INDUSTRIAL AND ENGINEERING CHEMISTRY
392
reduced pressure, P / P , R gas constant (equal to 1.986), gram cal./(gram mole) (" K.), or B. t. u./(lb. mole)(" R.) V = molal gas volume, liters/mole T = absolute temperature T, = critical temperature, K. To = critical temperature of reference substance (370" K. for propane) 6 = correlation factor (T,/370)" P p
= =
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(6) Beattie, J. A., Simard, G. L., and Su,G. J., J. Am. Chem. SOC., 61, 24 (1939). (7) Ibid., 61, 26 (1939). (8) Ibid., 61, 924 (1939). (9) Ibid., 61, 926 (1939).
(10) Edmister, W. C., IND. ENG.CHEX.,30, 352 (1938). R. V., and Scheeline, H. W., Am. Inst. Mining Met. Eng., Tech. Pub. 1060, 14 (1939). (12) Keenan, J. H., and Keyes, F. G., "Thermodynamic Properties of Steam", New York, John Wiley & Sons, 1936. (13) Lindsay, J. D., and Brown, G. G., IND.ENG. CHEM.,27, 817 (11) Gilliland, E. R., Lukes,
Literature Cited
(1935). (14) Michels, A., and Nederbragt, G. W., Physicu, 3, 569 (1936). (15) Michels-Veraat, G. A. M., Ph.D. thesis, Amsterdam, 1937. (16) Sage, B. H., Webster, D. C., and Lacey, W. N., IND.ENG. CHEW,29, 659 (1937). (17) Smith, L. B., Beattie, J. A., and Kay, W. C., J . Am. Chem. SOC., 59, 1587 (1937). (18) Watson, K. M., and Smith, R. L., Natl. Petroleum News, July 1, 1936. (19) York, R., IXD. ENG.CHEW,32,54 (1940). (20) York, R., So.D. thesis, Mass. Inst. Tech., 1938.
(1) Beattie, J. A., and Bridgeman, 0. C., PTOC. Am. Acad. Arts Sci., 63, 229 (1928). (2) Beattie, J. A.. Hadlock, C., and Poffenberger,N., J . Chem. Phus., 3, 93 (1935). (3) Beattie, J. A., and Kay, W. C., J. A m . Chem. SOC.,59, 1586 (1937). (4) Beattie, J. A., Kay, W. C., and Kaminsky, J., Ibid., 59, 1589 (1937). (5) Beattie, J. A., Poffenberger, N., and Hadlock, C., J . Chem. Phvs., 3, 96 (1935).
Color Changes in Green Vegetables FROZEN-PACK PEAS AND STRING BEANS
G. MACKINNEY AND C. A. WEAST Division of Fruit Products, University of California, Berkeley, Calif. VEGETABLE, whether prepared for canning, freezing storage, or immediate consumption, is ordinarily given the simplest of treatments-hot water or steam (in canning also a final sterilization in dilute brine) for specified times. The changes taking place in the vegetable are complex, those of chlorophyll alone necessitating many distinct and devious lines of research. Repeated examination of many species of plants offers the best safeguard against faulty generalization. Under ordinary conditions the degradation of chlorophyll, although complex as to detail, follows one or more of three courses: (a) hydrolysis, whereby phytol is removed; ( b ) oxidation, as a result of which the phase test is no longer obtained, nor are chlorin e and rhodin g the products of hot saponification; and (c) pheophytin formation, by removal of magnesium from the chlorophyll molecule. I n the string beam and peas examined, the changes are apparently limited t o the last mentioned cause. In general, vegetables prepared for freezing storage or for canning are given a preliminary blanching; i. e., they are treated in hot water for a specified time. Then they are packed into suitable containers and frozen, or they are sealed in cans in dilute brine under partial vacuum and sterilized. I n the former event they must be cooked after thawing prior to consumption. Blanching is necessary to prevent formation of off-flavors and discoloration. The relation between enzyme activity and development of certain undesirable qualities has been studied by Joslyn and Marsh (4) of this laboratory and by Diehl and co-workers ( 2 ) . Joslyn and Marsh found that blanching or scalding resulted not only in a greener appearing vegetable, but also in lessening to a marked degree the yellowish-brown discoloration on subsequent cooking so noticeable with inadequately blanched samples. This was confirmed by
Diehl and his group. In addition, Campbell (1) found that too high a storage temperature favored discoloration owing to a progressive conversion of chlorophyll to pheophytin, even though the vegetables were apparently adequately scalded. KO quantitative studies of this phenomenon were reported. Some of the many effects of treating living cells with hot water have been recognized for over 25 years as an interrelated function of time and temperature. Lepeschkin (6) expressed thus the leaching of pigment from the cells of red beet in hot water: T = a - blogt time to produce an arbitrary degree of color in the bathing solution T = temperature a, b = constants
where t
=
This expression is useful for a range of' temperatures which will cause impairment of the cell membrane with loss of its characteristic semipermeability. The equation has general applicability to the loss of water-soluble constituents as long as these were present originally and are not hydrolytic products of more complex cell constituents. Mestre ( 8 ) explained the shift in absorption spectrum for chloroph$l (maximum in the living leaf about 6820 A., and around 6750 A. in the killed leaf) on a similar time-temperature basis. We have a t various times confirmed the above authors' results. The well-known change to bright green when a frond of brown seaweed is dipped in boiling water may be brought about a t much lower temperatures-. g., 50" C. for 5 to 7 minutes. The change was correlated with a shift in absorption maximum, again confirming Mestre.' The importance 1 These observations are based on collaborative norh of one of the authois with Harold T. Byck.
MARCH, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRI-
A substantial part of the chlorophyll in frozenpack peas and string beans is converted to pheophytin. In simple cases, where the vegetable is treated for successive times in water at various temperatures, the formation of pheophytin is an interrelated function of time and temperature. Translated to industrial blanching practice, this step in itself is not prolonged to the point w-here serious impairment of color is obtained. This happens during subsequent handling prior to consumption. Since blanching does in many cases have a beneficial effect upon color retention, it is suggested that an adequate blanch must remove a large proportion of those volatile and water-soluble constituents which would react with the chlorophyll during subsequent cooking. The evidence for pheophytin formation lies not only in the spectroscopic data but also in the behavior of ethereal solutions to hydrochloric acid and to dilute alkali, and finally upon the cleavage products obtained on hot saponification-namely, chlorin e and rhodin g. The frozen-pack string bean, on cooking, has between 60 and 85 per cent of its chlorophyll converted to pheophytin, regardless of pretreatment. The canned beans examined apparently contain no unchanged chlorophyll. Frozen-pack peas, after cooking, may still appear bright green if they were adequately blanched previous to freezing. A minimum blanching temperature for 2 minutes is approximately 73” C. If inadequately blanched, the cooked pea contains about 80 per cent of its green pigment in the form of pheophytin; if adequately blanched, the value is between 50 and 60 per cent after cooking.
of this change as related to time-temperature effects is in demonstrating that the chlorophyll in the blanched vegetable is probably in substantially the same physical state of dispersion, regardless of minor modification in the blanching procedure. This state is considered to be a true solution of the pigments in the lipides of the plastid.2 Although the nature of the coagulum will t o some extent be determined by the previous heat treatment, the chlorophyll in all cases is affected to the same extent, as evidenced by identity of absorption maxima. Blanching results in a rapid expulsion of air from the vegetables, so that subsequent treatment minimizes possible oxidative changes. We have been unable to find any evidence of the presence of the enzyme chlorophyllase in either string beans or peas, which would cause hydrolytic changes, and no products of hydrolysis have been observed in the chlorophyll degradation products. We therefore explain the changes in chlorophyll in these vegetables on the basis of pheophytin formation caused by interaction of plant acids with the chlorophyll which, although still located in the chloroplast, is no longer protected by the plastid’s own membrane from its aqueous environment. Examination of a large number of microscopical sections shows that the chloroplast retains its identity throughout processing. There is no evidence of the leaching of pigment from these bodies a t any time. According to Willstatter and Stoll (IO,page 61), the effect of boiling water is to deform or rupture these plastids so that their masses fuse and spread 2 Recent evidence suggests the chlorophyll-protein complex, “chloroplaetin” may merely be denatured.
393
throughout the cell. We have found, on the contrary, that the plastids, originally turgid and arranged around the periphery of the cell, become shrunken and clumped in the center of a mass of coagulated protoplasm. The analogy of grape and raisin roughly illustrates this difference. With material subjected to low-temperature blanching and with living sections, great care must be exercised, since even slight pressure on the cover glass results in the discharge of the contents of the plastids into the cell which becomes uniformly green throughout. These observations are best made with chlorophyll-rich material, such as spinach leaves. Similar results however were obtained with string beans. After one hour in water at 100’ C. the plastids of the bean were recognizable though deformed in shape. They were still the only observable pigmented bodies on the microscope slide. We believe, there fore, that Willstatter and Stoll’s results are incorrect except possibly with leaves of high oil content. These observations do not apply to unblanched frozen material. In the case of several different species of fresh leaves that have been subjected to freezing, the chloroplast is ruptured, and we have not observed any that are intact. String beans show marked discoloration at all times after cooking when compared with the original material; this is particularly true of the string beans available on the market during the winter months because of their low chlorophyll content. In the case of peas this is markedly less evident in material adequately blanched prior to freezing, in which the vegetable after thawing and cooking may still be of bright green appearance. These vegetables are highly buffered, but as Marsh (7) showed, the p H of the juices is affected by heat. Where there are not too many complicating factors, it may be shown that with a given vegetable there is a progressive increase in pheophytin content with time a t a given temperature level, as Table I will show. Joslyn and Mackinney (3) found that the conversion of chlorophyll to pheophytin may occur a t B measurable rate. However, this is markedly slower than the rate a t which water-soluble constituents are leached from the cell by the hot water. In a highly buffered vegetable such as the pea, therefore, an adequate blanch requires, prior to freezing, a substantial removal of constituents which would otherwise react with the chlorophyll during subsequent thawing and cooking. The cell structure of the frozen pea is apparently less d i 5 rupted by ice crystals than is that of the string bean, and this was particularly true of our samples owing to the longer storage of the beans which were thawed and refrozen a t least once during this time.
Determination of Pheophytin In an earlier paper (3) the absorption coefficients of solutions of pure chlorophyll and pure pheophytin were determined. Measurements were made by the same technique as before, but different standards were chosen for the following reasons: (a) because of minor differences in the ratio of chlorophyll components and in the presence of carotenoids in our crude extracts, and (b) because of the absence of satisfactory data on the absorption coefficients of the pure components (6). Furthermore, the experience of Schertz (9) indicates how difficult it is to obtain chlorophyll with analytical data approaching normal standards of chemical purity. If, for example, the ash content is 5 or 10 per cent low, it is difficult to determine whether this is due to ash-free pigment or to colorless impurity. Careful and rapid extraction of the fresh material, particularly if the pH of the sap is near neutrality, gives a solution readily duplicable and as free from pheophytin as can be obtained without laborious
INDUSTRIAL AND E N GINEERING CHEMISTRY
394
The curve for an unknown mixtuze of chlorophyll and pheophytin, when superposed a t 5600 A. on the parent curves for chlorophyll and pheophytin, must a t all other wave lengths occupy an intermediate position between the extremes represented by the respective values for log k p and log k,, the effect of concentration being nullified. We hope to remedy soon the lack of satisfactory absorption data, but it is not yet possible t o predict how complex a system can be solved in dealing with crude extracts or the limits of accuracy attainable. It must be realized that, in fact, the unknowns contain four components, the two chlorophylls giving rise a t different rates to the two pheophytins. Necessarily these unknowns are treated as a two-component system. Considerable trouble has been taken to verify (3) the photographs of absorption spectra by Willstatter and Stoll (IO) which show that both pheophytins cgntribute substantially to the increased absorption at 5350 A.; and in this region no serious error is introduced in regarding the pheophytins as a single component. Because there are more ronounced differences in the absorption at 5800 and 6300 and in the rates of conversion of chlorophylls a and b (3), these regions are less suitable unless there has been at least a 50-60 per cent cpversion to pheophytin as calculated from the data a t 5350 A. This is well shown in curve 111, Figure 1. However, $he measurements have served as a useful check on the 5350 A. calculations which are made as follows: From transmission ( T ) measurements on our standards of concentration c, we calculate l/d log 1/T, the extinction coefficients (numerically 2, y) for wave length X:
.6 .5 .4
.3
.z
u
* .I .OJ
.07
.06
2
.05
.02
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I
and time-consuming effort. Such a preparation containing chlorophyll and carotenoids shows a maximum transmission near 5200 d.,is bright green in color, and in acetone solution is stable for several days if air and light are excluded. If we take two aliquots of such a preparation and to one we add a small quantity of saturated oxalic acid in 90 per cent acetone, it is possible to convert the chlorophylls to the respective pheophytins quantitatively. (In previous work on a larger scale, the isolated pigments were found to be ash-free.) To the second aliquot we add an equivalent amount of solvent, and we now have two solutions; the one contains chlorophyll and carotenoids, the other an equivalent quantity of pheophytin and the same carotenoids. The absolute concentrations are unknown and for present purposes immaterial. The tendency for the pheophytin solution to become cloudy may be overcome by adding the oxalic acid solution (2-3 cc.) to the aliquot when nearly made to volume. The solvent used is 90 per cent acetone. The transmissions are then determined and from the expression, log 1/T = kcd the values of kc are plotted logarithmically for the two solutions (Figure 1, curves I and 11). Since the concentration, c, is the same, kch = k p a t approximately 5600 A. where the two curves intersect. As long as Beer's law is obeyed for solutions containing concentrations c', c", c"', either of pure chlorophyll or pure pheophytin, the plots are merely displaced vertically in toto by a constant quantity: (log k ~ m f 1%
C)
- (1% kssao f 1%
C')
i. e., log c/c' and similarly log c / c C , log c/c'". This quantity is added to each curve so displaced, so that the curve for concentration c' is exactly superposed on the key curve for concentration c. The logarithmic plot permits a rapid inspection for identity of such curves.
x
k,k
=
y =
kchXC
An unknown contains two components for which c' =
cp'
Z'
Icp'Cp'
$.
(3) (4)
Coh'
f keh'Cd'
The total pigment concentration, c', is related t o c a t 5600 A. by the factor Z(SWO)/Z'(Q,X,). If performed graphically, curve I11 is simply displaced in toto up or down, so that the curve is superposed on the key curves I and I1 a t 5600 A. The z' values may now be written z, for which, a t wave length X, 2 = kp% f ksh'cch where Cp Ceh = C c = same value as in Equations 1 and 2
(5)
+
(6)
From Equations 1, 2, and 6 we substitute kpXlkch', and cp in Equation 5 : z= whence
Similarly
(c
- cch)
---
C
Cch
2
f -Y
-z
Cch
(7)
y--s
(8)
5 = -2 - Y
(9)
c
c
s--y
Thus for any convenient unknown and arbitrarily selected concentration c', we can determine the proportion of pheophytin. The x, y, z values may be read directly from the plot of log x, log g, etc., since kc has been plotted on semilogarithm paper. I n earlier work (with Harold T. Byck) the curves for absorption coefficients for pure chlorophyll, pheophytin, and synthetic mixtures invariably intersected in the range 56505600 A. so that, regardless of the proportions of the four components, the error introduced in this step is small. The curves in Figure 1 were repeated four times, and the error in the relative values was found to be of the order of
MARCH, 1940
INDUSTRIAL I N D ENGINEERING CHEhlISTRY
*2 per cent, all instrumental factors being kept constant. The unknowns could be duplicated with an average error of less than * 7 per cent. The error inherent in treating an approximately 3 to 1 mixture of a and b components as a two- $stead of as a four-component mixture iz small a t 5350 A., and of the order of 10-20 per cent a t 5800 A. or longer wave lengths, if the percentage conversion is below 50 per cent. It should be emphasized that the unknowns invariably gave, on hot saponification, only degradation products of lorn hydrochloric acid number; 5 and 9 per cent hydrochloric acid extracted the approximately correct amounts of pigment, none being left in the ether. There was never a positive test for chlorophyllide or for pheophorbide. Therefore, the results seem to be correctly interpreted on the basis of the suggested chlorophyll-pheophytin transformation. Both pea and string bean samples had been prepared by Joslyn and co-workers for enzyme inactivation studies already reported (4). The peas (Laxton Progress variety) were blanched and frozen in May, 1937, and stored approximately one year. The beans (Pink Seed Kentucky Wonder, KO. 2 sieve) were treated in July, 1937, and had been in freezing storage for about 15 months. Table I gives the percentage of pheophytin found in fresh string beans immersed in boiling water for increasing periods of time. There is a small lag during the first few minutes, caused probably in part by slowness of heat penetration in killing the cells and in destroying heat-precipitable buffers ( 7 ) . Table I1 shows a series of tests made on string beans after freezing storage for some months and after subsequent cooking for 20 minutes a t 100” C. TABLE I. EFFECT OF TIME ON PHEOPHYTIN FORMATION IN FRESH STRING BEANS AT 100” c. Time, Min. 0
% Pheophytin
Appearance of Ext. Green
0 7
i
Green -.-...
16 37.5 Green Brownish green 20 72.5 86.5 Yellowish green 30 Yellowish 1ooa 60 a Products of hot saponification were completely extracted with 5 and 9 per cent HCI; i. e., they were chlorin e and rhodin g. TABLE 11. EFFECTOF BLANCHING ON PHEOPHYTIN IN STRING BEANS -%
-Treatment0
c.
hlin.
09 thawing
FORMATION
PheophytinoAfter subsequent cooking
a All values are the average of a t least three determinationu. T h e identity of several values is to some extent fortuitous. The plot of the whole band was taken in making t h e necessary adjustment. Several plots were virtually superposable, hence z values were identical. Uncertainty from thia c a w e is about 2 per cent. b Samples directly from the can.
Considerable difficulty was encountered in obtaining representative samples; close examination of the beans revealed lack of uniformity in the coloring, even with individual beans. Those with definitely brownish areas were rejected. All the blanches listed in Table I1 gave individual results within the limits of variation in column 2. Excluding the untreated samples and after cooking for 20 minutes, all samples whether
3 95
previously blanched or not showed from 60 to 90 per cent conversion of the chlorophyll. This is in accord with the data of Table I, where 70 per cent conversion is found for the fresh bean after a 20-minute cooking. The data are similarly tabulated for peas in Table 111. TABLE 111. EFFECT O F BLANCHING ON PHEOPHYTIN FORMATION IN PEAS -yo PheophytinbA . After B. After Treatment, C.0 thawing cooking Appearance of Vegetable No blanch 10.5 89 Off-color 60 35,s 89 Off-color 71 44 85 Off-color 76.5 66 Sli,ghtly (off-color 82 27:5 53 Still green 87.5 19.5 53 Still green 93.5 27 63 Still green 100 7 51 Still green 0 The time for this series was 2 minutes. b Except for t w o cases in column A , all results are a t least in duplicate.
As Table I11 shows, there is a definite minimum temperature a t the particular time involved, below which a bright green color is not obtained in the cooked product ( 4 ) ,and the retention of green color is definitely associated with lower pheophytin content. There is a suggestion of a similar trend in the blanched cooked beans, but i t is not nearly so striking; and from casual observation without quantitative data, this trend appears to be more readily obscured by time of storage than is the case with peas. The appearance of the vegetable is altered by blanching owing to the expulsion of air and collapse of intercellular spaces. In specific cases, such as spinach, clover, and sunflower, an instantaneous dip into boiling water causes no apparent change in the structure of the chlorophyll molecule since there is complete identity of absorption curves of suitably prepared extracts (6). This is also true of peas. Apart from this change in appearance caused by increased opacity of the vegetable, the color will be determined by the nature of the incident light, the visibility function, the reflected light for the vegetables, and the transmitted light for extracts. A decrease in chlorophyll content by conversion to pheophytin causes substantial decrease in absorption in the red and orange and a sharp increase in the green. The samples from which the data in Table I were compiled were readily arranged in the order of increasing pheophytin content, and the abrupt color change came a t 20 minutes with about 70 per cent conversion of the chlorophyll. Viewed, therefore, under a given light source, the vegetables will become noticeably brown when the proportion of pheophytin is sufficiently high so that it reduces the total luminosity in the green and blue to a magnitude comparable with that in the red and yellow. Changes in chlorophyll in canned peas cannot be ascribed solely to pheophytin. More drastic degradation has taken place. The prolonged heat treatment in a strongly buffered medium causes changes which indicate the formation of weakly basic cleavage products of chlorophyll.
Literature Cited (1) Campbell, H., Food Research, 2, 55 (1937). (2) Diehl et al., Ibid.,1, 61 (1936); Western Canner and Packer, 29, 18, 22 (1937). (3) Joslyn and Mackinney, J . Am. Chem. SOC.,60, 1132 (1938). (4) J o s l y n and M a r s h , Science, 78, 174 (1933); Westmn Cantur and Packer, 30, 21, 35, 37 (1938); Food Industries. 10. 379. 435, 469 (1938). (5) L e p e s c h k i n , Ber. deut. bot. Ges., 30, 703 (1912). (6) M a c k i n n e y , Plant Physiol., 13,123 (1938). (7) M a r s h , Hilgardiu, 11, 317 (1938). (8) Mestre, d i s s e r t a t i o n , S t a n f o r d Univ., 1929. (9) S c h e r t a , F. M., IND.ENG.&EM., 30, 1073 (1938). (10) W i l l s t a t t e r a n d Stoll, “ U n t e r s u c h u n g e n uber C h l o r o p h y l l ” , B e r l i n , Julius S p r i n g e r , 1913
