 min. +++++++ ++++ asparagus flavor and left a watery-tasting tissue. 3 min. 5 rnin. + I n general, scalding for 3 minutes in boiling 1938 series water resulted in inactivation of the "gum87.5' C., 3 min. +++++ ++ 92O C., 4 min. guaiacum peroxidase" and good flavor retention. +++ ++ 100' C. (HaO), 1 min. + ++++ ++ Other factors besides enzyme inactivation are -2 min. ++++ ++ apparently involved in flavor retention. Storage 3 min. of the asparagus either before or after blanching 100' C. (steam), 1 min. +++ ++++ 2 min. ++++ ++ for several hours had little effect on flavor in view 3 min. ++++ ++ of the natural variability of the material. Thus, of ten lots of asparagus blanched for 3 minutes, eight were only fair in flavor and only two were of The texture of the former was somewhat better than that of good flavor; the latter two were lots stored for 5 hours in the the latter samples. Scalding in steam resulted in an unexsun before blanching and for 4 hours after blanching. I n view pected impairment in texture and flavor as compared with of the fact that the asparagus had been cut some 12-24 hours boiling water. This was not found in all lots and is not the previously, i t is surprising than any variations in holding usual commercial experience. appeared, particularly of the type found. Canning before The type of package used affected flavor retention, best freezing a t -29" C. reduced rate of freezing sufficiently to flavor being found in asparagus packed dry in hermetically cause appreciable softening in texture. At - 17" C. increassealed containers. Freezing in brine had a detrimental effect ing surface exposed to air and consequently increasing rate of on both flavor and texture. heat transfer had but little effect on texture. Immersion freezing in brine, which had been planned, was not used in Acknowledgment these series so that a further comparison of rates of freezing apart from temperature was not made. The authors are grateful to G. L. Marsh for his aid in The 1938 series again indicate that even for a short storage preparation of the samples, and for the nontechnical assistperiod the gum guaiacum test is a good indication of sufficient ance furnished by WPA projects 8876-B5, 10481-B5,and enzyme inactivation for flavor retention. Slight positive test 11589-B5. for peroxidase using this reagent was observed in samples Literature Cited blanched for 3 minutes a t 85" C., and these samples had de(1) Bisson, C. H., Jones, H. A., and Robbins, W. W., Calif. Agr. veloped a slight off-flavor in 3 months a t -17" C. Scalding Expt. Sta., Bull. 410,l-28 (1926). for 4 minutes a t 92" C. gave products of fair to good flavor, (2) Bitting, A. W., "Appertiaing or the Art of Canning: Its History but asparagus so blanched was not appreciably superior to and Development", pp. 367-88, San Francisco, Trade Pressthat blanched for 3 minutes in steam or in boiling water. room, 1937.
+++ ++
++++
++++
++
++
INDUSTRIAL AND ENGINEERING CHEMISTRY
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(3) Bitting, A. W.,U. S . Dept. Agr., BUT. Chem. Bull. 196, ( 1916). (4) Bitting, K. G., K’atl. Canners’ Assoc., Bull. 11 (1917). (5) Culpepper, C. W., and Moon, H. A , , U. S. Dept. Agr.. Tech. Bull. 462, 1-23 (1935). (6) Diehl, H . C., Pentser, W. T., Berry, J. A, and Asbury, C. E., Western Canner and Packer, 26, No. 5, 31-33; No. 6, 39-41; NO. 7, 33-35; NO. 8 , 43-44 (1934). (7) Fellers, C. R., Young, R. E., Isham, P. D., and Clague, J. A., Proc. Am. SOC.Hort. Sci., 31, Suppl. 145-51 (1934); Mass. Agr. Expt. Sta., Bull. 305 (1934). (8) Fyler, H. M., and Manchesian, J. T., Hilgardia, 11, 295-314 (1938). (9) Joslyn, M. A,, and Marsh, G. L., Fruit Products J . , 11, 327-31 (1932). (10) Joslyn, M. A . , and Marsh, G. L., Western Canner and Packer, 30, NO.5, 21-22; NO.7, 35-37; NO.8 , 37-40 (1938).
VOL. 32, NO. 5
Joslyn, M. A. et al., IND.ENG.C H E Y . , 28, 595 (1936): (1938); 31, 751 (1939).
30, 1068
Magoon, C. A., and Culpepper, C. W.,U. S. Dept. -\gr., Bull. 1265, 1-47 (1924).
Morse. F. W., Mass. Agr. Expt. Sta., BdZ. 171 (1916).
(17) (18) (19) (20) (21)
Ibid., 172, 297-307 (1917). Pentaer, W.T., Perry, R. L., Hanna, G. C., Wiant, J. S., ana ilsbury, C. E., Calif. Agr. Expt. Sta., Bull. 600, 1-46 (1936). Stewart, E. D., Food I n d . , 1. 705-10 (1928). Thornton, N. C., Contrib. Boyce Thompson Inut., 8, 2 5 4 0 (1936); 9, 1 3 7 4 8 (1937). Tressler, D. K., and Evers, C. F.. “Freezing Preservation of Fruits, Fruit Juices and Vegetables”, pp. 236-7, New York, -4vi Pub. Co., 1936. Wassin, G. F., S. Dak. Agr. Expt. Sta., Bull. 261 (1931). Wiegand, E. H., Oreg. Agr. Expt Sta., Circ. 116, 1-12 (1936). Working, E. B., Aria. Agr. Expt. S t a . , Tech. Bull. 5,87-124(1924).
Vitamin A Destruction in Fish Liver Oils RELATION TO E. J. SIMONS, L. 0. BUXTON, .4ND H. B. COLRlAN Nopco Vitamin Laboratories, Harrison, N. J.
E
DIBLE oils and fats develop rancidity on storage. The most common type of rancidity is due to the action
of air on the unsaturated components of the fat, and its rate of development varies widely with the conditions of storage and the type of oil or fat. I n general, the highly unsaturated fats are most susceptible to atmospheric oxidation. Rancid and oxidized fats have disagreeable flavors and odors and decreased nutritive value. This is particularly true of fish liver oils and is very important when these oils are used for their vitamin content. Fish liver oils are the best natural source of vitamins A and D, and are extensively used to supply these essentials. Thorough studies on the stability of these products are needed. Since vitamin D has been found more stable to atmospheric oxidation than vitamin A, most of the work has been devoted to studies of the stability of vitamin A. Wokes and Willimott (22), Jones (8),Huston et al. (T), Evers (a), Norris and Church ( I C ) , Marcus (IS),Dann (I), MacWalter ( l a ) , Holmes et al. (6),and Fraps and Kemmerer (4) reported wide variations in the stability of vitamin A under different conditions; Fridericia (6) and Powick (15) showed that rancid fats destroy vitamin A and were the first to suggest that the destruction might be due to organic peroxides. Rosenheim and Webster (17) found peroxides in samples of cod liver oil when most of the vitamin A had disappeared. Lease et al. (IO) observed that, if oxidized fat and vitamin A were fed separately but a t the same time, the combination resulted in lower storage of vitamin A in the liver, and they suggested that the preformed peroxides in the fat destroy the vitamin A between the time of feeding and storage in the liver. Whipple (21) reported a drop in vitamin A potency of cod liver oils as the peroxide value increased, and Lowen et al. (11) found that vitamin A in halibut and salmon oils was rapidly destroyed after the termination of the induction
PEROXIDE FORMATION period as judged by the peroxide value. While our work was in progress, Smith (19) reported that vitamin A dissolved in rancid fats containing peroxides was destroyed in the absence of air a t a rate approximately proportional to the peroxide concentration. From the previous work it is difficult to compare the effect of rancidity on the vitamin A content of the various oils since the experimental conditions employed varied widely, and only a limited number of oils was studied. This work was undertaken to compare the oxidative changes of several oils when stored under like conditions.
Materials and Procedure High-grade commercial fish liver oils were selected from large lots so that the samples could be considered representative of the species. The content of free fatty acids was below one per cent in all cases, and the oils were substantially free of moisture and foreign materials. The samples of the U. S. P. reference oil were obtained from the usual source. Several chemical tests have been proposed for measuring oxidative changes in fats. Since the estimation of peroxides is one of the most sensitive and widely used methods (9), it was chosen as the criterion for evaluating the state of oxidation of the oil. Under the usual conditions of storage, oxidation of the oils would have been too slow to complete the tests within a reasonable time; therefore accelerated tests were run. High temperature, light, and catalysts accelerate the oxidation of oils, though these agents were avoided so as t o make the tests more comparable with usual storage conditions. After several trials the following method of exposure during storage was adopted since it satisfied the above conditions and gave reproducible results:
