BROMELIN
0
Properties and Commercial Production
A. IC. BALLS, R. R. THOiMPSON1, A ~ U M. W. KIES Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C.
T
HE existence of bromelin, a protein-digesting enzyme bromelin is destroyed by the temperatures commonly used in pasteurization procedures (75-80' C.) . in pineapple, was probably first established in 1891 by Marcano ( l a ) and also by Chittenden (8) who salted it Willstatter, Grassmann, and Ambros found that old prepaout of the juice and studied its action in considerable detail. rations of bromelin had changed in their relative activities Later the enzyme was studied by Vines (IS), Caldwell ( 7 ) , toward gelatin and peptone. They concluded, however, and more recently by Willstatter, Grassmann, and Ambros that such differences could be explained as readily by the accumulation of substances relatively inhibitory to one activity (14). The properties of the pineapple proteinase lead to the conclusion that it is similar to papain and certain other plant as by the existence of more than one ferment in the preparaproteinases, among them asclepain from milkweed ( I @ , the tions (14). proteinase in Lima beans (9), and that in wheat flour (2). Bromelin is completely precipitated from pineapple juice Like these enzymes, bromelin is activated by hydrogen sulfide by ammonium sulfate and by alcohol (7,8,14). Such prepaand by cyanide, and deteriorates in aqueous solution, prerations are ten to twenty fold purer (on a protein-digestsumably because of oxidation. ing basis) than the original sugar-rich juice. Our work has Like other proteinases, bromelin clots milk. Such obserbeen largely confined to the protein precipitated from prevations are of practical interest, for if a proteinase is to be viously heated low-grade pineapple juice (second or third used technically, it will probably be either as a milk-clotting pressing). It is probable that only such preparations can or a protein-digesting agent. [The recently discussed ancompete with cheap proteolytic material such as papain. thelmintic property of bromelin (6) may, of course, prove to High-grade pineappIe juice is too costly for such a purpose. be a third property of practical interest.] Proteolytic enThe precipitated material may not possess all the enzymes zymes possess milk-clotting power to different degrees, compresent in the fresh juice. It afforded no evidence for the pared with their respective abilities to "digest" proteins. existence of two distinct enzymes. However, the behavior For example, trypsin digests casein about as rapidly as does of this bromelin toward acid, alkali, and heating is curious. chymotrypsin, but it is much less powerful as a milk-clotting There is evidence that the enzyme protein is relatively stable agent. The ratio of milk-clotting t o protein-digesting acbut inert in alkali, and active but unstable in acids. It untivity may, perhaps with some reservation, be taken as typical dergoes denaturation on heating. This denaturation is of a proteinase. A comparison of the milk-clotting power of partly reversible when the heating occurs a t pH levels where crude papaya latex with that of crystalline papain indicates the enzyme tends to be stable, but the reversion is not demonthat two enzymes exist in the latex, one of which hydrolyzes strated unless the material is later brought t o a pH where the proteins faster (or farther) than the other, in proportion to enzyme is not stable. Since the first work of Anson and the milk-clotting activity (4). I n this case a partial separaMirsky ( I ) , the reversion of denatured protein has been recogtion may be made by fractional precipitation with ammonium nized as a frequent phenomenon, but the present case seems sulfate. Chittenden as early as 1894 made similar claims to exhibit some uncommon features. for pineapple juice. He found that sodium chloride precipitated two proteins-one insoluble in TABLEI. BROMELIN CONTENT OF PINEAPPLE PLANTS~ water and able to clot ---Not ActivatedActhatedbTitration inmilk but not to digest Titration increase after crease after protein, the other soluble Milk unitsc digestiond per Milk units digestion per % Juice per cc. cc. enzyme cc. per cc. cc. enzyme, cc. in water and proteolytic Obtained of juice 0.10 A; K 6 H of juice 0.10 h' KOH Variety Material (8). Ca enne Caldwell believed that Leaves & &een 10 0.G 0.4 0.6 0.0 stems fresh pineapple juice con36 ... ... ... 0.3 Shell Green 69 ... ... 0.3 Inside Green tained two enzymes-one 4 1 0 . 5 0.4 0 . 6 1.1 Shell Ripe active in alkaline solutions, 62 1.5 0.7 0.9 0.5 Inside Ripe the other active in acid .. ... 0.4 1.0 ... Shell Very ripe media and destroyed by .. ... 0.5 0.5 Inside Very ripe heating to 65" C. in salt Pernambuco Leaves & Green solution ( 7 ) . 45 ... 0.7 1.4 ... stems 42 ... 0 9 2.2 ... Shell Eckart and Cruess (IO), Green ... 57 . . . l . G 3.6 Inside Green who studied various means ... 53 ... 0.7 3.1 Leaves Ripe of preservation of pineapple 45 1.0 .1 ... Shell Ripe 60 ... 1.2 . 23.1 ... Inside Ripe products, concluded that
--
I..
I
1 Located a t and cooperating with t h e Honolulu Agricultural Experiment Station, Honolulu, T. H.
.
.
aPress juice from a hand press. bActivation with 6 mg. cysteine per cc. of enzyme for 30 minutes a t 30' C. just prior t o t h e digestion. CDetermined on 10 cc. of milk r t t 40" C. according to,Balls and Hoover (8). dDetermined a t p H 5 after 20-minute digestion of ossein a t 40° C. according t o Balls, Swenson, and S t u a r t (6).
950
-
July, 1941
..
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
Stability of Bromelin IN PINEAPPLE JUICE. As Table I shows, bromelin, unlike papain, does not disappear as the fruit ripens. It might be concluded that the quantity of ferment increased on ripening, but this may be nothing more than its greater solubility in the juice a t that stage. The Pernambuco variety of pineapple appears to be richer in enzyme than the Cayenne, but the latter is the only variety in industrial use (in Hawaii). The enzyme is present in the leaves and stalks as well as in the fruit. The fate of bromelin in the INFACTORY MID-PRODUCTS. factory was observed by following the material (except the fruit for canning) through the several treatments that consist essentially of pressing, screening, and heating (Table 11). It is evident that the bromelin follows the juice, not the solid matter. The enzyme is remarkably stable toward heat, as shown by the fact that press juice heated to 60' C. and screened thereafter still contained a large proportion of the
The fate of bromelin, the proteolytic enzyme of pineapple juice, has been followed throughout the factory operations of pineapple canning. The enzyme appears in the juice and is there remarkably resistant to heat. A method is suggested which laboratory experiments only have indicated might be used to recover bromelin from low-grade juice without decreasing the yield of alcohol. The curious behavior of bromelin toward heating and alkali has been studied, and it was found that the activity of a bromelin preparation at a fixed pH depended upon the previous pH at which the preparation was held. The facts appear to be best explained by the assumption that the enzyme protein easily undergoes reversible denaturation.
original enzyme in the active state. Such juices also kept well in the cold. No material change in total enzyme content and very little reversible inactivation were observed during storage in glass in the ice box for 10 days. Eventually some of these stored juices were partially fermented by yeast. Vigorous alcoholic fermentation reduced the quantity of enzyme only by about a third. Neutralization of the juice did not affect its activity immediately but hastened deterioration.
Proposed Rlethods of Obtaining Bromelin The economic advantages in the manufacture of bromelin over papain are apparent. The material would be the byproduct of another industry and always obtained from a single variety of the plant; under the agricultural conditions of Hawaii, it would therefore be unusually uniform. The quantity made could be easily adjusted t o suit the market, and manufacture would require very little addition to the existing facilities for pineapple canning.
951
However, there are disadvantages in the manufacture of bromelin in place of papain. The material used must be factory waste, since other pineapple products are too valuable. The amount of enzyme present is small, and it would not pay t o destroy the sugar or citric acid even in the waste in order to get the bromelin. TABLE 11. BROMELIN CONTENTOF FACTORY MID-PRODUCTS~ iMilk Units per Cc. Before After activation activation 1.1 1.6 0.8 1.2 0.9 1.2 0.1 0.6 0.7 0.7 a Enryme determinations as in Table I. b This pulp was largely juice; the results when compared t o those obtained on t h e juice itself indicate no conoentration in the pulp.
I n practice] therefore] the problem reduces itself to one of obtaining the enzyme without destroying the value of the raw material for other purposes. A scheme is possible whereby this can be done; whether it would be profitable we cannot say. Bromelin may be precipitated by alcohol and the alcohol recovered later. The residual juice would thereafter be as available for the usual alcoholic fermentation as it was before, since no sugar would have been lost. The same idea is apparently feasible with ammonium sulfate, although the process may then be too roundabout. It would entail precipitation of the enzyme by a salt such as ammonium sulfate, recovery of most of the sulfate by precipitation with alcohol, recovery of the alcohol by distillation] and finally fermentation of the juice as usual. No method of precipitating the enzyme can be satisfactory in the presence of so much solid matter as exists in the screened juice. Some form of filtration would be necessary, and this may prove a serious technical objection. For the purposes of this experiment the juice was filtered in the laboratory through paper but did not filter readily. Table 111 describes several preparations made by the methods outlined here. The enzyme was precipitated from the heated, screened] and filtered third-press juice. Five volumes of 95 per cent alcohol or 0.6 saturation with ammonium sulfate were used, respectively, to precipitate the protein. The precipitate was filtered off and dried in vacuum at about 50" C. in each case. The proteolytic and milkclotting activities of these preparations were, in general, found to be about the same as those of ordinary commercial papain.
Behavior of Protein Precipitated by Ammonium Sulfate After the moist filter cake hdd stood for several months in sealed lacquered tin containers; abgut half the activity then observed resided in a fraction &soluble in water, from which i t was incompletely eluted with alkaline or neutral 0.02 M cyanide. The water-soluble fraction was usually capable of further activation by cyanide, while that bound to the solid was not. In experiments on the moist filter cake, milk-clotting activity was determined] not as in the previously reported experiments, but by using 5 cc. of Klim suspension at 30" C . and 1 cc. of enzyme, as described in recent experiments with apain (4). Since the latter milk-clotting technique employs Righer concentration of enzyme a t a lower temperature, the values for milk-clotting activity are not very different from those obtained by the earlier method, but are naturally not quite comparable t o those re orted in Tables I and 111. Hroteolytic activity was determined at pH 7.1-7.3 by the formol titration technique of Northrop (11) with casein
Vol. 33, No. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
952
TABLE III. PREPARATIONS OF BROMELIN FROM PINEAPPLB JUICE” Yield
7
Juice taken, cc.
Precipitant
dry ppt., grams
2000 1000 1000 1750
Alcohol Sulfate Sulfate Sulfate
4.7 1.: 4.? 1 .a
E
5000
Alcohol
11,9
F
2000
Alcohol
5.4
L(
C
D
Not activated Milkunits Cc. KO 0.1 HN
wt.
‘a Enzyme quantities determined 6 This preparation was analyzed c Almost fully active.
activated Milkunits Cc. K O0.1 H N
Remarks
per mg.
per mg.
per mg.
per mg.
.....
0.0 0.063 0.0 0.075
o:i5
0.05 0.20 0.065 0.20
0:32 0.0 0.20
0,035
0.0
0.063
0.16
0.22
0.18
..,..
Prior fermehktion: filtered; sulfits 2 mg. per 0 0 . ; 3rd press Prior fermentation. supercentrifuge: sulfite’ 2 mg./cc. Juice screened a t 60° C.: filtered: sulfite 1 mg./cc.
0:25
% recovery based on milk units Before After activaactivation tion 12 0 14
..
13 30b 39 34
0.0
18
32
0.20
51
5SC
as in Table I. as representative of this type of material, and was found t o contain 40% of ammonium sulfate.
(Hammersten) as a substrate, using the same enzyme quantity
aa for its milk-clotting determination. With bromelin, as with papain, the use of herno lobin for a test protein requires the presence of cyanide or a simfiar reducing agent. Otherwise the hemo-
lobin inactivates part or all of the bromelin by oxidation. bromelin solutions capable of digesting casein were found to be inactive toward hemoglobin unless cyanide was present. When such bromelin solutions were mixed with hemoglobin in the absence of a reducing agent and then added t o casein, they were inactive toward the casein but they could be reactivated by the addition of cyanide. The effect of alkali was determined on a suspension of the filter cake in water, and therefore on a mixture of the soluble and insoluble proteins. As Table IV shows, both proteolytic activity and milk-clotting activity decreased greatly when the enzyme was made akaline, and increased again when it was reacidified. The ratio of milk-clotting to proteolytic activity was significantly not changed by the exposure to alkali. There is thus no evidence from these experiments that our protein preparations, made from heated pineapple juice, contain two enzymes of different milk-clotting powers (as
TO PROTEIN-DIGESTING TABLE IV. RATIOOF MILK-CLOTTING ACTIVITYON TREATMENT WITH ALKALI
Treatment 2.5% bromelin suspension adjusted t o p H 2.9 (HC1) (assayed immediately) 2.5% bromelin suspension adjusted t o pH 8.8 (XaOH) assa ed after 1 hr. a t
as.&
A . Increase in Formol Titration Recip’rocal ? after 30&fin. Diges- of Milk-ClotB/A, ting Time, Ratip pf tion, Cc. Activities hhn. 0.01 W XaOH 4.36
1 61a
0.37
1.05
0.12
0.07
After standing 1 hr alkaline suspension resdjuaied t o pH 3.2 (HC1) and assayed immediately
2.73
0 71
0.26
2.5y0 bromelin suspension adjusted,to pH.2.7 (HCI) (assayed immediately)
4.04
1 . 22a
0.30
2 . 5 7 bromelin suspension adjuited t o p H 8.5 (NaOH) (assayed after 1 hr. a t 25’ C.)
1.39
0.12
0.09
After standing 1 hr., alkaline suspension readjusted t o p H 3.0 (HCl) and assayed immediately
3.00
0.71
0.24
Only the ratios found for the ori inal and the reacidified reparations can be compared with assurance. The &we, value of B / A for t t e enzyme while alkaline ia preaumably due t o t h e fact t h a t milk-clotting activity is a straightline function of enzyme concentration (unpublished data) whereas formol titrations made a t a fixed time may not be. Such a situation precludes e comparison of d a t a obtained with the enzyme in two very different activity ranges 0
FIGURE 1. MILK-CLOTTING ACTIVITY AT PH 5.6 OF 2.5 PER CENTPROTEIN SCSPENSIGN PREVIOUSLY HELDAT VARIOCS PH LEVELS
compared to their proteolytic activity), one of which is destroyed by alkali. If such an enzyme exists in fresh pineapple juice, it must be easily destroyed also by heat. It is notable, however, that measurements of both milkclotting and proteolytic activities were made on well-buffered substrates. KOimportant change in pH occurred when enzyme preparations, themselves as acid as p H 2.5 or as alkaline as p H 9, were added to the milk or to the casein solution used for the tests. Because the digestion and milkclotting determinations were respectively made a t essentially the same p H in each instance, i t follows that the activity of the bromelin varied with the pH at which the enzyme stood prior to its addition to the substrate. This peculiar behavior was accordingly investigated over the usual p H range, but since it had already been observed
July, 1941 100
INDUSTRIAL AND ENGINEERING CHEMISTRY
953
The permanent destruction of about half of the enzyme by alkali is shown in the difference between curves I and 11. The remainder of the enzyme, however, is shown by the similarity between curves I1 and I11 to be of nearly the same t activity when brought to the same pH, irrespec70 tive of whether that pH had been approached from u < the acid or from the alkaline side. -I 60 The reversibility of this p H effect suggests the 2 denaturation and reversion of the enzyme protein so !as a possible explanation. This hypothesis is z 40 fortified by the behavior of bromelin on heating, a as shown in Figure 2. By heating for 30 seconds W a 30 from 65' to 80" C. much of the enzyme was inactivated, no matter what the pH. A point of 20 maximum stability was observed near pH 5.2. I n acid and alkaline ranges the destruction of milk(0 clotting activity was practically complete in less than one minute, and did not return in one hour. However, as soon as the pH was brought to about PH 3.2, a noticeable fraction of the activity reappeared in the preparations that had previously FIQWRE 2. STABILITY OF BROMELIN TOWARD HEATAND THE RETURN OF been more alkaline. The return of activity was ACTIV~TY AFTBR HEATINQ practically instantaneous. Strangely enough, 0 Milk-olotting activity of unheated preparations, previously kept at various pH levels. bromelin heated at pH 3.2 (in the range of its 0 Activity of the same reparations immediately after heating to SO0 C. (pR not maximum activity) wa.s permanently lost. materially altered by geating). x Activity of the same preparations after heating and then adjusting the pH t o 3.2 The enzyme acts as though it were reversibly in all cases. denatured at certain pH levels but does not regain its activity until adjusted to an acid p H where the protein is much less stable. It is also possible that values for milk clotting and protein digestion run parallel, that heating the alkaline enzyme simply accelerated the inonly determinations of milk-clotting activity were made. activation produced by the alkali, and there is thus no esI n order to eliminate variations in the amount of natural sential difference between the inactivation observed on activator present, the enzyme was prepared by suspending heating and that reported from the previous experiment where the ammonium sulfate precipitate in water half saturated the enzyme was merely kept a t pH 9. with hydrogen sulfide. Under these conditions it was again The experiment shown in Figure 2 represents the behavior found that the @,ivity of the enzyme varied with the p H a t of the greater part of the original plant enzyme, for the reason which the enzyme was kept prior to its addition to the milk. that fresh pineapple juice behaved in the same manner as the The activity was greatest when it had been previously kept precipitated protein. (Since the fresh juice contained a at p H 2.5-3.6. At more acid levels permanent destruction sulfhydryl activator other than hydrogen sulfide, i t seems occurred. At more alkaline levels partial inactivation took unlikely that a difference in the ionization or in the oxidationplace, the extent of inactivation depending upon the p H emreduction potential of the activator a t various p H levels. ployed; but partial reactivation occurred when the enzyme could have been responsible for the effect.) was readjusted to a more favorable acidity. The change in milk-clotting activity took place rapidly Fresh pineapple juice is usually near pH 3.5; therefore when the pH of the enzyme solution was changed. The the protein is in the range of maximum activity. The application of heat to raw juice thus causes irreversible rather shortest time in which our measurements were made was about 1-2 minutes, and this appeared to be ample. It is therefore than reversible inactivation, and it is evident that any attempt to lessen the acidity of the juice w d d tend to protect curious that the enzyme did not again change its activity on the enzyme'ggainst heating. being introduced into the milk, which was always at pH 5.6. There is a possibility that observations of this type might Literature Cited give considerable information on the nature of enzyme substrate combinations. Evidently, once bromelin has comAnson, M. L., and Mirsky, A. E . , J . Gen. Physiol. 9,169 (1925). B d s , A. K.,and Hale, W. S.,Cereal Chem., 15, 622 (1938). bined with its substrate, its activity is no longer susceptible to Balls, A. K.,and Hoover, S. R., J. Biol. Chem., 121,737 (1937). the effect of the pH. Balls, A. K.,and Lineweaver, H . , Ibid., 130,669 (1939). Neglecting considerable permanent inactivation that ocBalls, A. K.,Swenson, T. L., and Stuart, L.S.,J . Assoc. Oficial curred in alkaline media, the effect of pH on the activity apAgr. Chem., 18,140 (1935). Berger, J., and Asenjo, C., Science, 90, 299 (1939). pears to be reversible. The data are shown in Figure 1. Caldwell, J. S.,Botan. Gaz., 39,409 (1905). A bromelin suspension in half-saturated hydrogen sulfide Chittenden, R. H . , Trans. Connecticut Acad. Sci., 8 (Dec. water was brought from the original pH level (4.3) to pH 2.3. 1891); J. Physiol., 15, 249 (1894). Separate portions of this suspension were then brought to the Davis, W. B., Food Research, 4, 613 (1939). Eckart, T. G., and Cruess, W. V., Fruit Products J.. 10, 364 desired p H levels with alkali, and the milk-clotting activity (1931). measured as usual (curve I). A similar suspension was made Northrop, J. H., J . Gen. Physiol., 16,41(1932). alkaline (pH 9) ; then separate portions were brought to difRussell, J. B., Bull. Pharm., 5, 77 (1891). ferent pH levels by adding acid, and the milk-clotting acVines, S.H., Ann. Botany, 17,597 (1903). WillstBtter, R., Grassmenn, W., and Ambros, O., 2. physiol. tivity was determined (curve 11). The same suspension Chem., 151, 286 (1926). kept at p H 9 for 90 minutes longer was then made acid (pH Winnick, T., Davis, A. R., and Greenberg, D. M., J . Gerr. 2.3), separate portions were again removed, and each portion Physwl., 23, 275 (1940). was brought t o a desired p H with alkali. The milk-clotting CONTRIBUTION 61 from the Enayme Research Laboratory, Bureau of Agriactivity is shown in curve 111. cultural Chemiitry and Engineering. 90
e
