Enzyme Activity in Frozen
Vegetables
ARTICHOKE HEARTS M. A. JOSLYN,C. L. BEDFORD, AND 0 . L. MARSH University of California, Berkeley, Calif.
The activity of catalase, phenolase] ascorbic acid oxidase, and peroxidase enzymes of artichoke hearts was studied in relation to their discoloration in air. The time necessary for the complete destruction of catalase and ascorbic acid oxidase was closely related to that necessary to prevent discoloration. The phenolase and peroxidase activity was easily destroyed by heating although there is some evidence that the peroxidase activity cannot be adequately measured by the method used. The acetaldehyde content is closely related to catalase activity. Attention is called to the very high tannin content of the artichoke and to the presence of leucoanthocyanins. Because of these factors the enzyme-substrate system in the artichokes is quite complex and similates that of fruit rather than that of other vegetables. This is understandable because botanically the globe artichoke (Cynara S C O I ~ I I Z U S ) is a flower or bud.
M
OST of the vegetables that are commercially preserved
by freezing usually undergo pronounced changes in flavor rather than color when underblanched, as shown in the first paper in this series (1). Changes in color that may occur, particularly when the product is stored a t relatively high temperatures, are usually due to nonoxidative decomposition of chlorophyll (7), although oxidative transformation of the plant pigments present may occur aIso. No discoloration occurs such as results during the enzymecatalyzed oxidative transformation of the chromogens present in fruits (2, 8, IS,17,19,24, 26, SI). The artichoke, however, discolors rapidly in the presence of oxygen and differs from the other common vegetables in its high content of tannins which apparently serve as the chromogens. Artichoke hearts were frozen-packed commercially in California during the 1937 season and were marketed successfully. After being trimmed, the artichokes were blanched in a citric acid solution, cooled, packed loosely in cartons, and frozen. Such packaging necessitated careful control of discoloration during preparation and freezing storage, and presented several problems that were not envisaged in the early work; in that work whole artichokes, after a comparatively light blanch in boiling 1 per cent solution of citric acid, were cooled and frozen in an acidified brine which effectively prevented discoloration during freezing storage (16).
The discoloration of artichokes during trimming and preparation for canning is well known, and the literature in this field was surveyed by Bitting (6). Appert in 1810 described blanching of artichokes in boiling water, but the first mention of the use of acid was made by Faucheux in 1851 who soaked artichoke hearts in a dilute vinegar solution; Corthay (1899), Pacrette (1890), and Rossate (1910) described the sulfurous acid or sulfite method of controlling discoloration; Roques (1906) mentioned the then rather common use of citric acid brine. I n the present California practice of canning artichoke hearts, the artichokes are trimmed and cut to shape, the hearts being plunged into water or a brine acidified with citric acid or vinegar to prevent oxidation during preparation; they are then blanched for 10 to 20 minutes in a salt brine acidified with citric acid and are canned in a brine acidified with citric acid. The careful selection of buds which should be freshly picked and a t the optimum maturity is stressed by Bitting (6). In the preparation of the artichoke hearts for freezing, it is necessary to use a shorter period of blanching in order to conserve more of their fresh flavor. Prolonged blanching results in a rather flat tasting product. The acid concentration in blanching must be carefully controlled also, as changes in color as well as flavor result when too high a concentration is used. The effect of kind and concentration of acid and length of blanching period on the color, flavor, and freezing quality of artichoke hearts was studied, and the results obtained are reported in this paper. Some observations on the nature and the activity of the enzymes present in the artichokes are presented also. The enzyme-substrate system of the artichoke is different from that of other vegetables and is of interest not only in connection with the control of discoloration but also because of possible contributions to our knowledge of the oxidative enzymes of plants. A more detailed study of this system will be presented elsewhere.
Preparation and Storage Several crates of globe artichokes grown in Salinas were purchased through the local commission merchant. The outer bracts were rapidly pulled off until the light yellow or white inner bracts free from all green were reached. The tops of the buds were cut off and the butt trimmed to acone, according to the usual practice. The yield of hearts obtained varied from 25 to 30 per cent by weight. Stainless-steel knives were used, and the hearts were submerged in water as quickly as prepared. The artichokes were trimmed in lots usually consisting of twentyfour buds and wei hing approximately800 grams. The prepared hearts were blancted in boiling water, in 0.5, 0.75, and 1.0 per cent solutions of citric acid, and in 0.5 per cent lactic acid solution, respectively, for 3, 4, 5, 7, and 9 minutes. Five lots were blanched together in each acid solution, removed after the given time period, and then cooled in tap yater. In addition, two lots were prepared by blanching in water and in 0.6 per cent citric acid, respectively, for 3 minutes, were partially cooled, and were then submerged in a solution of potassium metabisulfite containing 250 p. p. m. of available sulfur dioxide.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Each lot was packed in 500-cc. (1-pint) tin cans which were closed with a single friction top and. immediately placed in freezing storage at - 17" C.
Analytical Methods CATALASE activity was determined as in the previous investigation ( 1 ) by a modification of the Balls and Hale procedure (s).. A representative sample of the frozen artichokes (six t o eight hearts selected at random) was ground in a food chopper while frozen, and then mixed, and an 8-gram aliquot of the mixture was further triturated with sharp quartz sand in 40 ml. of glycerol-phosphate buffer mixture. PEROXIDASE activity was determined by the Balls procedure
(4)*
PHENOLASE activity was measured by the determined rate of oxygen absorption by l cc. of a 0.075 M catechol solution buffered to pH 5.5 in presence of 1 cc. of enzyme extract prepared as for peroxidase. The oxygen uptake was measured in a Warburg-Barcroft manometer as suggested by Wieland and Sutter (do), Dixon (9), Sarnisch (29), Sutter (34),and others. ASCORBIC ACIDOXIDASE activity was measured by TABLE I. the rate of oxidation of added ascorbic acid, as was done by Saent-Gyorgyi (36), Tauber e2 al. (98), Kertesz et al. (do), and others (10, 83). A lo-gram Minutes sample was ground with sand and 50 ml. of water. Heated in To a 5-ml. aliquot of the extract were added 40 ml. Boiling of 8 per cent sulfuric acid and then 1 ml. of an ascorWater bic acid solution containing 0.5 mg. of ascorbic acid 0 per ml.; the mixture was titrated at once with 0.01 N 3 5 iodine solution with starch indicator. Another 5-ml. 7 aliquot of the extract was heated for 5 minutes at 9 100" C.,cooled, and mixed with 1 ml. of ascorbic acid solution and stored at room temDerature for 15 minutes. It was then acidified *and titrated as above. A third aliquot was treated with l ml. of ascorbic acid solution, allowed to stand for 15 minutes, and acidified and titrated as above. The iodine solution was preferred t o the 2,8dichlorophenol-indophenol dye because of the badly fading end point obtained with the latter, particularly with the unblanched samples [cf. Joslyn et al. (18) and others
(23, 2711.
ACETALDEHYDE was determined as in previously reported investigations, although subsequent tests indicated incomplete recovery by this procedure (15). ASCORBIC ACID. A 10-gram sample was ground with sand and 25 ml. of the sulfuric acid-metaphosphoric acid solution recommended by Mack and Tressler (26). The supernatant liquid was decanted off, and the residue washed with 15 ml. of the acid solution (8 per cent sulfuric acid-2 per cent metaphosphoric acid), and the combined solutions were made up to 50 cc. Then 10-ml. aliquots of this solution were mixed with 40 ml. of 8 per cent sulfuric acid and titrated with 0.01 N iodine solutions.
Preliminary Observations During the preparation of the artichokes they discolored extremely rapidly and showed a tendency to turn reddish brown and then black, which was similar to the discoloration of potatoes rather than to that of fruit, such as apples, which turn a light to dark brown color (9,8, IS, 17, 19, 94, 25). Since the first visible product of the action of tyrosinase on tyrosine is a red substance (96), it was thought that the artichokes might contain a tyrosinase. However, enzyme extracts prepared from leaves and other portions of the buds had no effect on tyrosine solutions which were quickly discolored by potato extracts. The center bracts of the artichokes, particularly a t the base, were often pink in color, and the pink regions became redder after blanching in acid solutions. Some apparently white bracts also became pink in acids. This pink coloration did not always occur but, if present, was accentuated by treatment with acid, much like the behavior of leucoanthocyanins (28). The presence of anthocyanins in the globe artichoke, however, is apparently due to certain cultural conditions and is not a normal phenomenon. The artichoke hearts had an extremely active peroxidase which was quite resistant to destruction by heat, but they had a comparatively weak catalase (Table I). The cut tissue and extracts of the tissues blued solutions of guaiac
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quickly and blued more slowly those of benzidine, both in This the presence and absence of added hydrogen indicates that the tissues contained a peroxidase and the oxygenase of Onslow (24) or that they contained a n organic peroxide or substance capable of forming an organic peroxide on autoxidation, which would be activated by the peroxidase present (cf. 5,11,19,34). Extracts of the artichokes also discolored catechol and pyrogallol solutions. It is likely that is due to the activity of discoloration that the a polyphenolase (21, 34, 40,41) or a phenolase such as catechol phenolase (34) or dopa oxidase (32), and that the peroxidase reaction which is given by many enzyme extracts containing phenolases is due to the presence of peroxidases of the hematochromogen type ( 1 1 ) . The latter are Present in plant tissues in addition to phenolases and other oxidative enzymes. 1
REACTIONS OF TISSUES FROM CENTER OF ARTICHOKEHEARTS ~ E HEATING R IN BOILING WATER Reaction
Reaction with.0.5% Gua,aoum
Reaction with Benzidine 0.5%
Oxygen
with o.5% Guaiacum Soln.
Soln. in Presence of 0.5% HzOz
S o h in Presence of 0.5% HzOn
Liberation from 0.5% HzOz
Color after Standing 1 Hour
,,, , ,
Verydarkbrown Dark reddish brown Lightbrown Very light brown No discoloration
+ ++ ++ + ++ ++ ++ ++ + ++ + ++ ++ ++ ......
......
++ ++ ++ ++ ++ ++ ++ + + + + .... .. . +...... +
The artichoke heart tissue is extremely sensitive to iron, and cutting, even with a bright untarnished steel knife, results in a prompt bluing. The tannins present are the iron-bluing type and may be catechol-tannins. The blue-black discoloration in the presence of iron is similar in color to that which occurs naturally. A preliminary investigation was made of the intensity of enzyme activity in various portions of the whole globe artichoke, with the average results shown in Tables I1 and 111. It is quite evident that although the outer bracts, which are probably more active physiologically, contain more catalase and also somewhat more acetaldehyde, the enzyme content of the whole heart is not very different from that of the bracts composing it. ACTIVITYAND ACETALDEHYDECONTENT TABLE 11. CATALASE OF FRESH ARTICHOKES Portion Green outer scales Top portion of white scaleles (bracts) Bottom (edible) portion a t base of white scales Heart (trimmed) Stem (with base or cheese)
Catalase Conbent Acetaldehyde k unit/gram Mg./kg. 0.402 26.7 0.206 I . .
0.272 0.242 0.149
24.2 13.3
...
CONTENT OF FRESH ARTICHOKES TABLE111. PEROXIDASE Top Portion of White Soales P. &?.@/gram 0.00319 0.00344 7 0.00319 10 0.00308 15 0.00270 Peroxidase units. Time Min. 2 5
5
Bottom Portion of White Scales P. E./grarn 0.00454 0.00362 0.00319 0.00276 0.00227
Heart P . E./orarn 0.00780 0.00588 0.00510 0.00460 0,00380
The peroxidase content as measured by the ability of the extracts t o catalyze the oxidation of pyrogallol by hydrogen peroxide a t p H 8.0 is surprisingly low, particularly in respect to the intense reaction with benzidine. This behavior was thought to be due to the use of apoor substrate. However, similar results were obtained when catechol was used instead
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of pyrogallol and when the runs were made at a lower pH. Furthermore the k values are not constant and decrease with time even though the experimental conditions were so chosen that less t.han a third of the initially present hydrogen peroxide was reduced. This falling off in the k values of peroxidase has been noticed for extracts of all plants containing pheriolases (e. g., potato, apple, apricot, grape) and not for the purely peroxidase plants (radish, turnips, etc., 14). The enzyme system involved is apparently of a different type. In the presence of either catechol or pyrogallol the solutions became badly discolored and render the deterniination ai residual hvdrogeii peroxide difficult. This did not happm uith radish, t.urnip, or sqiiasli extracts or with tlie plants listed hy Balls ami Hale (4).
was approximately 6.5 when tested by spot test on tissue althongli the small hairs a t center which were not wet readily by the test solution tended to be a t pH 7.0. After blanching in acid for 3 minutes or more and cooling in water, the pH value was 4.44.6, depending on concentration of acid. As stated before, t.he artichoke hearts were blanched in lots of about twenty-four, weighing some 800 grams, in 45.5 liters of acid solution. The final sample of the acid solution was taken after it was brought to initial volume. The pH values of the acid solutions increased somewhat during blrmcnching:
Blanching Tests
In the 0.5 per cent solution of citric acid, the acid content was initially 0.47 per cent and after use, 0.43 per cent, showing n redoction of acid at tlie rate of 0.005 gram per 454 grains of lieart,s I~lancherlper 100 cc. of acid solution. In the 0.75 per cent solution the initial acidity was 0.749, final 0.736, or a reduction of acid at the rate of about 0.007 gram per 454 grams per 100 cc. of solution. In the 1 per cent citric acid solution the initial acidity was 0.94, bhe final 0.885, or a rediiction of 0.007 gram per 454 grams per 100 ce. of solution. 111 the lactic acid fiolution there was a reduction in acidity from 0.504 per cent initially to 0.480 per cent, or 0.002 gram per 4% grams per 100 cc. This is relatively rapid pickiip of acid and indicates t.he necessity for constant replenistiing of acid iii the blanching solution.
In tlre destruction liy heat
the enzymes present in a large \,eget,ahle suell as artichoke hearts, the Ileating period necessary depends not only on the rate a t whicli the enzyme of
It&m hiaschine .Aitei blsnchinc
Water ... 8.6
.- -X i t r i o Arid-.---0.5% 0.75% 1.0% 2.42 ... 2.22 2.54 2.38 2.sa
0.5%
1.sctie Arid
2.44 2v2
Data and Discussion After storage at -17°C. for 3 nionths tlie treated artichoke hearts were removed a,nd analyzed. Representative samples were removed, cut in half, and allowed to stand a t room temperature for 4 hours; the underBlanched hearts becanre markedly discolored as slio~min Figures 1 and 2. The discolored FlQURE 1. EFFECTON Tt1E APPEARANCE OF FROJEN ARTICHOKE^ Ox’ areas decreased with time of b~anching,correBLANCEING IV l i o r ~ i ~WATER o FOE THE NUMDEIL 01 MINCTESINDICATED, snoniiine to destruction of enzvnes. Fieure 1 4 H ~ U L SA ~ E RI)EWROEITIN~ s h m &tit a 7-minute bl&h in w&r is the minimum necessary to reduce discolorais destroyed by lieat but also on the rate of heat perretiatiou tion and that a Y-miilute blanch does not entirely ininto the tissues. The latter was determined by stringing fine hibit it. In neitlier case did the tissue ]lave as desirable copper and constantan wires through the heart, soldering a, junction between them, and pulling the wires through until this junction was at the geometrical center of the base. The rate of tentperatwe change in boiling sater for hearts of various sizes is shown in Table 1V. The dimensions given are length multiplied by greatest width, taken just at the cheese or edible base portion. The temperature rise depends (I 24 15.5 21 22 13.3 ZG.5 28 0 25 44 . . 27.7 ... ... ... on size of the heart and the compactness of its leaves or 0 50 5U 40.5 34 . 4 i4 4 411.5 90.5 63.9 0.75 54.5 ... ... ... ... ... ... bracts; the time to reach 88”C . varied from 4 to 6 minutes. 1 56.8 48.8 58.9 50.5 56 92.8 67.8 1.5 The catalase and peroxidase activity of the artichoke 66.5 24.5 o2.n 56.8 58.9 Y?. 3 72.8 2 66 68 08 81.6 62.8 94.4 78.8 hearts WLIS determirietl qiialitatively from the intensity of 2.5 7G.8 83 70 68.6 80.5 65.5 85.0 :i 7 2 . 8 m 0 68 7 3 . 3 6 7 . 8 , . . 82.8 decomposition of 0.5 per cent hydrogen peroxide and from -, 8.5 83,s 71.5 75.6 ,A6 71 ... 85.8 the intensity of bluing of a 0.5 per cent solution of bennidine 4 88 75 75 5 78.8 80 . . . 87.8 4.5 87.8 79.5 82.8 81 G 78.3 . . . 90.0 in 50 per cent alcotiol in tlie presence of hydrogen peroxide. 89.4 82 xi 85.5 82.2 ... 41.6 5 .5 91,6 S5.5 87 86.6 85 ... 0a.n Two hearts were removed from ertch lot after blanching and G 9:
