Enzyme Activity in
FROZEN VEGETABLES The catalase content of peas and spinach varies with the temperature of scalding. I n a series of samples blanched at constant time, it is greater in those blanched at 40" C. than in those at 20" and 50". The quality of the product is slightly affected in proportion to the catalase activity, but it is necessary to heat to higher temperatures than those required to inactivate the enzyme in order to secure a product of good quality.
The acetaldehyde content of peas is apparently related to the catalase activity as the two curves are decidedly similar. The acetaldehyde content of spinach remains constant and is not related to the enzyme content. A satisfactory product has beep obtained by scalding peas for 2 minutes at 80" to 90" C. They have retained their good quality for over 2 years in freezing storage.
A. L. ARIGHI, M. A. JOSLYN, AND 0 . L. MARSH University of California, Berkeley, Calif.
HAT changes occur in the flavor of most vegetables during freezing storage is common experience (2-6, 8, 10, 12-18, 20). The changes in flavor have been ascribed to the activity of enzymes which were not entirely inhibited by low temperatures and ice formation. Kohman (13) in 1928 pointed out that "cooking vegetables merely enough for serving" preserves the flavor of vegetables in freezing storage. Barker (2) in 19301 also recommended partial cooking; peas were cooked for about 8 minutes in water. Joslyn and Cruess (10) in 1929 favored blanching for a short period and Joslyn (9) pointed out that the destruction of the enzymes by heat should be carried out under conditions which would result in the minimum of injury to flavor and texture. It is well known that prolonged blanching is detrimental to flavor (7, 11, 16). Although today the preliminary blanching of the raw product to destroy the enzymes present prior to freezing is a n accepted practice, Magoon (16), in 1931, questioned the reliability of the process. To establish the process of blanching and chilling prior to freezing on a sound basis, it is necessary to know the nature of the enzymes or other agencies involved and the type of changes brought about in the product. Kohman (16) believes that the changes in flavor are brought about by a process of anaerobic respiration and more directly by respiratory enzymes. Barker (2) speaks of the process as autolysis. Tressler (90) believes that the reactions are both oxidative a n d hydrolytic in nature and implies that enzymes such as catalase and tyrosinase are involved.2 Diehl et al. (6) found that the catalase activity in Alderman peas served as a n index 1 Subsequently he reported that as little as 1 minute of blanching of pea8 prior to storage at -10' C. gave promising results [see T. N. Morris and J. Barker, Brit. Dept. Sci. Ind. Research, R e p t . Director Food Iwestigation, 1933, 77-80 (1934)]. 2 In a private communication he pointed out that little scum forms and no foaming oocurs during the cooking of properly blanched peas; during the cooking of underblanched peas, much foaming occurs and much mum forma.
for adequate blanching. Peas blanched a t sufficiently high temperatures and for a sufficiently long time to destroy catalase remained unaltered in flavor when stored for several months a t -6.5" C. (20' F.) or lower. Peas showing positive catalase activity developed haylike flavors and odors with attendant changes in color. Joslyn and Marsh (12) found that peroxidase activity was not involved in changes in flavor; the inactivation temperature (and time) for peroxidase was considerably higher than the temperature necessary to inactivate the more thermolabile enzymes responsible for flavor changes. Because of the close relationship between respiratory enzymes and catalase, it is possible that catalase activity may serve as an index of the activity of these enzymes. It is well known that acetaldehyde is formed in appreciable amounts during anaerobic respiration; therefore acetaldehyde content should serve as a n index of respiratory activity. To determine whether respiratory enzymes are involved and to test their relation to catalase activity, the acetaldehyde content and catalase activity of California garden peas and spinach, blanched a t various temperatures for various lengths of time and stored a t 0" F. (- 17.8" C.) for over 2 years were determined, and the results are reported in this paper.
Preparation and Storage of Material Locally grown TeIephone variety peas were shelled by hand, and, after mixing and washing; aliquots were treated (blanche?) in water for 2 minutes at 20 , 40") 50', 60", 65', 67.5', 70 , 72.5", 75', 77.5", 80",85", go", 95", and 100' C., respectively. The pees were immersed in a 12-galIon (45-liter) water bath in loosely tied cheesecloth bags containing approximately 3 pounds (1.4kg.) of peas. Each successive lot was blanched in the same water. After blanching, the peas were rapidly cooled in running cold water, packed into 8-ounce (237-ml.) tin cans, sealed, and stored at -17' C. Peas were also blanched in water for various lengths of time from 30 seconds to 60 minutes at 55', 65', and 75" C.; and in boiling water and in flowing steam for 15 and 30 seconds, and 1, 2, and 5 minutes, respectively.
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
596
OF BLANCHING ON CATATABLE I. EFFECTOF TEMPERATURE LASE CONTENT
-Catalase Peas 0.03468 0,03905 0.01877 0.01163 0.00000
Temp., C. 20 40 50 60 65-100
Factor-Spinach 0.02600 0.03526 0.02627 0.00940 0.00000
VOL. 28, NO. 5
Only one series of spinach samples was tested for acetaldehyde content because it was found that the aldehyde content of spinach remained constant throughout the series. However, the complete series of peas was analyzed.
Organoleptic Observations
Samples of the canned frozen material were removed from freezing storage, allowed to thaw for several hours, opened, boiled in 2 per cent sodium chloride solution for 10 minutes, TABLE11. EFFECTOF PERIOD OF BLANCHINQ ON CATALASE and observed for color, odor, texture, and flavor before and CONTENT after cooking. I n the samples of peas blanched at a constant time of 2 minutes, decided color change occurred in the Catalase Factor 1000 C. 1000 c. sample blanched at 70" C. Samples blanched below this Time 55'C. 65' C. 75'C. (HzO) (steam) temperature were a dull olive green color, those blanched at higher temperatures were the bright grass green characteristic of properly blanched peas. On cooking, the 70" and 75" C. samples turned to olive green whereas the sample blanched at 77.5" C. and above retained the green color. Texture changes 8 0.02480 0.02000 occurred in the sample blanched a t 90" C. Samples blanched l2 0.01732 below this temperature were of crisp texture and remained 20 l6 0.01480 60 0.00000 so on cooking. The go", 95", and 100" C. samples had tough Spinach 0.25 0.5 1 2 4
a!
26
0,00000
0 . 6%20
0.04080 0.03718 0.02557 n- . 01x76 - - -. -
0.6ii68 0.01521 0.00180 0.00000
0.02?90 0.02220 0.00650 0.00000
0.00778 0.00000
0.01632
I n this set of tests the quantities of peas needed for the samples were tied in separate cheesecloth bags. All the bags that were to be held a t a constant temperature were then immersed in a 12-gallon water bath; at definite prearrangedjntervals a bag of peas was removed, cooled in running water, packed, sealed, and stored. The water in the bath was changed for each succeeding temperature interval. In addition, in order to remove air from intercellular spaces and to destroy organic peroxides, samples of peas were impregnated in water, 3 per cent salt solution, 1 per cent hydrochloric acid solution, and 1 per cent tartaric acid solution by a process of vacuumization followed by release with air which was repeated for several cycles. The peas were under the various solutions mentioned when air replaced the vacuum. The spinach, after trimming and washing, was treated in like manner. The material was held at about -17" C. for over 2 years before examination.
PEAS - CATALASE 2 - ACETALDEHYDE I
Analytical Methods CATALASE.The catalase was determined by a slightly modified Balls and Hale procedure ( I ) , which is itself a modification of the German method of Stern (19). Four grams of material were used instead of 2 grams, the catalase activity was measured at 20" C. instead of 0" C., and 4 N sulfuric acid was used instead of 2 N: Precautions were taken to avoid loss of iodine by volatilization. In other respects the method was that described by Balls and Hale, and the results are reported in terms of the catalase factor, Kf or k units per gram of fresh material. ACETALDEHYDE DETERMINATION. Acetaldehyde was determined by the iodometric bisulfite procedure. A can of peas or spinach removed from freezing storage was finely ground cold, in a food chopper, and 50 grams of the ground sample were then steam-distilled. (It was necessary to use 5 to 10 cc. of crystal oil to prevent excessive foaming during the distillation.) The distillate was collected in a 600-cc. Erlenmeyer flask, and distillation was continued until 300 cc. of distillate had been collected. The receiving flask was kept in an ice bath during the distillation to prevent vaporixation losses. As soon as 300 cc. of the distillate had been collected, the flask was removed from the ice bath and enough ethyl alcohol added to bring the total percentage t o 10 per cent by volume. Then 10 cc. of 0.1 N potassium acid sulfite were added, The flask was then ti htly stoppered and allowed to stand for exactly 30 minutes. i t the end of 30 minutes 10 cc. of 0.1 N iodine were added, and the sample was titrated at once with 0.1 N thiosulfate, using starch indicator. A blank determination was made in the same manner using 300 CC. of distilled water in place of the distillate. The difference between the amount of thiosulfate used by the blank and the amount used by the distillate is the amount e uivalent to the acetaldehyde present in the distillate. This me&od is subject to the criticism that other volatile sulfite-fixing and iodine-reducing matters may be involved. However, the method is commonly used for this purpose.
I
40
60
80
FIQURE 1. CATALASE ACTIVITY AND ACETALDEHYDE CONTENTOF PEASAND SPINACHBLANCHED AT VARIOUSTEMPERATURES
MAY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
skins, and the cotyledons were of mushy texture, mushiness increasing with temperature. The skins separated from the cotyledons on cooking. Noticeable differences in flavor and odor occurred in the ranges of temperatures from 20" to 77.5" C., 80" to 85" C., and 90" to 100" C. Before and after cooking, the odor and flavor of the samples blanched in the temperature range of 20" to 77.5" C. were decidedly haylike and disagreeable. It was more pronounced in the sample blanched a t 40" C. than in the unblanched sample at 20" C., and decreased in intensity as the temperature'of blanching increased. The samples blanched a t 80" and 85" C. had a clean, fresh pea odor and flavor comparable in all respects to those of the fresh product. In the cooked go", 95", and 100°C. samples it was possible to detect an off-odor and off-flavor not usually associated with cooked peas; these effects increased in amount as the temperature increased. Samples of peas blanched for various periods of time a t 55", 65", and 75" C. were all found undesirable in color, odor, and flavor. Samples blanched for periods of time longer than 10 minutes a t these temperatures were also decidedly mushy in texture on cooking. The samples of spinach blanched a t various temperatures for constant time were similar in behavior to peas in regard to the effect of temperature of blanching on color, odor, and flavor, but to a less marked degree and over broader temperature ranges. The samples blanched from 20" to 85" C. were of poor color, odor, and flavor. Those blanched above 85" C. were satisfactory in color and flavor and were equal to freshly cooked spinach in all respects.
TABLE 111. EFFECT OF DEAERATION IMPREGNATION ON CATALASE CONTENT -Catalase Peas
Impregnating iigent HzO
Z$'iP
TABLE IV.
FactorSpinach
0.03050 0.01370 0 00000 0.00000
,
0.02780 0.09800 0.00000
I
Tartaric acid
0~00000
EFFECTOF TEMPERATURE OF BLANCBING ON ACETCONTENT OF PEAS A N D SPINACH
ALDEHYDE
Temp. C. 20
'
Aoetaldehyde Peas Spinach P,p,m. P.p.m.
26.4 61.6 52.8 44.0 35.2
40 50 60 65 70 72.5
TABLE V.
26:4
Min.
Temp. O
cl
..
75 80 85 90 95 100
Acetaldehyde Peas Spinach P.p.m. P.p.m.
17.6 -17.6 17.6 17.6 17.6 17.6
1611 8:s S:8
..
..
..
Acetaldehyde Content 1000
c.
1000
c.
55' C.
65' C.
75' C.
(HzO)
P.p.m.
P.p,rn.
P.p.m.
P.p.m.
(steam) P.p.m
26:4 52.8 44.0 44.0
35:2
4i:O 61.5 52.8
61.6 44.0 35.2
70.4 61.6 44.0 36.2
61.6 26.4 17.6 8.8
39:6 35.2 26.4
24:6 17.6 4.4
l7:6
0:oo
4i:O
1?:6
010
..
0.25 0.5 1 2 4 5 6 8 10 12 15 16 20 60
TABLE VI.
17.6 17.6 17.6 17.6 17.6 17.6
Temp. C.
EFFECTOF LENGTHOF BLANCHINQ PERIOD ON ACETALDEHYDE CONTENT OF PEAS 7
Time
Discussion of Data The catalase activity of peas and spinach scalded a t various temperatures for 2 minutes is shown in Table I, at various temperatures for different periods of time in Table 11, and impregnated with various agents in Table 111. The samples of peas blanched a t 40" C. contained more catalase than those blanched at either 20" or 50" C. This behavior is in keeping with the organoleptic observations which also showed that this sample was inferior to the others. Impregnation with water did not inhibit catalase but sodium chloride seemed to reduce the activity of the enzyme. Hydrochloric and tartaric acids completely inactivated it; however, the vegetables impregnated with hydrochloric and tartaric acids were so badly disintegrated by these agents that they were not fit for use. The results obtained with spinach were similar to those obtained with peas. The acetaldehyde content, in mg. per kg. of peas and spinach, is shown in Tables IV and V. The trend for acetaldehyde content in peas corresponded rather closely to that for catalase activity, but in spinach the acetaldehyde remained constant throughout the series (Figure I). The acetaldehyde content of peas is apparently a good indication of quality, since it was found that the amount of acetaldehyde decreases as the quality of the samples improves. However, in the case of spinach, acetaldehyde cannot be used as an index of quality because it remains constant. Apparently the acetaldehyde found was formed as a result of enzyme activity during freezing storage, for the freshly shelled peas had practically no acetaldehyde and neither did fresh peas directly after blanching. The results on unfrozen peas were obtained on another lot. Table VI shows the relation of quality of samples to the amounts of acetaldehyde and catalase present. Table VI shows that the catalase content is not a reliable index of quality although it decreases as the quality of the samples becomes better. However, in order to inactivate the agencies producing off-flavor, it is necessary to scald a t temperatures considerably higher or for longer times than those necessary to inactivate the catalase. There is a decrease in the amount
591
..
2e:4
26:4 17.6 0.0
EFFECT OF ACETALDEHYDE AND CATALASE CONTENTS ON QUALITY OF PEAS Color
Texture
Remarks
20 40 50 60 66 67.5 70 72.5 75 77.5
Olive green Olive green Olive green Olive green Olive green Olive green Better Better Better Greener
Solid Solid Solid Tough Tough Tough Firm Firm Firm Firm
80
Good color
Firm
Poor flavor Worse flavor Poor flavor Poor flavor Poor flavor Poor flavor Poor flavor Poor flavor Fair flavor Good flavor, slightly off Good flavor
85
Good color
Firm
90
Good color
Slightly mushy
95 100
Good color Good color
Mushy Very mushy
Catalase Factor
Acetaldehyde
0.03468 0.03905 0.01877 0.01163 0.00000
P . p . m. 26.4 61.6 62.8 44.0 35.2
.... ... .
26:4
,...
..
1s:1
Good flavor
.... .. . . ... .
Good flavor
....
8.8
Fair flavor Poor flavor
... . ....
8:s
..
of acetaldehyde in peas as the quality of the samples becomes better. In the last three samples the mushiness and poor flavor are undoubtedly due to overblanching. Figure 1 shows that the acetaldehyde and the catalase cantents of peas follow a curve of the same general type. The curve for catalase activity of spinach is similar to that of peas. The results for catalase activity determined by rate of oxygen evolution from hydrogen peroxide, as followed manometrically in a Warburg apparatus, were closely similar to those obtained by the iodometric procedure. While making the preliminary determination of acetaldehyde, it was noted that determinations made on the same sample before and after it had been allowed to stand for several hours did not agree. The latter determination was
INDUSTRIAL AND ENGINEERING CHEMISTRY
598
invariably higher in samples blanched a t low temperatures. T o determine the extent of this increase, three lots of frozen peas, which had been blanched for 2 minutes a t 20", 65", and 77.5" C., respectively, were defrosted and brought to room temperature in a water bath, ground, and their acetaldehyde contents were determined immediately and after various intervals of time. The data obtained are as follows: Time of Storage at Room Temp., Hours 0 3.25 4.50 5.0 9.33 10.25 13.0
Acetaldehyde Content of Peas Previously Treated in Water, Mg./Kg. 200 c . 65" C. 77.50 c. 26.4 17.6 17.6 127.6 145.2
..
:
127 6
...
...
2i:4
44.0
26.4
The acetaldehyde content of samples of peas which were active in catalase increased on standing after defrosting. The increase was less in samples with the lower catalase activity.
Literature Cited (1) Balls, A. K., and Hale, W. S., J . Assoc. Oficial Agr. Chem., 15, 483-90 (1932). (2) Barker, J., Brit, Dept. Sci. Ind. Research, Rept. Director Food Investigation, 1930, 69 (1931).
VOL. 28, NO. 5
Barker, J., and Morris, T. N., Ibid., Investigation Leaflet 2, 1-8 (1934). Diehl, H. C., Western Cunner & Packer, 24 (lo), 24-7 (1933). Diehl, H. C., and Berry, J. A., Proc. Am. SOC.Hort. Sei., 30, 496-500 (1933). Diehl, H. C., Dingle, J. H., and Berry, J. A., Food I n d . , 5, 300-1 (1933). Gowen, P. L., Canner, 68 (1778), 100, 103 (Feb. 23, 1029). Joslyn, M. A., Calif. Agr. Expt. Sta., Circ. 320, 1-35 (1930). Joslyn, M. A., Fruit Products J., 13 ( 5 ) , 142-5, 153 (1934). Joslyn, M. A., and Cruess, W. V., Ibid., 8 , 9 (April, 1929). Joslyn, M. A., and Marsh, G. L., Calif. Agr. Expt. Sta., Bull. 551, 1 4 0 (1933). Joslyn, M. A., and Marsh, G . L., Science, 78, 174-5 (1933). Kohman, E. F., Cunner, 68 (1778), 147 (Feb. 23, 1929). Kohman, E. F., and Sanborn, N. H., Ibid., 74 (ll), 64-6, 132-4 (Feb. 27, 1932). Kohman, E. F., and Sanborn, N. H., IND.ENG. CHEM.,26, 773-6 (1934). Magoon, C. A., Ice and Refrigeration, 80, 39-41 (1931). Magoon, C. A., and Culpepper, C. W., U. S. Dept. Agr., Bull. 1265 (1924). Morris, T. N., and Barker, J., Brit. Dept. Sci. Ind. Research, Rept. Director Food Investigation, 1931, 129-33 (1932). Stern, K. G . , 2. physiol. Chem., 204, 259 (1932). Tressler, D. K., IND.ENQ.CHEM.,24, 082-6 (1932).
RECEIVED January 7, 1936.
SULFITE TURPENTINE m A L
A
CHARLES A. M A " , R. E. MONTONNA, AND M. G. LARIAN' University of Minnesota, Minneapolis, Minn.
S
ULFITE turpentine is a by-product obtained from the pulping of spruce by the sulfite process. From 0.36 to 1.0 gallon of this by-product is obtained per ton of pulp, depending upon the conditions of pulping and the quality of the wood, with an annual supply of 1.5 to 2 million gallons from the mills of the United States and Canada (8). Most of this is not recovered on account of lack of any appreciable demand for p-cymene, the main constituent, at the price required to obtain p-cymene of a fair degree of purity from recovered sulfite turpentine. The purification of p-cymene is so tedious and expensive that Bert (5) claims that his synthetic process will produce p-cymene a t least at the same price as p-cymene obtained from sulfite turpentine. This is important only to countries where sulfite turpentine is not available from domestic sources. Even then, some of the existing demand for p-cymene may be supplied by dehydrogenation of monocyclic terpenes as reported in the patent literature (19). These terpenes are themselves by-products in some industries, such as in deterpenation of essential oils or in the manufacture of synthetic camphor. Various suggestions have been made to utilize sulfite turpentine. A fraction within close range of the boiling point of p-cymene (175' to 176" C.) may be used as a high-boiling solvent. According to Groggins (10)p-cymene is a solvent for many gums. Pure p-cymene may be used in certain organic syntheses. Wheeler and his co-workers (%', 23, 24) studied the nitro derivatives of p-cymene and prepared various dyes from aminocymene. Nitrocymene can be partially reduced to cymyl hydroxyl amine which can be made to rearrange to form aminothymol. Aminothymol is then diazotized and 1 Present
address, State College of Michigan, East Lens&
Mich.
finally converted to thymol. I n the electrolytic reduction of nitrocymene in acid solution by either the Cole (7) or the Austerweil (3) process, the rearrangement of cymyl hydroxyl amine takes place by itself. In chemical reduction with aluminum amalgam (4), cymyl hydroxyl amine is subsequently rearranged by warming its dilute acid extraction for half a n hour a t 60" C. or by boiling a few minutes. Thymol is converted by catalytic hydrogenation into menthol which is identical in all respects with the natural product except in its lack of optical activity. The annual world consumption of menthol is about 300 tons ( 3 ) . Thymol and menthol, therefore, represent a potential outlet for p-cymene depending on whether thymol can be prepared a t a price competitive with the price of thymol prepared b y other methods. Thymol is prepared synthetically, according to various patents, by condensing m-cresol, in the presence of an inorganic acid, with propylene, with isopropyl alcohol, with isopropyl bromide, and with acetone-hydrogen mixture. It can also be prepared from the condensation product of m-cresol with acetone. This condensation product is decomposed and then partially hydrogenated. In the absence of cost data, the fact that most of these synthetic processes are patented by companies with headquarters in Germany (Rheinische Kampfer-Fabrik Ges. ; Schering-Kahlbaum A.-G. ; Chemische Fabrik auf Actien vorm. E. Schering), where sulfite turpentine is not a domestic by-product, suggests that the preparation of thymol starting with p-cymene may be successful in countries where it can be obtained in large quantities from sulfite turpentine. With this possibility in mind as one step towards the utilization of sulfite turpentine, pure p-cymene was prepared from it for nitration purposes. In the course of this preparation and nitration, certain observations were made, and therefore it
