l ‘l M “1 | lki I I‘I‘MJ‘J‘ " \j (I “IN “H V m NM "i! H! \ > “i L‘ in l,‘ ‘ x l‘ m .1 1 IWI ' H I‘ ; jl W tl ‘ l > M 144 865 ‘z‘HE EFFECT OF SULFUR DIOXIDE ON THE @EGERADATRCN OF THE TART CHERRY ANTHOCYANEN BY TYROSNASE “19325 for the Degree of M. S. MECEISAN STATE UNEVERSITY Louis P. Goodman 1963 LIBRARY Michigan State . University AN ABSTRACT THE EFFECT OF SULFUR DIOXIDE ON THE DEGRADATION OF THE TART CHERRY ANTHOCYANIN BY TYROSINASE BY Louis P. Goodman This study was carried out using both red tart cherry (Prunus cerasus L. var. Montmorency) juice and the chromato- graphically and electrophoretically purified mecocyanin pig- ment isolated from this juice. The degradation of the pig- ment was measured Spectrophotometrically.w The rate of the degradation of the mecocyanin pigment by tyrosinase, in a citrate-phOSphate buffer, at pH 6.5, decreased rapidly as the concentration of sulfur dioxide was increased. The half life of the mecocyanin pigment, degradation at pH 6.5, with no sulfur dioxide added, was 40 seconds. There was almost no decolorization at the level of 8 ppm sulfur dioxide during the two-minute period of observation. Cherry juice, at pH 6.5, exhibited the same trend as the mecocyanin pigment, but 25 ppm of sulfur dioxide were required to inhibit the reaction for the 75 minutes of observation. The half life of the cherry juice anthocyanin, at pH 6.5, with no added sulfur dioxide was 8 minutes. At lower pH levels than 6.5, less sulfur dioxide was required to inhibit the decolorization reaction both in the purified pigment system and in the cherry juice. Louis P. Goodman Preincubation of enzyme and sulfur dioxide at pH 6.5, before addition of the purified pigment or the juice, further decreased the subsequent rate of decolorization. Again, a higher concentration of sulfur dioxide in the preincubation enzyme-sulfur dioxide mixture was required for the juice than for the pure pigment. Experiments conducted-with model systems using sulfur dioxide to inhibit the enzymic oxidation of tyrosine and catechol showed that the rate of oxidation by tyrosinase of both tyrosine and catechol decreased as the concentration of sulfur dioxide was increased. Complete inhibition of both the catechol and tyrosine oxidation was obtained with 4 ppm sulfur dioxide. If the enzyme and the sulfur dioxide were preincubated, lower levels of sulfur dioxide were re— quired for inhibition of the reaction than without pre- incubation. THE EFFECT OF SULFUR DIOXIDE ON THE DEGRADATION OF THE TART CHERRY ANTHOCYANIN BY TYROSINASE By Louis P. Goodman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree bf MASTER OF SCIENCE Department of Food Science 1963 1géfnuvctlvi (EzszéZ; 4gaakufliéuéé; IN MEMORY of my mother Helen Goodman ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. Pericles Markakis for his guidance, aid, and en- couragement during the research for this project and in the preparation of this manuscript. He is deeply indebted to the following people: to Dr. Clifford L. Bedford for his beneficial suggestions and advice; to Dr. Donald H. Dewey for his help in the preparation of this manuscript; to Mr. Chung-Yen Peng for his assistance in the necessary techniques used in this experiment; and to Dr. Clarence H. Suelter, of the Department of Biochemistry, for the use of his equipment. The author also expresses his appreciation to the United States Public Health Service for the financial support of this project. iii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . METHODS AND MATERIALS . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . LITERATURE CITED . . . . . . . . . . . APPENDIX . . . . . . . . . . . . . . . iv Page 17 29 31 34 Figure 1. LIST OF FIGURES Effect of various levels of $02 con- centration on the rate of mecocyanin degradation by tyrosinase . . . . . Effect of various levels of $02 con- centration on the rate of cherry juice anthocyanin degradation by tyrosinase . . . . . . . . . . . . . Effect of various levels of 502 con- centration on the rate of tyrosine oxidation by tyrosinase . . . . . . Effect of various levels of 502 con- centration on the rate of catechol oxidation by tyrosinase . . . . . . Page 18 22 26 27 INTRODUCTION Enzymatic degradation of the anthocyanin pigments found in red tart cherries (Prunus cerasus, L. Var. Mont— morency) is one of the major causes of loss in color of the fruit. The discoloration, known as ”scald” in the cherry industry, is exhibited on the fruit by the appearance of a light colored area on the cherry skin. In severe scald these areas turn brown. Van Buren ££_§l. (27) showed that the anthocyanin pigment, in the areas of the cherry which ex- hibited scald, was actually decolorized by an enzyme present in the cherry. An enzyme Specifically attacking anthocyanins_ has not yet been found. However, anthocyanins are poly- phenols and polyphenolases are widely distributed in the plant kingdom without being very substrate-specific. There- fore, the enzyme chosen for this work was a commercial mush- room polyphenolase which Peng (20) showed decolorized the purified anthocyanin pigment of cherries. Bedford (13) has subsequently shown that polyphenolase activity is present in red tart cherries. Sulfur dioxide (502) has been widely used in the food industry both as a microbial inhibitor and for the preser- vation of color in fruits (1,5,7,lO,12,13,l6,26,28,29). It is also used extensively in the Maraschino cherry industry for bleaching the natural cherry pigments (4). 1 The purpose of this study has been to investigate the effects of sulfur dioxide on the degradation of the tart cherry anthocyanin pigments by a mushroom tyrosinase. REVIEW OF THE LITERATURE Enzymatic Degradation of Anthocyanins The enzymatic degradation of anthocyanin pigments from various fruits and flowers had been studied by a number of workers. In 1917, Nagai (l8),working with the anthocyanin from Chrysanthemum, showed that the plant itself contained an oxidase which decolorized the anthocyanin of the flower at room temperature. The same result was produced by dilute solutions of hydrogen peroxide. Boiled plant juice did not exhibit this decolorization action. Continuing his work, in 1921 Nagai (19) showed that the red anthocyanin pigments of scarlet Papaver were destroyed by a peroxidase obtained from soybean seedling hypocotyls and rootlets. The decolorization of blackberry anthocyanins was studied by Huang (8) in 1955, who used crude enzyme extracts from Aspergjlli. He found that the decolorization was rapid at 30°C. over a pH ranging from 3.0 to 4.5. The antho- cyanins were hydrolyzed by the enzyme to anthocyanidin and sugar followed by a Spontaneous transformation of the aglycone-to colorless derivatives. This enzyme acted as a glycosidase. In 1956, Huang (9) studied the kinetics of the de- colorization of Chrysanthemum anthocyanin by a fungal 3 anthocyanase at 30°C. and pH 3.0. An apparent first order rate constant for the enzymic hydrolysis of the glucoside was shown. An enzyme extracted from the leaves of Coleus hybridus by Bayer and Wegmann (2),in 1957, was capable of degrading the anthocyanin of red roses to yellow colored products. The enzyme, which they called cyaninoxidase, had an optimum activity at the pH range of 7.0 to 7.5. Catechol and oxygen were necessary for the enzymic reaction to occur. Van Buren g£_al. (27),in 1959, reported the presence of an oxidizing enzyme isolated from Montmorency cherries. Using the cyanidin 3-rhamnoglucoside of cherries as a sub- strate, they found that this anthocyanin was decolorized by the enzyme upon the addition of catechol. Scheiner (24), in 1961, showed that a crude enzyme preparation obtained from cherries decolorized the cherry .anthocyanins. He found that catechol oxidase activity and anthocyanin decolorizing activity followed each other closely during the purification and that catechol was oxidized by all the preparations that decolorized anthocyanins. Purified preparations of the enzyme had little decolorizing effect on the anthocyanin unless catechol or some other o-dihydroxy- phenol compound was present in the reaction mixture. p-Benzoquinone and o-benzoquinone, the first oxidation pro- ducts of the catechol-catechol oxidase system, decolorized the anthocyanins non-enzymatically. Peng (20), in 1962, used a commercial preparation of mushroom tyrosinase and showed that it decolorized the meco- cyanin 3-gentiobioside anthocyanin pigment from cherries when catechol was added. The Use of Sulfur Dioxide to Inactivate Enzymes of Fruits The enzyme-catalyzed oxidative browning of fruit and fruit products was reviewed by Joslyn and Ponting (14) in 1951. This review was continued by Joslyn and Braverman (15) in 1954. More recently (1959), Ponting used Spectro- photometric methods to Show the effect of 502 on the poly- phenol oxidase activity when different concentrations of 502 were added to a buffered catechol solution. The enzyme was added last. Even one part per million (ppm) 502 caused a significant drop (20%) in activity. Ten ppm 802 inactivated the enzyme almost completely. His studies with apple juice containing added catechol showed that if five ppm of 502 were added to the apple juice after oxidation had proceeded long enough to form quinones, the 802 reacted instantaneously with the quinones to lighten the color of the juice as was shown by a drop in optical density. It had no effect on the activity of the enzyme, presumably because the 502 had been oxidized before it had a chance to react with the enzyme. From these two experiments, Ponting concluded that polyphenol oxidase was very sensitive to $02, but to be most effective, the 502 had to react with the enzyme before any enzymatic oxidation occurred. Diemair §£_§l. (6) in 1960, found that there was no direct relationship between enzymatic activity in grape juice used for wine and the amount of 502 required to inactivate it. They showed that the inactivation was reversible and was not connected with the oxidation of H2503 to H2804, Preincubation of 802 and enzyme decreased the activity of the enzyme as measured by the purpurogallin test. Sastry §t_al, (23),in 1961, added peroxidase obtained from the fruit of the custard apple to various quantities of potassium metabisulphite and let the two incubate for ten minutes. After the addition of guaiacol and hydrogen per- oxide, a gradual decrease in the development of browning was noted with increasing $02 concentrations. A concentra- tion of 350 ppm of $02 completely inhibited both color pro- duction and enzymic activity. The purpurogallin test was I used to determine enzymic activity. METHODS AND MATERIALS Preparation of Purified Anthocyanin Frozen, pitted Montmorency cherries were partially de- frosted and placed in boiling 95 per cent (v/v) ethanol in such a proportion as to achieve a 70 to 75 per cent (v/v) final ethanol concentration. The mixture was boiled for five minutes and allowed to cool. This treatment extracted the anthocyanins, inactivated the enzymes, and precipitated the pectins. The mixture was filtered through a milk filter and the filtrate was concentrated under reduced pressure in a rotatory flash evaporator thermostated at 37°C. The concentrated anthocyanin extract was applied to a 2 x 10 cm. column of Dowex 50W—X8 (100 to 200 mesh, H+ form) resin which retained the pigments along with other basic components of the extract. The column was eluted with 50 to 100 m1. of 0.2 HCl in methanol. The eluate was concentrated in vacuo and applied as a narrow band to Whatman 3 MM paper. The paper was placed in a chromatography cabinet and irrig- ated ascendingly with l N acetic acid for 25 to 35 minutes. At the end of this time the two Montmorency cherry antho- cyanins appeared as well separated zones. These zones were cut out and eluted separately with methanol containing a trace of concentrated hydrochloric acid. 7 The eluate of the pigment with the higher Rf value (mecocyanin pigment) was concentrated in vacuo and further purified by zone electrophoresis. A Reco Model B apparatus was used for this purpose. Whatman cellulose powder, stand- ard grade, was made into a past with l N acetic acid solution. A filter paper long enough to connect the two electrode vessels, containing 1 N acetic acid, was placed over the plate of the apparatus. The cellulose paste was then Spread evenly over the paper. Pigment was applied as a narrow band on the paste at five places across the electric field and 700 volts of direct current, approximately 50 milliamperes, were applied for about six hours. At the end of this time the pigment had moved 2 to 3 cm. The colored zones of the cellu- lose paste were removed and eluted with methanol. The eluate was concentrated in vacuo and the dry pigment was dissolved in water. Preparation of Cherry Juice Frozen, pitted Montmorency cherries were partially de- frosted and pressed through a cheesecloth to extract the juice. The juice was heated in 1-1/2 x 15 cm. test tubes for 3 to 4 minutes in boiling water, with constant shaking, to inactivate the enzymes. It was cooled and filtered through No. 2 Whatman filter paper using a Buchner funnel and suction. It was then diluted with demineralized water using approximately one part water to three parts juice. Preparation of Enzyme The tyrosinase preparation used in this study was ob- tained from Worthington Biochemical Corp., Freehold, New Jersey. Its activity was found to be 417 units per mg. of dried enzyme. A unit of tyrosinase activity equals an in- crease in absorbance of 0.001 per minute under the Specified conditions of the assay. Yasunobu (30) distinguished between a true tyrosinase and polyhenoloxidase in that the former catalyzed the oxid- ation of both mono and diphenols while the latter only catalyzed the oxidation of o-diphenols. The enzyme used in this study of pigment degradation was a true tyrosinase which exhibited a low substrate Specifity. The stock solution consisted of 10 mg. of enzyme di— luted in 10 ml. of water. No subsequent dilutions were made for the work on the purified pigment and cherry juice. It was necessary, however, to make dilutions of the stock solution for experiments involving the inhibition of tyro- sinase by sulfur dioxide using catechol and tyrosine as substrates. Preparation of Sulfur Dioxide $02 was generated from a 5 per cent sodium bisulfite solution by adding concentrated sulfuric acid. The gas was bubbled into a flask containing boiled distilled water which had been cooled. Boiling removed air dissolved in the water, while cooling increased the solubility of 802. The stock 10 solution contained approximately 1700 ppm 502. Due to oxidation of the sulfurous acid when the container was opened, the stock solution had to be titrated with 0.02 N iodine, us- ing starch as an indicator, every four or five days to deter— mine the exact 502 concentration. The required dilutions were made from the stock solution. Other Reagents Three buffer solutions were employed. The reaction mixture contained a buffer consisting of equal volumes of 0.1 M citric and 0.1 M phOSphoric acids adjusted to the de- sired pH level with sodium hydroxide at the time of preparing the reaction mixture. The two other buffers employed were 1.0 M citric acid solution adjusted to pH 2.0 and 7.0, reSpectively, with sodium hydroxide. These were used in the determination of anthocyanin concentration by means of differential color development, as will be explained later (p. 12), A 0.1 M solution of potassium cyanide was used to stop the enzymic reaction at the necessary time intervals during the reaction. A 0.2 mM catechol solution was used to accelerate the enzymic reaction causing pigment degradation as shown by Peng (20). Mecocyanin and Cherry Juice Enzyme, catechol, and 502 were allowed to react in a citrate-phOSphate buffer with either the purified 11 mecocyanin pigment, or cherry juice and the progress of the reaction was followed colorimetrically in a Beckman D U Spectrophotometer. Two factors affecting the reaction were studied: 802 concentration and pH. Temperature, enzyme con- centration, and catechol concentration were kept constant. The pigment concentration in the reaction mixture differed within_:lSper cent, but it was found that these differences did not affect the degradation rate of the pigment. In the case of cherry juice, 1.0 ml. of water was added instead of catechol, since the juice contained enough phenolic compounds to act as substrates for the enzymic reaction. The reaction was carried out in a 30 ml. beaker. The mixture consisted of the following: 3.0 ml. of the citric- phOSphoric acid mixture, 1.0 m1. of 0.2 mM catechol, and 1.0 m1. of the mecocyanin solution or cherry juice. The pH was adjusted to the desired level by adding 4 N sodium hydroxide solution from a graduated 1.0 ml. pipette and noting the quantity of base added. At this point 1.0 m1. of SO2 solu- tion was added followed by distilled water to make a total volume of 6.8 ml. A 0.5 ml. aliquot of the reaction mixture was transferred to a test tube containing 2.5 ml. of the citrate buffer at pH 2.0 and 0.1 ml. of 0.1 M potassium cyanide, and another 0.5 ml. of the reaction mixture to a tube containing 2.5 ml. of the citrate buffer at pH 7, and 0.1 m1. of the potassium cyanide solution. These tubes served for determining the anthocyanin concentration at zero time. Following the removal 12 of these 0.5 ml. aliquots, 0.2 m1. of the enzyme solution was added to the remaining 5.8 m1. of the mixture and a stop watch was started. At various time intervals, two 0.5 m1. aliquots of the reaction mixture were transferred to tubes containing the citrate-cyanide solutions. After a 15-minute development time, at room temperature (22 to 25°C.), the contents of these tubes were transferred to one cm. matched cuvettes for absorbancy measurements in a Beckman D U Spectrophotometer at the wavelength of 520 mp for the mecocyanin pigment and 518 mp for the cherry juice. The pH 2 buffer was used as a blank. The concentration of the mecocyanin and the cherry juice were expressed in absorbancy difference between pH 2 and pH 7. Sondheimer and Kertesz (25), working with strawberry products, showed that the difference in absorbancy at two pH levels was a more accurate measurement of the antho- cyanin concentration than the absorbancy at one pH when interferring substances were present. The main source of interference in the system presently used came from browning products. The absorbancy at 520 mp of these brown products did not change with pH and cancelled out when the difference of absorbance at two pH levels was calculated for the re- action system. The zero time reading was multiplied by a factor of 0.966 to correct for the dilution of the reaction mixture by the addition of enzyme, after the removal of the two 0.5 ml. l3 aliquots. All subsequent values were then subtracted from the corrected zero value to give absorbancy units of destroyed pigment. Preincubation of Tyrosinase and Sulfur Dioxide using the Mecocyanin Pigment and Cherry Juice The preincubation of tyrosinase and $02 was carried out in a 5 ml. beaker, with constant stirring, for 30 minutes. To 0.8 m1. of an enzyme solution, containing 1.0 mg. tyrosinase/m1., 4.0 ml. of a 13.2 ppm 502 solution was added. The final concentration of $02 in this mixture was 11 ppm. During the preincubation period the reaction mixture was prepared by adding 1.0 ml. of the purified mecocyanin pigment, 1.0 ml. of 0.2 mM catechol, and 3.0 ml. of the citrate-phosphate buffer to a 30 ml. beaker. The pH was ad- justed with 3 N sodium hydroxide to 6.5, noting the amount added, and distilled water was added to make a total volume of 5.8 ml. From the above reaction mixture, two 0.5 ml. aliquots were pipetted into the pH 2 and pH 7 developing tubes, previously described. At zero time, 1.2 ml. of the enzyme- 502 mixture was pipetted into the remaining 4.8 ml. of the reaction mixture and a timer was started. At 15 minute intervals, two 0.5 m1. aliquots were pipetted into the pH 2 and pH 7 developing tubes. The readings were taken after a 15 minute developing time, measuring the absorbancy at 520 mp in a Beckman DLJSpectrophotometer. The final concentration 14 of $02 in the reaction mixture was 2.5 ppm. Preincubation experiments with cherry juice were carried out in the exact manner as indicated above, with the exception that the SO concentrations were changed to 11.7 2 ppm in the enzyme-$02 mixture and 2.9 ppm in the final re- action mixture for the first experiment and to 16.8 ppm and 3.2 ppm for the second experiment. The zero time reading was multiplied by a factor of 0.8 to correct for the dilution of the reaction mixture by the addition of the 1.2 m1. of enzyme—502 mixture. Tyrosine-Tyrosinase-Sulfur Dioxide These experiments were conducted with a Beckman D U Spectrophotometer equipped with a Gilford cuvette changer and automatic recorder. The reactions were carried out in one cm. matched cuvettes. Two reactions and a blank were run simultaneously. All three cuvettes contained 1.0 ml. of loO mM tyrosine, and 1.0 m1. of 0.5 M phOSphate buffer, pH 6.5. The blank (first) cuvette contained 0.9 ml. of $02 solution and 0.1 ml. of water. The second cuvette contained 0.9 m1. of distilled water, while the third cuvette contained 0.9 m1. of 502 solution of the same concentration as the blank. To the second and third cuvettes, 0.1 m1. of enzyme solu- tion, containing 0.5 mg. tyrosinase/ml. were added simul- taneously. The recorder was started and the reaction was followed at the wavelength of 280 mp. 15 Catechol-Tyrosinase-Sulfur Dioxide These reactions were carried out in one cm. matched cuvettes. Two reactions and a blank were run simultaneously. The blank (first) cuvette contained 1.0 ml. of 0.5 M phos- phate buffer, pH 6.5, 1.1 m1. of water, and 0.9 ml. of 502. The second cuvette contained 1.0 ml. of buffer, 1.0 ml. of catechol, and 0.9 ml. of 502 solution of the desired concen- tration. To the second and third cuvettes 0.1 m1. of enzyme solution, containing 0.1 mg. enzyme/m1. were added simultane- ously at zero time. The recorder was started and the re- action was followed at the wavelength of 420 mp. Various 802 concentrations were employed, using 0.2 mM catechol as a sub- strate. Preincubation Experiments with Tyrosinase and Sulfur Dioxide Using Tyrosine and Catechol For these experiments 1.8 ml. of 802 solution of the desired concentration was allowed to incubate in a 5 ml. beaker with 0.2 ml. of enzyme solution, containing 0.5 mg. tyrosinase/ml. for 30 minutes, with constant stirring. After the allotted time, 1.0 ml. of the mixture was transferred to a cuvette containing 1.0 ml. of 0.5 M phOSphate buffer, pH 6.5, and 1.0 ml. of 1 mM tyrosine. The reaction was followed at the wavelength of 280 mp. These results were compared with the no preincubation experiments. Preincubation experiments were also conducted using 0.2 mM catechol instead of tyrosine as the substrate. In this case the enzyme solution was diluted to give 0.01 mg. 16 enzyme/ml. water. The reaction was followed at the wave- length of 420 mp. These results were also compared with the no preincubation experiments. RESULTS AND DISCUSSION Mecocyanin Pigment The results on the effect of $02 concentration on the rate of mecocyanin degradation by tyrosinase at various pH levels are presented in Appendix Tables I, II, and III, and they are graphically summarized in Figure 1. Presented in tabular form are the absorbancy readings at the two pH levels used for the estimation of anthocyanin concentration, their difference corrected for the dilution by the enzyme addition, and the amounts of pigment destroyed in absorbancy units. From these data graphs were drawn (not shown) to determine the initial rate of pigment destruction per minute, in absorbancy units. These rates were then plotted in Figure l as the rate of mecocyanin decolorization versus 502 concen- tration in ppm. The rate of mecocyanin decolorization, at pH 6.5, was highest with no 502 present. The reaction rate decreased slightly with 2 ppm 802. At a concentration of 4 ppm 502, the decolorization rate decreased to approximately one—third the rate as compared to the no 802 level. Very little de- colorization occurred at the 6 and 8 ppm 502 levels. At pH 5.5, the highest rate of mecocyanin degradation occurred with no 802 present. Using a concentration of 4 17 Rate of Mecocyanin Degradation (A520 mu, pH 2 - A520 mu, pH 7) min. -1 .09 .080. .070 .060 .050 .040‘ .030 .020 —» .010 - pH 4.5 I W 502 Concentration (ppm) Figure 1. Effect of various levels of 802 concentrationg - on the rate of mecocyanin degradation by tyrosinase. 19 ppm 502 the reaction rate decreased to approximately one- quarter of the rate at the no 802 level. A level of 6 ppm 502 showed a very low decolorization rate, while 8 ppm 502 stopped the reaction completely for the period of two minutes during which the reaction was followed. The reaction was completely inhibited by 6 ppm 502 at pH'4.5. While the concentration of 502 played an important role in determining the rate of mecocyanin degradation at any one pH level, it is evident from Figure 1 that pH also played an important role in determining the amount of enzymic decoloriz- ation of the pigment. Less 802 was needed to decrease the decolorization rate at the lower pH levels. Tyrosinase has been shown to have an optimum pH range of 5.0 to 7.8 (30). Yasunobu (30) showed that the variation of activity with pH is due to a change of ionization of the enzyme and not the substrate (catechol), since the substrate did not ionize in the pH range studied (pH 5 to 7.8). Ingraham (ll) pointed out that at pH levels much below 4.0, the tyrosinase enzyme was not active. This work is in agreement with Ingraham's. To show that 802 at the concentration level of 10 ppm could inhibit the enzymic destruction of the mecocyanin pig— ment, at the Optimum pH 6.5, for longer periods than two minutes, the reaction was followed for 75 minutes. No de- colorization was observed during this extended period (Table I). 20 Preincubation experiments of enzyme and 502 showed that 11 ppm of $02 in the enzyme-802 mixture was adequate to inhibit the subsequent mecocyanin decolorization, at pH 6.5, even though the concentration of $02 in the reaction mixture was only 2.5 ppm (Table IV). The reaction was followed for one hour. From this it can be concluded that the enzyme was quite sensitive to 802' Comparing the results of the no preincubation experi— ment at pH 6.5, and 2.5 ppm of 502, shown in Figure l, with the results of the preincubation experiment at pH 6.5 and 2.5 ppm 502 shown in Table IV, it is apparent that there was no decolorization of the mecocyanin pigment with preincub- ation while there was a decolorization rate of 0.063 absorb- ancy units per minute with the no preincubation experiments. The question of other substances in the reaction mix- ture, competing for the 502 with the enzyme, or reacting with it, now becomes evident. The proposed mechanism for the destruction of anthocyanin pigment was that tyrosinase with oxygen oxidased the catechol to o-benzoquinone, and the quinone destroyed the pigment non-enzymatically (24). Pont- ing (21) has shown in his work on apple juice that if 802 was added after some o-benzoquinone had formed, the 502 was readily oxidized by the o-benzoquinone and browning resulted. Extending this reasoning, if 802 is present from the begin- ning of the reaction in very small quantities, o—benzoquinone may be formed and oxidize the 502 before the latter has the chance to inactivate the enzyme. Larger quantities of 802, 21 however, may inactivate the enzyme even if part of the former had been oxidized by any o-benzoquinone that may have been formed. Cherry Juice The results of experiments with cherry juice are given in Tables V, VI, VII, VIII, and Figure 2. Before complete inhibition of the enzyme resulted in cherry juice, the SO2 concentration had to be tripled over that used in the pure mecocyanin experiments. This was probably due to sugars and other carbonyl compounds present which combined with 502 (7). Since it is only the free $02 which inactivates the enzyme, more $02 was required to compensate for that 502 bound by the sugars and other carbonyl compounds. The highest rate of anthocyanin decolorization occurred at pH 6.5 with no 802 present. At a level of 5 ppm of 802 the decolorization rate was decreased to approximately one- half of the rate at the no SO2 level. Concentrations of 15 and 20 ppm 502 further decreased the decolorization rate, and 25 ppm 502 stopped the reaction completely for the 75 minutes of observation. The results at pH 5.5 were not very different from those obtained at pH 6.5. A possible explanation for the similarity of these data is that there is a broad pH optimum range for the tyrosinase oxidation of the juice substrates and pH 5.5 and 6.5 are within this range. Rate of Anthocyanin Degradation (A518 mp pH 2 - A518 mp pH 7) min.”1 22 .014 .012 O H O O O 00 O O O‘ O O b .002 fw. 5 i _ l L 0 5 10 15 20 25 502 Concentration (ppm) Figure 2. Effect of various levels of $02 concentration -on the rate of cherry juiceianthocyanin degradation by tyrosinase. 23 At pH 4.5, the highest rate of anthocyanin decoloriz- ation was obtained with no 502 present. At a level of 2 ppm 502 the decolorization rate decreased to one-third the rate of the no 502 level. A concentration of 5 ppm 502 decreased the rate even further, while 10 ppm 502 almost completely inhibited the degradation when the reaction was followed for 75 minutes. At pH 3.5 duplicate results were difficult to obtain since the decolorization reaction was very slow. An average of four runs were taken to obtain the results of the no 802 level. A concentration of 5 ppm 502 almost completely stopped the reaction when followed for 75 minutes. What is meant by complete inhibition holds true only for the time of observation. In cases where 25 to 30 ppm 502 were added to the juice, at pH 6.5, complete inhibition of the degradation reaction was observed for the 75 minutes of observation. However, when 30 ppm 802 was added to the juice, at pH 6.5, and the reaction was followed for six hours, a small but noticeable decolorization rate of 0.004 absorbancy units per hour was obtained after the second hour of observation (Table V). The no 302 sample at pH 6.5 showed a rate of 0.014 absorbancy units per minute during the ten minutes of observation. During the above experiments, two blanks were also followed for six hours. One blank contained the usual re- action mixture containing 802, except that water was added in place of enzyme. This blank showed that no decolorization 24 took place due to the direct effect of 802 on the pigment even after six hours of observation. This may appear contrary to the common experience of SO2 bleaching of fruits pigmented with anthocyanins. It appears, therefore, that a certain minimum quantity of $02 is necessary for the bleach- ing effect to occur and the concentrations of 502 used in this work were below this minimum. The second blank contained no 502 and no enzyme. Water replaced the latter two sub- stances. This blank showed that there was no decolorization due to non-enzymatic degradation of the juice during the six hours of observation (Table 5). Preincubation experiments with enzyme and 502 for the cherry juice, showed that 11.7 ppm of $02 in the enzyme-SO2 mixture did not stop the subsequent reaction in the final reaction mixture which contained 2.9 ppm 802. This was in contrast to the results obtained with the mecocyanin pig- ment. It took 16.8 ppm 802, in the enzyme-$02 mixture, to stop the reaction in the final mixture which contained 3.2 ppm 502, when the reaction was followed for 75 minutes. These results indicated that even though the enzyme was pre- incubated with $02 for 30 minutes, the final reaction de- pended upon the substances present in the reaction mixture. Since the cherry juice contained sugars and other carbonyl compounds, it may have been these substances which "pulled" the $02 away from the enzyme. Results appear in Table IX. 25 Tyrosine-Tyrosinase-Sulfur Dioxide Results of these experiments are presented in Figure 3. At an 502 concentration of 3.6 ppm, the reaction was com- pletely stopped when followed for 75 minutes. At lower levels, there was a lag period followed by a small increase in the re- action, and finally the reaction proceeded as with the no 802 sample. The straight line portions of the graph then began to level off (this portion not shown) and became parallel to the time axis. The reaction rates were almost the same for the 0.9 and 1.8 ppm SO2 levels. Lag phases of the graph may be explained as was previously stated for the mecocyanin pig- ment. If there was not an excess of 802 to completely in- hibit the enzyme, then some DOPA quinone was formed which oxidized the 802 during the lag phase. When enzyme and SO2 were preincubated with 5.4 ppm 502 in the enzyme-$02 mixture, and 1.8 ppm 802 in the final reaction mixture, no reaction took place when followed for 40 minutes. Catechol-Tyrosinase-Sulfur Dioxide Results of these experiments appear in Figure 4. At an 502 concentration of 4 ppm, the reaction was completely in- hibited when followed for 30 minutes. At lower concentrations a lag phase was noticed and then the reaction proceeded. In contrast to the experiment with tyrosine, the reaction rates were not the same for all levels of SO2 concentrations. A.mm:fionooou 6MPHEOpsm scum czmupomv .A.Uoom .NOm Ema o.m one .w.H .o.o .o ..HE\ommcwmouuu .me m.o .ommcfimou>u 28 Av omwcflmou>u >9 coapdpaxo - Honooumo mo open may no cowuwuucoocoo Now mo mHo>oH mzowud> mo «oommm .m ousmflm 26 Ammpsefizv mafia we Om mm om ma OH filllluuu-u _ _ _ _ m Om.EQQ ital: v0.0 6 m ma.o o .o mow sag m.H m NOm and 6.0 Now and o wm.o .om.o V dw 088 notieptxo BSBUISOJAL go 3123 27 A.mmcw6nooou uwuweopsm scum czmuo -mmv .A.oomm .mOm and e 6:“ .m .m .H .o ..HE\mmR:Hmou>p .me Ho.o .Honooumo SE m.ov ommefimouxu >9 COMDmoaxo Honoopdo mo opmu may no coflpmupnoucoo Now mo mHo>oH m30flud> mo poommm .e ousmflm Amoudcaev mefiH Om OH w o v N Inul - d u _ . II I“ mOm sad M. New and m mom and A Now and o UOIIEPIXO TOHDGJPO I0 9123 28 Preincubation of enzyme and 502 with 5.4 ppm 802 in the enzyme-SO2 mixture, and 1.8 ppm in the final reaction mixture gave no reaction when followed for 30 minutes. SUMMARY AND CONCLUSIONS The effect of various concentrations of 802 on the enzymic decolorization of the purified mecocyanin pigment was studied at various pH levels. At pH 6.5, which was in the optimum range of the reaction, the rate of enzymic decoloriz- ation of mecocyanin was decreased rapidly as the concentra- tion of $02 was increased to 8 ppm. The decolorization rates at the lower pH levels of 5.5 and 4.5 with no SO2 added, were less than the rate at pH 6.5. The same trend of decreased mecocyanin decolorization as the concentration of $02 was increased to 8 ppm was noticed for the lower pH levels. Preincubation of $02 with enzyme further decreased the degradation rate of the mecocyanin pigment in a model system. In studying the effects of various concentrations of 802 on the enzymic decolorization of the cherry juice at various pH levels, the same trend was noticed as with the purified mecocyanin pigment. However, the Concentration of 802 had to be increased to 25 ppm before inhibition resulted during the period of observation. More $02 was required for the cherry juice presumably because of the reaction of SO2 with carbonyl compounds of the juice. 29 30 If the enzyme and 502 were preincubated, lower levels of 502 had to be added to the juice in order to obtain in- hibition similar to that of no preincubation experiments. The enzymic oxidation of tyrosine by tyrosinase was shown to proceed rapidly with no 502 present. At a concen- tration of 3.6 ppm 502, the reaction was completely in- hibited for the time period during which the reaction was followed. Lower levels of S02 inhibited the reaction for a short period of time, and then the reaction proceeded at the same rate as the no 802 reaction. Preincubation of enzyme and 502 showed that the subsequent enzymic oxidation of tyrosine was completely inhibited for the time period during which the reaction was followed. Experiments with catechol showed that a concentration of 4 ppm SO2 completely stopped the enzymic oxidation of catechol for the time period observed. Lower levels of SO2 inhibited the reaction for a short period of time and then the reaction proceeded at different rates depending upon the concentration of 502 used. Preincubation of enzyme and 802 showed that the subsequent enzymic oxidation of catechol was completely inhibited for the time period observed. LITERATURE CITED 1. Arthur, J. M. and M. S. Benjamin. Preservation of whole fruit with sulfur dioxide. Agr. Gaz. N.S. Wales. 38:873-5. (1927). 2. Bayer, E. and K. Wegmann. Enzymic degradation of antho- cyanins. Z. Naturforsch. 12B:37-40. (1957). 3. Bedford, C. L. Unpublished data and private communic- ation. Department of Food Science. Michigan State Univ., East Lansing, Mich. (1963). 4. Bullis, D. E. and E. H. Wiegand. Bleaching and dyeing Royal Anne cherries for Maraschino or fruit salad use. Ore. Agr. Expt. Sta. Bull. 215:3-29. (1931). 5. Dickinson, D. and C. L. Hinton. Fruit preservation with sulfur dioxide. Food Sci. Abstr. 28. Abstr. No. 1584. (1956). 6. Diemair, W., J. Koch, and D. Hess. Influence of sul- furous acid and L-ascorbic acid in wine-making. II. Inactivating the polyphenoloxidase. Z. Lebensm.- Untersuch, u. Forsch. 113:381-7. (1960). (In German7 7. Heintze, K. Behavior of sulfur dioxide in fruit pre— serve. Deut. Lebensm.-Rundschau 58:224-7. (1962). (In German) 8. Huang, H. T. Decolorization of anthocyanins by fungal enzymes. Jr. Agr. Food Chem. 3:141-6. (1955). 9. . The kinetics of the decolorization of anthocyanins by fungal anthocyanase. J. Am. Chem. Soc. 18:2390-3. (1956). 10. Hussein, A. A., E. M. Mrak, and W. V. Cruess. Effect of pretreatment and subsequent drying on the activity of grape oxidase. Hilgardia 14:349-57. (1942). ll. Ingraham, L. L. Polyphenol oxidase action at low pH values, in Gorden, M., ed., Pigment Cell Biology, Academic Press, Inc., N. Y., p. 609. (1959). 31 32 12. Johnson, G. and D. K. Johnson. Natural flavor retained in new frozen uncooked apple pulp. Food Tech. 6: 242-5. (1952). 13. Joslyn, M. Color retention in fruit products. Ind. EngJ Chem. §§:242-5. (1952). l4. and J. D. Ponting. Enzyme-catalyzed oxid- atIve browning of fruit products, in Mrak, E. M. and G. F. Stewart, eds., Advances In Food Research, Vol. 3,-Academic Press Inc., N. Y., pp. 1-44. (I951). 15. and J. B. S. Braverman. Sulfur dioxide treat- ment of fruit and vegetable products, in Mrak, E. M. and G. F. Stewart, eds., Advances in Food Research, Vol. 5,-Academic Press, Inc., N. Y., pp. 127-9. (1954). l6. Kyzline, V., D. Curda, and.M. Curdova. Discoloring of fruit preserves and effect of antioxidants. Con- fructa No. 3:91-112. (1961). 17. Li, K. D. and W. C. Wegenknecht. The anthocyanin pig- ments of sour cherries. J. Am. Chem. Soc. Z8; 979-81. (1956). 18. Nagi, I. Action of oxidase on anthocyanin. Botan. Mag. Tokyo 31g65-74. (1917). l9. . A genetico-physiological study on the formation of anthocyanin and brown pigments in plants. g. College Agr. Tokyo Imp. Univ. 8:2-12. (1921). 20. Peng, Chung-Yen. The degradation of a cherry antho- cyanin by tyrosinase. Michigan State Univ., M.S. Thesis. (1962). 21. Ponting, J. D. The control of enzymatic browning of fruits, in Schultz, H. W., ed., Food Enzymes, The Avi Publ. Co., Inc., pp. 105-123. (1960). 22. Reyes, P. and B. S. Luh. Characteristics of browning in Fay Elberta freestone peaches. Food Tech. 14: 570-75. (1960). 23. Sastry, L. V., B. S. Bhatia, and G. Lal. Custard apple peroxidase. J. Food Sci. ggzz44—7. (1961). 24. Scheiner, D. M. Enzymic decoloriation of anthocyanin pigments. Cornell Univ., Ph.D. Thesis. (1961). 25. 26. 27. 28. 29. 30. 33 Sondheimer, E. and Z. I. Kertesz. Anthocyanin pigments. Anal. Chem. 293245-8. (1948). SorbenlD. G. The relation of sulfur dioxide and total sulfur content of dried apricots to color change during storage. Fruit Products J. 23:234-7. 251. (1944). _ Van Buren, J. P., D. M. Scheiner, and A. C. Wagenknecht. Newly discovered enzyme may cause color loss in small fruits. N. Y. Agr. Expt. Sta. Farm Res. Bull. 25:15. (1959). - Vas, K. Preservation of fruit preparations and pulps by sulfur dioxide. Magyar Kem. Lapja 4:280-5. (1949). (In Hungarian) Vas, K. and M. Ingram. Preservation of fruit juice with less sulfur dioxide. Food Manuf. 24:414-16. (1949). Yasunobu, K. T. Mode of action of tyrosinase, in Gorden, M., ed., Pigment Cell. Biology, Academic Press, Inc., N. Y., pp. 583-608. 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