March, 1935
INDTJSTRIAL AND E N G I N E E R I N G CHEMISTRY PRACTICAL VALUE O F THE
GRAPH
From the many articles on the storage of raw sugar in humidity-controlled warehouses to prevent bacterial growth by maintaining the factor of safety, and consequent inversion a n d deterioration, the practical value of this type of graph can readily be seen. I t may also be used for soft (brown) sugars, granulated sugars, and products of high sugar content. T h e graph may be within practical limits for honie3, candies, chocolates of high sugar content, corn sugar (glucose) products, etc. Those attempting to prepare levulose in crystalline form for commercial use mill do well to give particular attention to the rather low percentage relative humidity a t which this Droduct absorbs water. There is n o doubt but that this type of graph is not the
335
last word on the absorptive properties of sugar. There are many factors in this problem that are not yet known or understood; it is hoped that this article will stimulate further work to obtain definite information.
BIBLIOGRAPHY (1) (2) (3) (4) (5) (6) (7) (8)
Browne, C. A,, J. IXD. EXG.CHEM.,14, 712 (1922). Edgar and Swan, J . Am. Chem. Soc., 44, 570 (1922). Honig, R., Intern. Sugar J., 31, 214 (1929). King, R. H., and Suerte, D., Ibid., 32, 542 (1930).
Owen, W. L., Ibid.,24, 581 (1922). Sandra, K., Listy Cukrovar., 51, 314-18 (1933). Smith and Menaies, J. Am. Chern. Soc., 32, 1412 (1910). Whittier, E. O., and Gould, S. P., IND. ENQ.CHEM., 22, 78 (1930).
RECEIVED November 15, 1934.
Peroxidase in the Darkening of Apples A. K. BALLS AND W. S . HALE,Bureau of Chemistry and Soils, Washington, D. C.
A
that the tissue, nhen f e hlY LTHOUGH other reactions Darkening of the freshly cut surfaces of apples are necessary preliminacut, darkened more rapidly and is a reaction that is catalyzed by peroxidase. c o n s i s t e n t l y than that of the ries to the formation of The formation of hydrogen peroxide by a respiraother common varieties availt h e brown pigment 011 the surtion enzyme which uses molecular oxygen is a able.) face of freshly cut apple tissue, necessary preliminary step. Both the fruit tissue the steps which culminate diDECOMPOSITION OF PEROXIDH rectly in the production of pigand the juice darken in the absence of air, but BY CATALASE m e n t are accelerated by peronly until the peroxide present is complelely Ten grams of peeled apple tiso x i d a s e . A theory of tissue utilized. The reaction continues on further sue were ground in a mortar with oxidation which involves per100 grams of ice, the pul was addition of peroxide or on reexposure to air, oxidase was long ago proposed filtered rapidly, and the &trate when peroxide is again produced enzymically. by Bach and his co-workers (2) was kept in an ice bath during the experiment. From 20 cc. of this a n d was used by Onslow in exPigment formation is accelerated by horse-radish diluted juice, 2-cc. portions were plaining the darkening of fruits peroxidase. I n boiled apple pulp the addition of removed at intervals and mixed (4). It seems to fit the facts with one drop of a 1.5 per cent both peroxide and peroxidase is required. observed on apples very well, alcoholic solution of guaiacum. The inhibition of peroxidase is therefore of A positive reaction, the rapid as long as pigment formation is formation of an intense blue color, importance in delaying the discoloration of cut regarded as subsequent to a procwas observed undiminished in the ess of direct oxidation such as fruit. Peroxidase inhibitors fall into two classes: untreated juice for 15 minutes. t h a t regulated by the respiraAfter 25 minutes the color was substances that affect ihe enzyme directly and subappreciably weaker than a t the tion ferments. It is unnecessary stances that accelerate its inactivation by hydrogen beginning. The apple peroxidase t o discuss here whether or not itself had, therefore, no rapid effect peroxide. The former class, which includes the such a Fystem corresponds to at this temperature on the perthe o x y g e n a s e s of Bach and sulfhydryl compounds, is more important f o r the oxide present. To 20 cc. of the same diluted C h o d a t . In addition t o t h e inhibition of fruit darkening. Treatment of juice, 0.25 cc. of catalase solution evidence to be found in earlier sliced apples with a dilute solution of glutathione was added [l.O gram of Hennich' work, the behavior oi' apple tiscrude liver catalase (3) was susor cysteine salts permils drying or long-continued sue and apple juice observed in pended in 100 cc. of water and the keeping wilhout discoloration. The su[Shydryl insoluble material was then rethe f o l l o w i n g e x p e r i m e n t s moved in the centrifuwj. One strongly supports this view. The dericaiiee occurring in pineapple juice as the minute after the introdurtion of d a t a given are typical of a much natural actitlator of bromelin has the same effect. the catalase (at 0" C.) the blue larger amount which it does not color was weak, and after 10 minutes it was absent. Hydrogen seem necessary to cite. Oxidation by peroxidase requires the presence of a per- peroxide in the proportion of 0.10 cc. of 0.1 N hydrogen peroxide to 10 cc. of juice was addpd 25 minutes after the catalase addioxide; the ferment cannot use molecular oxygen. Both per- tion. This caused a temporary recurrence of the reaction with oxide and peroxidase are known to be present in apple tissue. guaiacum tincture, which was again very strong at first but became negative after another 10 minutes. This reaction indicated The former mag be recognized by the usual potassium iodidestarch method,-and thepresence of both may be detected by that t h e peroxide, not the peroxidase, had varied during the the coloration of tincture. peroxide is also experiment. The addition of peroxidase to the juice had no effect at 0" C. except t o intensify the guaiacum reaction. found in the press juice and in the water extract of fresh The peroxide in apple tissue is therefore hydrogen peroxide, apples, where it m a y be decomposed by catalase. as shown in the following experiment with a water extract of Paragon or is capable of changing into hydrogen peroxide which is apples. (This variety and the almost identical Arkansas the only peroxide decompoeed by catalase. The presence of Black Twig variety were used in all the experiments reported hydrogen peroxide is to be expected, since Wieland and his here. They were chosen because preliminary tests showed co-workers (6) have shown by quantitative measurements
336
INDUSTRIAL AXD ENGINEERING CHEMISTRY
that i t is the product of the oxidation of organic hydrogen by certain direct oxidases. The first step in the formation of the brown pigment is therefore the production of hydrogen peroxide by the normal action of a ferment of direct oxidation, This conclusion is borne out by the observation that apple pulp or apple juice darkens a t room temperature in a vacuum as well as in air, but only so long as the peroxide reaction persists. After the darkening has stopped, the addition of hydrogen peroxide permits it to start again, as does exposure to air. Following the admission of air, the peroxide test again becomes positive, indicating a reduction of oxygen by the oxidase system. The experiments are easily made, either in Thunberg tubes or under a thick layer of vacuum-boiled oil, using the diluted juice prepared as in the experiment with catalase and boiled for a short time in vacuum to remove dissolved oxygen. After shaking the peroxide-free juice for 2 or 3 minutes with air, it again begins to darken and the color reactions for peroxide are again strongly positive. Besides peroxide and peroxidase, there is present in apple tissue a chromogenic substance capable of rapid oxidation by the peroxidase-peroxide system. Its presence can be demonstrated in apple pulp which has been boiled until neither peroxide nor peroxidase remains. A pigment of the proper color is rapidly formed in the boiled pulp on the addition of both hydrogen peroxide and horse-radish peroxidase (a crude peroxidase preparation, after Willstiitter and Stoll, 6), but is not formed on t'he addition of either alone. Horseradish peroxidase is also able to accelerate the pigment formation in fresh apple tissue which contains peroxide. The chromogen was concentrated by the following method : The apples were reduced to pulp in a dilute solution of sulfurous acid, and the liquid was filtered off. The acid filtrate was then treated with an excess of lead acetate and acetic acid, and the precipitate was discarded. A second precipitate which contained the chromogen was formed by adding ammonia to the filtrate. This precipitate was removed, washed with water, suspended in water, and decomposed by hydrogen sulfide: After removal of the lead sulfide, the liquid was concentrated in a vacuum to a sirup. More water was removed by distilling absolute alcohol frord this residue, which was finally suspended in absolute alcohol and filtered. The filtrate contained the chromogen. From this a t r a t e the alcohol was distilled off, and the residue was dissolved in a little %-butyl alcohol. The butyl alcohol solution was mixed with four volumes of dry ether and a precipitate containing some chromogen, but also a considerable amount of impurity was removed. The solution was finally poured into five volumes of cold petroleum ether, and the precipitate was filtered off, washed with ether, and dried in vacuum. The product, one gram of which was obtained from 10 kg. of apples, was a white, slightly hygroscopic powder, very soluble in water. It contained no nitrogen which could be detected when 50 mg. were fused with sodium. The solution was acid to Congo red and contained considerable titratable acid. It gave a green coloration with ferric chloride. Attempts to crystallize the material or to prepare crystalline derivatives have been unsuccessful as yet, so no quantitative data are given here. There is reason to think that this product is still impure. The material oxidizes in alkaline solution in the air. In neutral or slightly acid solutions it is rapidly oxidized by peroxidase and hydrogen peroxide with the formation of a deep brown color. Apple tissue contains a complete peroxidase system. Darkening does not occur when this system is incomplete (lacking peroxide). But it can occur with the complete system, without air (experiments in vacuum). It appears, therefore, that the pigment formation is a direct result of peroxidase activity. I n an attempt to delay the darkeuing of freshly cut apples, a study was made of the inhibition of horse-radish peroxidase. The theoretical aspects of this work have been published separately (1). The practical object was to seek a means of preventing the fruit from darkening during canning and drying without recourse to the use of sulfur dioxide.
Vol. 27, No. 3
Horse-radish peroxidase was found to be inhibited (3) by two classes of substances: One class inactivates the enzyme directly, the other class only in the presence of hydrogen peroxide. [Hydrogen peroxide itself inactivates peroxidase (7) but much more slowly in the concentrations used.] Substances of the second class are more active the higher the concentration of peroxide present, and are less active Then an easily oxidizable substrate of peroxidase is present in addition. Such inhibitory substances resemble the acceptable substrates of the enzyme in chemical constitution. Among them are phenol, aniline, and meta diamino or dihydroxy compounds. The miters have suggested that these inhibitors resemble the substrates of the ferment in structure sufficiently to permit them to combine with it but not sufficiently to undergo the type of oxidation that the enzyme accelerates (one step seems to be the formation of a quinone). Perhaps the transformations that lead to the oxidation of an acceptable substrate result in the inactivation of the enzyme when carried out with a substance that is a kind of incomplete substrate. T h a t the ferment is really inactivated and not merely reversibly bound to the inhibitor follows from the fact that, while the presence of a substantial concentration of a very reactive substrate such as pyrogallol protects the enzyme fairly well from inhibition, yet once it is inhibited in the absence of pyrogallol, no amount of this substance added subsequent~lycan restore the activity. This is a peculiar phenomenon, arid the fact, that the pigment-forming enzyme from apples behaves towards phenol in the same manner as does horse-radish peroxidase, supports the view that the ferment which produces the pigment is a peroxidase.
EFFECT OB PHEKOL os APPLE PEROXIDASE The phenolic chromogen was removed from apples by the method of Onslow (4which , consists of grinding and extracting the tissue in alcohol. The enzyme remains bound to the plant fibers, which are dried. Enough of this material to represent 5 grams of original apple tissue was placed in each of a series of test tubes together with 5 cc. of 0 . 2 111 phosphate buffer (pH = 6.5). In addition, some of the tubes contained phenol and 0.1 N hydrogen peroxide as shown in the table. after 10 minutes in one case and after 2 hours in another, 5 cc. of boiled apple pulp (10 grams per 100 cc.) were added to each tube as a substrate for the enzyme, together with 0.25 cc. of 0.1 N hydrogen peroxide. The color development in the tubes was read after 2 hours more at room temperature. The sign indicates that much color was formed, 0 indicates that no color change occurred, as followvs:
+
PRETREATMEST OF EVZYUE PERIOD OF PRETREATXENT
Min. 10 120
10 mg.
None (control)
++++ ++++
10 mg.
phenol
+++ +++
phenol and 0.25 cc. HxOa
0.25 cc. H202
++o++
Blank (sub-
strate only)
;
The results show that the enzyme was uninjured by phenol alone, rapidly inactivated b y phenol and hydrogen peroxide, and slowly inactivated by peroxide alone. Horse-radish peroxidase has been shown to behave in exactly the same manner. Inhibitors of the phenol type would be of little value in delaying the activity of fruit peroxidase during a factory process. -On the other hand, those inhibitors which affect the enzyme directly might find considerable application. Among these are the sulfhydryl compounds, some of which occur in common foods. Glutathione and cysteine are powerful inhibitors of peroxidase action and prevent the darkening of freshly cut apple surfaces when applied to them in very dilute solutions. As far as laboratory experiments can show, apples dried under conditions similar to those used in commercial practice retain their original color during the drying and for
March, 1933
I N D U S T R I A L A K D E N G I N E E R I N G C H E hl I S T R Y
at least 9 months thereafter, if sprayed with a solution of cysteine hydrochloride when the fruit is first cut. An interesting application of this principle is based upon the fact that pineapple juice often contains a sulfhydryl compound (probably glutathione) which serves as the natural activator of the bromelin. It should therefore be possible to substitute a pineapple juice of high proteolytic acti\;ity for the glutathione and cysteine. Laboratory experiments have indicated that this is the case. Pineapple juice may be fermented, freed from alcohol, and concentrated without losing its property of inhibiting apple darkening. In these experiments, slices of apple dried after treatment with pineapple juice were always lighter than control slices ‘which underwent no treatment. As a rule they did not look as well as fruit treated with cysteine or glutathione. There was a great difference between various samples of pineapple juice in respect to the color of the dried apples; until the reason for this variation is better understood, it would be unwise for anyone to attempt a technical process depending on pineapple juice to prevent fruit darkening. The under-
337
lying principle of such a process, however, seems to be established. The applicability of peroxidase inhibitors as deterrents of fruit discoloration lays the foundation of many possible processes for the technical handling of those fruits and vegetables whose darkening is partly or wholly accelerated by this ferment.
LITERATURE CITED (1) Balls, A. X., and Hale, W. S., J.Biol. Chem., 107, 767 (1934). (2) Chodat, R., in Abderhalden’s Handbuch der biochemischen srbeitsmethoden, Vol. 3, p. 42, Vienna, 1910. (3) Hennichs, S., Biochem. Z., 145, 286 (1924). (4) Onslow, M. W., Biochem. J., 13, 1 (1919) : 14, 535, 541 (1920). (5) Wieland, H., and MacCrae, T. F., Ann., 483, 217 (1930). (6) WillstRtter, R., and Stoll, A., Ibid., 416,21 (1918). (7) Willstitter, R., and Weber, H., Ibid., 449, 175 (1926). RECEIVED November 5, 1934. Presented before the Division of Agricultural and Food Chemistry at the 88th Meeting of the American Chemical Society, Cleveland, Ohio, September 10 t o 14, 1934. This paper is Contribution 237 of the Food Research Division, Bureau of Chemistry and Soils.
Properties of I-Octadecene, n-Octadecane, and I)i-m- tolylethane V
M
ETHODS of synthesis are recorded for a large num-
n-Octadecane by hydrogenation was prepared under from
M. V. DOVER‘AND W. A. HENSLEY~
a pressure of 3 atmospheres of hydroUniversity of Missouri, Columbia, MO. ber of hydrocarbons whose gen. The platinum black-platinum oxlde catalyst used for the hydromolecular weights are in excess of enation Was Prepared according to the method recommended by 200. However, the data concerning the physical properties i d a m s (2). The apparatus used for hydrogenation was similar of these hydrocarbons of higher molecular weight are quite to that suggested by Adams meager. This investigation was undertaken with tW0 obDi-m-tolylethane n-as prepared by the oxidation of mxylene jects: (1) to synthesize a small number of hydrocarbons with potassium persulfate as recommended by Moritz and whose properties would resemble those of lubricating‘ oils of Wolffenstein (13). Di-mtolylethane prepared by this method is identical with that Prepared by Carre (S), using the reaction below viscosity and (2) l;o study the chemical and prop- tween magnesium and m-xylyl bromide. erties of these hydrocarbons in a n effort to determine whether or not any of these properties are related to “oiliness.” It was desired to include heptadecene in this study, and It is hoped that the work recorded in this report may be of two attempts were made to synthesize this hydrocarbon. are I n the first attempt the method of Mai (11) Some service in helping to determine which followed, and valuable in reducing friction, and that it may also aid in the a fraction distilling betn,een 1 4 8 and ~ 1530 C. (6 mm, presidentification of Some of these compounds which may be sure) was obtained. Elementary analysis of this fraction isolated from petroleum. gave the following results: PREPARATION OF
HYDROCARBOSS
The three hydrocarbons chosen for this study were prepared as follows : I-Octadecene was prepared according to the method of Krafft (8) which consists in distilling octadecyl stearate under a pressure of 120 to 140 mm. of mercury. This distillation ca>usedthe decomposition of the ester into 1-octadecene and stearic acid according to the following equation:
+
Ci&MZ”OOCCiJL = CisH3~ CirI-4d2OOH Oxidation of the hydrocarbon with potassium permanganate and analysis of the oxidation products indicated that the hydrocarbon was practically pure 1-octadecene. Octadecyl stearate was prepared by treating octadecyl alcohol with stearyl chloride, allowing the reaction product to cool, ulverizing, and washing twice n
