III I 1 l \ 145 104 TH _ BRGWNE‘SG SF. §3ARSNEPS Thesis for the flame of M. S. MECHEWJ STATE. BNEVERSQTY MIKLGS S. KALDY 1369 LIBRARY TH ES'S Michigan State University " filuglua av HUM: & SONS' . Bnnr BINIJERY In. l Junv muons . :llfll ABSTRACT BROWNING OF PARSNIPS by Miklos S. Kaldy Parsnips, Pastinaca sativa L., develOp a browning coloration upon storage. The active principle for this browning was extracted from parsnips with water. 3, 4- dihydroxyphenylalanine (DOPA), catechol and chlorogenic acid were found to form colored products upon incubation with the extract. The optimum pH for the browning reac- tion with DOPA as substrate was 8.5 The Michaelis constant for the reaction with catechol was 1.7 x lO-ZM. The active principle of the water extract was heat resistant as it required 120 minutes at 100°C for complete inactivation. Gel electrophoresis indicated that the browning agent of the extract is a protein. Keeping the parsnips in a 0.48% NaHSO3 solution pre- vented the browning. Temperatures near 0°C delayed the browning considerably. BROWNING OF PARSNIPS BY 1( Miklos SfflKaldy A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1969 ACKNOWLEDGMENTS The author wishes to express his sincere apprecia- tion to Dr. Pericles Markakis for his guidance throughout this study and for his aid in preparing this manuscript. He is also indebted to Dr. David R. Dilley and Dr. Walter M. Urbain for their advice and help in preparation of this manuscript. The author feels deeply grateful to the Canada Departmentof Agriculture, Research Branch, for the edu- cational leave and financial assistance granted to him during his studies at this university. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . REVIEW OF THE LITERATURE . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . Parsnips . . . . . . . . . Preparation of Extracts . Spectrophotometric Studies Gel Electrophoresis Study Storage Studies . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . A. Spectrophotometric Studies . . . . . . . Determination of Maximum Absorption . Comparison of Substrates . . . . . . . Comparison of Extracting Liquids . . . Distribution of Enzyme in the Parsnip Enzyme Stability at Room Temperature Activation of Tyrosine . . . . . . . Effect of EDTA . . . . . . . . . . Effect of pH on Enzyme Activity . Effect of Substrate Concentration Heat Inactivation . . . . . . . . B. Gel ElectrOphoresis Study . . . . . . . C. Storage Studies . . . . . . . . . . . . Effect of NaHSO3 . . . Effect of Vacuum . . . . . . . . . . . Effect of Temperature . . . . . . . . S (MARY O O O O O O O O O O O O O O O O O 0 REFERENCES 0 O 0 O O O O O O O O O O O O O 0 iii Page ii iv LIST OF FIGURES Figure l. Absorbance shanges of phenolic compounds (0.33 x 10‘ M) by a water extract of parsnip 2. Absorbance changes of DOPA (0.33 x lO-ZM) by 3 different parsnip extracts: water, 0.5 M phosphate buffer pH 6.5, 1% Polyclar L 3. Absorbance changes of DOPA (0.33 x 10-2M) by water extracts of the whole parsnip, the peel and the peeled part of it . . . . . . . 4. Absorbance changes of DOPA (0.33 x lO-ZM) by water extracts of parsnip kept at 4°C and 22°C for 24 hours . . . . . . . . . . . 5. Effect of ascorbic acid (0.0017 M) and DOPA (0.00017 M) on the oxidation of tyrosine by parsnip extract . . . . . . . . 6. Effect of EDTA (0.03%) on the oxidation of DOPA (0.33 x 10'2M) by parsnip extract . . . 7. Effect of pH on the oxidation of catechol (0.16 x 10'2M) by parsnip extract . . . . . 8. Lineweaver-Burk plot for the catechol- parsnip extract reaction . . . . . . . . . . 9. Hofstee plot for the catechol- parsnip extract reaction . . . . . . . . . . 10. Woolf plot for the catechol- parsnip extract reaction . . . . . . . . . . 11. Heat inactivation of the parsnip phenoloxidase . . . . . . . . . . . 12. Polyacrylamide gel electrophoresis of water extract of parsnip. a. Aniline blue black staining of the proteins of the extract; b. DOPA staining; c. Catechol staining; d. Chlorogenic acid staining; e. Tyrosine stain- ing 0 O O O O O O O O O O O O O O O C O O 0 iv Page 12 14 15 16 18 19 21 22 23 24 26 27 Figure Page 13. Effect of NaHSO storage on the color of parsnip. a. Stared in 0.81% NaHSO solution for 4 months at 0°C; b. Stored in perforated polyethylene bag for 4 months at 1°C and 85% ROHI O O O O O O O O O O 29 INTRODUCTION Parsnips, the fusiform root of the biennial herb, Pastinaca sativa, L, was Europe's main vegetable before the introduction of potatoes. It came to the New World with the EurOpean settlers; and although today parsnips are not a staple for most people, it is a vegetable well-liked for its spicy flavor and characteristic aroma. Parsnips are white when they are harvested but they turn light brown soon after. The brownish color became accepted by the farmers and home gardeners. The sophisti- cated supermarket buyer, however, appears to prefer the light colored parsnips. Such selective consumer acceptance naturally penalizes growers who cannot supply parsnips with the right color. Mechanical or physical injury incurred during har- vesting, cleaning and packaging of certain fruits and vegetables may produce changes in their color. But pars- nips need experience no injury to undergo a color change. Once harvested and exposed to air, the white color of the parsnips turns brown within a few hours. While immediate cooling after harvest slows the process down, parsnips harvested in warm weather may begin browning even while the roots are still in the soil. Many factors may influence the degree of browning, such as soil type, parsnip variety and storage conditions. But there must be one primary factor that initiates the browning. The objective of this research was to determine the nature of this browning, study the conditions under which the discoloration develops and explore ways of pre- venting it. REVIEW OF THE LITERATURE It is a common observation that many fruits and vegetables develop a brown coloration after harvesting or when cut or bruised. In most instances the browning of fresh fruits and vegetables is undesirable. The literature distinguishes enzymatic from non- enzymatic browning reactions. While the latter fall into four broad classes: (1) the reaction of aldehydes and ketones, such as the reducing sugars, with amino compounds; (2) caramelization of polyhydroxycarbonyl compounds, such as sugars; (3) oxidation of ascorbic acid; and (4) reac- tions of metals with tannis (Meyer, 1960; Stadtman, 1948), enzymatic browning is an oxidative reaction requiring the presence of an enzyme, a substrate and oxygen. Lacking any one of these components, the mechanism is incomplete and the reaction will not proceed (Ponting, 1960). Can- taloupe and tomato do not discolor, for instance, because they lack both enzyme and substrate in any significant amounts (Ponting, 1960). The enzyme involved in this reaction is known as tyrosinase, catecholase, phenolase, polyphenoloxidase, cresolase and phenoloxidase. The name adapted by the International Union of Biochemistry is o- diphenolz 02 oxidoreductase (E.c.l.10.3.1) (Dixon and Webb, 1964). The phenolic compounds most commonly oxidized by this enzyme include catechol, 3,4-dihydroxypheny1alanine (DOPA), tyrosine, caffeic acid, and chlorogenic acid. Phenoloxidase purified from mushrooms (Mason, 1956; Kertesz and Zito, 1957) was found to have one atom of copper per molecule of enzyme. Removing the copper inactivated the enzyme; the inactivated enzyme may regain its activity when exposed to copper ions (Reed, 1966). The mode of phenoloxidase activity has been studied extensively (Dawson and Magee, 1955; Dressler and Dawson, 1960a; 1960b), and multiple forms of plant phenoloxidase identified (Constantinides, 1966). Researchers have par- ticularly sought to inhibit or prevent the browning reac- tion catalyzed by phenoloxidase (Guadagni 2E;§12, 1949; Reyes, 1960; Scott gt_al., 1960; Goodman and Markakis, 1965; Joslyn and Braverman, 1954; Ponting, 1960), by elim- inating the substrate or oxygen, or by inactivating the enzyme. While the enzyme can be inactivated by heat, of course, the most commonly used inhibitor is sulfite in or NaHSO either the SO 3 form. Embs and Markakis (1965) 2 studied the mechanism of inhibition of the phenoloxidase browning reaction. Ascorbic acid also prevents the comple- tion of the browning reaction, by reducing the o-quinone before it polymerizes (Hope, 1961; Meyer, 1964). And sodium chloride has reportedly inhibited enzymatic brown- ing (Reed 1966). Chubey studied the oxidative browning of carrot and found that storage duration increased the suscepti- bility to browning but browning was not affected by stor- auge temperature. Existing investigations on parsnips pertain to cultural and storage practices (Thompson and Kelly, 1957; Bleasdale and Thompson, 1966; U.S.D.A., 1968); apparently no one has yet studied the browning in parsnips. MATERIALS AND METHODS Parsnips Parsnips of the Harris Model variety were obtained from L. Campbell and Sons Company, Almont, Michigan, in the fall of 1968 at harvest time. The roots were washed, placed in plastic containers containing water and trans- ported to the laboratory in East Lansing where they were stored under different conditions. (See Storage Studies.) Preparation of Extracts In order to test the possible enzymatic nature of the browning of parsnips, tissue extracts with three dif- ferent solvents were prepared. Parsnips stored at 1°C for 3 to 4 months in polyethylene bags cut into cubes, approxi- mately 3/8 of an inch, and disintegrated in a Waring blender for one minute at high speed, with an equal weight of demin- eralized water, or 0.1 M phosphate buffer pH 6.5, or 1% Polyclar L (soluble polyvinylpyrrolidone supplied by General Aniline and Film Corp.). Two additional extracts were also prepared. In one of them 20 g of peel were disintegrated with 100 m1 of demineralized water and in the other 20 g of tissue without peel was disintegrated with 100 m1 of demineralized water. The slurry was centrifuged at 39,000 x g for 20 minutes in a Sorvall Superspeed RC-2 refrigerated centri- fuge. The supernatant solution was first filtered through a double layer of No. 42 Whatman filter paper, and then through 0.45 p and 0.30 p millipore filters. All filtra- tions were done at 4°C. To each 100 ml of freshly prepared extract 0.1 m1 of a thymol solution (1% thymol in toluene) were added to prevent microbial growth. Spectrophotometric Studies Spectrophotometric measurements were made with a Bausch and Lomb Spectronic 505 and a Beckman DU spectro- photometer at room temperature. The following reaction mixture was prepared for Spectrophotometric measurements: 1 ml 0.5 M phosphate buffer, of the desired pH (6.5-9.0), 1 ml substrate, 1 ml clear parsnip extract, 0.1 ml 1% ethylenediaminetetraacetate (EDTA) and 0.1 m1 of 0.5% gelatin (purified pigskin). Four substrate solutions were used, each at 0.33 x 10-2 M final concentration: catechol, chlorogenic acid, 3,4-dihydroxyphenyla1anine (DOPA) and L-tyrosine. For the heat inactivation tests the extract was placed in boiling water for various periods of time. In the spectrOphotometric tests the blank contained the same solutions with the samples, but no enzyme; the volume was made up with water. Readings of the absorbance were made at intervals of time up to 10 hours depending on the velocity of the reaction. Gel_Electropheresis Study Disc electrophoresis (Davis, 1964; Ornstein, 1964; Constantinides, 1966), using polyacrylamide gel, was em- ployed to separate the proteins present in the parsnip extract. Stock solutions were prepared as follows:* A. To 1N 48 m1 HCl, add 36.3 g tris (hydroxymethyl) aminomethane (TRIS), 0.23 ml N, N, N', N'-tetra- methylethylenediamine (TEMED), and H O to make 2 100 ml (pH 8.8-9.0). B. To 60.0 g acrylamide and 0.4 g N, N'-methylene- bisacrylamide (BIS) add H O to make 135 ml. 2 C. To 0.14 g ammonium persulfate add H20 to make 100 m1 of catalyst solution. D. To 6.0 g TRIS and 28.8 g glycine, add H O to make 2 1 liter (pH 8.3). This buffer solution must then be diluted 1/10 before use. E. To 1 g aniline blue black add 7% acetic acid to make 200 ml of protein stain. F. 7% glacial acetic acid was prepared for destaining. The working solution was made from 1.0 part A, 1.4 parts B, and 2.1 parts H 0. To form the gel, the working 2 solution is combined with the catalyst (C) in 1:1 ratio. Gel tubes, 3 x 1/4 in. I.D., were cleaned in acid solution, *All reagents used were from Eastman Chemical Co., Rochester 3, New York. rinsed in a Kodak Photo-Flo solution (diluted 200:1), dried and filled with gel solution to a height of 15/8 in. from the bottom. One drop of water was placed on top of the gel solution to ensure a flat surface on the gel as it solidified. When the gel had set, the drop of water was removed and one inch of buffer solution (D) was placed on top of the gel. The parsnip extract was diluted with 3 volumes of water. Then sucrose was added (2% of the diluted extract) to increase the specific gravity so as to prevent diffusion into the buffer above the gel, and 0.3 ml of this diluted extract was injected just above the tOp of the gel with a syringe. In the test for substrate specificity, catechol, chlorogenic acid, DOPA and tyrosine in 2 x 10-3M concentra- tion was used instead of the staining solution. In this process the gels were immersed in the substrate and left there until bands developed. To approximately 6 m1 of substrate 0.1 m1 of ethyl alcohol (95%) were added to facilitate the development of the band (Constantinides, 1966). The gels were then washed in water and stored in 30% ethyl alcohol. Electrophoresis was carried out at 4°C. The cur- rent for the protein separation was obtained from a Heath- kit Variable Voltage Regulated Power Supply Model PS-3.* *Manufactured by the Heath Co., Benton Harbor, Michigan. 10 The current was maintained at 4 milliamperes per tube, with a total running time of 2 hours. Storage Studies In an exploratory experiment regarding the effect of storage conditions on the condition of parsnips the following samples were prepared. Groups of 3 roots were immersed in 0.0%, 0.02%, 0.08%, 0.16%, 0.48%, and 0.81% NaHSO3 solutions for 2, 5, and 10 minutes. After the soak- ing the roots were dried with absorbant paper and stored in punctured polyethylene bags at 1°C and 4°C, R.H. 85%. Groups of 3 parsnips were also stored at 1°C and 4°C in jars filled with bisulfite solutions of the same concentrations with those used in the soaking tests. Parsnips were also stored at 1°C in polyethylene bags from which the air had been evacuated by a mechanical pump. A number of parsnips were stored in perforated polyethylene bags at 1° and 4°C. The observation on stor- age lasted for a total period of 6 months. RESULTS AND DISCUSSION A. Spectrophotometric Studies Determination of Maximum AbsorptiOn When a water extract of a whole parsnip was incu- bated with solutions of three different phenolic substrates and the absorption spectrum of the reaction mixture was taken in the 505 Bausch and Lomb spectrophotometer, the following absorption maxima were observed: catechol 500 mu 3,4-dihydroxyphenylalanine (DOPA) ‘ 420 mu chlorogenic acid 420 mu Comparison of Substrates Using the maximum absorption wavelength values, each substrate was tested at pH 6.5 with enzyme extract for activity on a Beckman DU spectrophotometer (Fig. l). The activities expressed in absorbance units per hour (AA/hr)are as follows: Substrate Concentration Activity (AA/hr) dopa .33 x lO-ZM .114 catechol .33 x lO-ZM .072 chlorogenic acid .33 x lO-ZM .052 tyrosine (at _2 420 mp) .33 X 10 M .000 11 i r , f . a. f, Absorbarce .400 an 12 Chlorogenic acid (420 mp) - I” ‘ DOPA (420 mp) catechol . (7'4 {500 mp) 025/) '4 .2004 tyrosine .16‘4 (“20 mp) O .1501 1 2 3 4 hrs Fin. 1 A sortance charges of phenolic compounds .. . -. -2 {’.35 x 10 V) ty a water extract of parsnip. 13 Comparison of ExtractianIquids Enzyme samples extracted with water, 1% Polyclar L, and 0.1 M phosphate buffer pH 6.5 were tested for phenoloxidase activity using DOPA as a substrate (Fig. 2). Activities of .038 AA420/hr. with water, .037 AA420/ hr. with 1% Polyclar L, and .033 AA420/hr. with the 0.1 M buffer were obtained. Since the water extract had the highest enzymic activity, water was used for extraction in subsequent experiments. Distribution of Enzyme in the Parsnips When the water extracts from the whole parsnip, the peel and the peeled part of it were compared for enzymic activity in terms of absorbance (AA420) changes in a DOPA solution (0.33 x 10-2M) the following activity were obtained (Fig. 3): Peel: 0.490 AA420/hr/g Peeled part: 0.170 AA420/hr/g Whole: 0.180 AA420/hr/g ‘Enzyme Stability at Room Temperature Enzyme extracts stored in a cold room (4°C) or at room temperature (22°C) for 24 hours showed no difference in activity (Fig. 4). In both cases the relative enzyme activity was .082 AA420/hr, Absortance at 420 mp 14 o q/‘O ‘ POE butfer (~——Polyclar 6 . q o)1 . . hrs Fig. 2. Absorbance changes of DOPA (0.33 X 10-2M) by 3 different parsnip extracts; water, 0.5M phOSphate buffer pH 6.5. 1% Polyclar L. Absorbance at 420 mu 15 peel whole .2CO 4 parsnip peeled part 0 ' 1 2 3 it 5 hrs Fla. 3. Absorbance changes of DOPA (0.33 x IO-ZM) by water extracts of the whole parsnip, the peel and the peeled part of it. U! r.) ,1 .1" 22°C 4°C ,._. N w 5-— U" .711"? Atsorlance changes of DOPA (0.33 x 10 ’N) by water extracts of parsnip kept at U°C and 22°C for 23 hccrs. 17 Activation of Tyrosine When tested with tyrosine (Fig. 1), the enzyme showed no activity. It is known, however, that mon0phenols, such as tyrosine, may be oxidized by phenoloxidase if a reducing agent is present in the reaction mixture (Bright gt_al., 1963). Additions of 0.05 ml of 0.1 M ascorbic 2M DOPA to the 3 m1 reaction mix- acid or 0.05 ml of 1 x 10‘ ture indicated the triggering action to a very small extent: ascorbic acid raised the activity from .0 to .004 AA420/hr. and DOPA from .0 to .014 AA420/hr. (Fig 5). Since a similar amount of DOPA in a reaction mixture contained all other reagents but tyrosine had an activity of .008 AA420/ hr., the net activity of the enzyme with tyrosine as substrate can be assumed to be .006 AA420/hr. Effect of EDTA Since the enzyme extract used in this study was not purified, EDTA was added to the test solution for the purpose of chelating metal ions which might inhibit the phenoloxidase activity of the extract. Tests with and without EDTA, showed that 0.1 m1 of 1% EDTA in 3 ml reac- tion mixture slightly enhanced the enzyme activity (Fig. 6): .063 AA420/hr. with EDTA and .060 AA420/hr. without. Absorbance at 420 mp .200 4 r 4 I?) O 18 tyrosine + DOPA tyrosine + ascorbic acid O 2 h 6 8 10 hrs Fig. 5. Effect of ascorbic acid (0.0017Ml and DOPA (0.00017M) on the oxidation of tyrosine by parsnip extract. mp 420 Absorbance at .SCOd 19 no EDTA EDTA (0.03%) j T I T '1 C 1 2 3 u 5 hrs F .03%) on the oxidation of DCPA (’ J J 4 4.1 ,1 X ». Effect of EDTA ( i“ “-3 (D M) iy parsnip extract. 20 Effect ofypH on Enzyme Activity Preliminary experiments with DOPA suggested that the Optimum pH for phenoloxidase in parsnips is in the higher pH regions. But since DOPA autoxidizes quite rapidly at higher pH values, cathechol was used as the substrate in these tests. Blanks, as in previous measure- ments, contained all components of the test solution ex- cept the enzyme. In addition, 0.5 ml 0.5% gelatin was used in the test solution for stabilizing the enzyme (Dawson and Magee, 1955). Test solutions remained the same in content, except for adding 0.5 m1 enzyme and 0.5 ml gelatin (instead of the usual 1 ml enzyme extract). The Optimum pH for the enzyme was found to be 8.5 (Fig. 7). Effect of Substrate Concentration Six concentrations, .42, .83, 1.33, 1.67, 2.50, 3M of catechol as substrate were used in and 3.33 x 10‘ order to determine the Michaelis constant of the reaction. The data were plotted according to the Lineweaver-Burk, Hofstee, and Woolf methods (Christensen and Palmer, 1967) and are illustrated in Figures 8, 9 and 10. From these graphs the following Km values were obtained: 1.67 x lO-ZM Lineweaver-Burk Plot 1.70 x 10_2M Hofstee Plot 2 1.72 x 10- M Woolf Plot nn/hr 5"" Absorbance at r\ A‘. I. Wop“ l ..x 21 1 1’ T Y j ‘ 6.5 7.0 7.5 8.0 ‘8.5 9-0 Fia.. . Effect of pH on the oxidation of catechol (0.16 x 10'2M) by parsnip extract. 22 I70: 15J 1 v 10 - Slepe = Km/Vmax 5 T fl T f 1 —T -05 To 05 1.0 1.5 2.0 05 1 Km (S) x C M Fig. 8. Lineweaver-Burk plot for the catechol-parsnip extract reaction. 23 Vmax Slope =-Km Vmax/Km 0 .C25 .050 .075 .100 .125 U3I<1 ) Fig. 9. Hofstee plot for the catechol-parsnip ( extract reaction. 211 .cofipomoa powapxc cacmawouficcomumo on» now BOHQ mace: .QH .afim zmIS x m8 ET 3 m o m- 2.. as- F 8.- [r n h L p 25 Heat Inactivation Studies of the time and temperature required to inactivate the enzyme sought to measure the stability of phenoloxidase in parsnip extract. Since initial trials revealed strong heat resistance, a temperature of 100°C was selected for the test. At this temperature complete inactivation was Observed after 120 minutes at pH 8.0. At various intervals during the heating period, samples Of the extract were tested and their activity plotted on semilogaritmic graph paper (Fig. 11). The activity de- clined rapidly during the first 10 minutes of heating, and decreased at a markedly slower rate upon further heat- ing. A similar high heat resistance was Observed by Jankov (1962) for the phenoloxidase of apples and plums. B. Gel Electrophoresis Study Figure 12 illustrates the results of the poly- acrylamide gel electrophoresis of the parsnip extract. It is apparent that the bands displaying the color reac- tion with DOPA, cathechol and chlorogenic acid also reacted with the protein stain. No color band can be seen with tyrosine. This confirms the evidence derived from the spectrophotometric studies concerning the enzymic nature of the browning agent. One can also Observe several isozymic forms of the parnship phenoloxidase. I t [(1 an '4 L Absorbance at 26 1.17 q C 30 60 90 120 minutes F12. 11. Heat inactivation of the parsnip phenoloxidase. Fig. 12. 27 11 Polyacrylamide gel electrophoresis of water extract of parsnip. a. Aniline blue black staining of the proteins of the extract. b. DOPA staining. c. Catechol staining. d. Chlorogenic acid staining. e. Tyrosine staining. 28 C. Storage Studies Effect Of NaHSO3 The storage study demonstrated that parsnips soaked in NaHSO3 for 2 to 10 minutes and subsequently stored in polyethylene bags showed no inhibition of browning com- pared to those stored in polyethylene bags without treat- ment during a 4 month period of straoge at 4°C. Also, no color difference was observed between NaHSO3 dipped and control parsnips stored at 1°C for 6 months. On the other hand, browning was completely inhibited in parsnips stored continuously in jars containing 0.02%, 0.08%, 0.16%, 0.48% and 0.81% NaHSO3 solution (Fig. 13). Bisulfite demonstrated some antiseptic properties for the parsnips stored in jars. Parsnips stored in 0.02% and 0.08% NaHSO3 concentrations disintegrated within one month at 4°C and two months at 1°C. Concentrations of 0.16% provided one additional month of preservation at both temperatures. Parsnips stored in 0.48 and 0.81% NaHSO3 concentrations (pH 4.85) kept well for four months at either temperature. After four months, molds started to grow on the surface of the solutions but not on the roots. When new NaHSO3 solutions were prepared with the same concentrations of NaHSO (0.48% and 0.81%) and the 3 parsnips were transfered into them no deterioration signs were apparent after an additional two months of storage. 29 Fig. 13. Effect of NaHSO3 storage on the color of parsnip. a. Stored in 0.81% NaHSO solution for 4 months at 0°C. 3 b. Stored in perforated polyethylene bag for 4 months at 1°C and 85% R.H. 30 Effect of Vacuum Vacuum packed parsnips retained their white color, but the roots could not be kept under these conditions longer than 10 days because of the develOpment of an off odor presumably due to anaerobic respiration. Effect of Temperature Parsnips stored at 4°C browned sooner and developed darker color than those stored at 1°C. Better quality was maintained at the lower temperature. Parsnips stored at 4°C, whether NaHSO3 treated or not, kept no longer than three months. On the other hand, all parsnips stored at 1°C in polyethylene bags remained in good conditions for 4 to 5 months. Generally speaking, after harvesting, parsnips slowly lose their white color and turn light to medium brown._ The rate at which the browning proceeds varies with the temperature and the availability of oxygen. COOling the roots soon after harvest can delay the brown- ing. Transporting :hi cold water is very effective since it also excludes the oxygen. Joslyn and Ponting (1951) reported that many plant tissues displaying such general patterns of darkening con- tain phenolic compounds with free o-dihydroxy benzene groups, e.g. catechol, which are oxidized by molecular oxygen under catalysis by the enzyme phenoloxidase. 31 Nelson and Dawson (1944) proposed the following oxidation reaction for catechol, which in the presence of oxygen produces o—benzoquinone: OH O OH O + H O ' 2 + 1/2 02 phenolox1dasei, catechol o—benzoquinone Upon further oxidation and condensation o-benzoquinone yields melanin, a dark-colored pigment: O + 1/2 0 polymerizationé. melanin 2 o-benzoquinone S UMMARY The reaction leading to the browning of parsnips was studied chiefly in extracts of these roots. Three different solvents were compared in order to find the best extraction method: demineralized water, 0.1 M phosphate buffer at pH 6.5, and 1% Polyclar L. Demineralized water appeared to be the best of the three because it caused the great— est browning Of 3,4-dihydroxyphenyla1anine (DOPA) as substrate. Among four possible substrates, DOPA, catechol, chlorogenic acid and tyrosine, tested for browning at the wavelength of maximum absorption of their solutions, DOPA resulted in the most rapid browning. No browning of the tyrosine solution was observed. When ascorbic acid and DOPA were used as triggering agents for the browning of tyrosine by the water extract, a slight reaction was observed. The catalytic nature of the peel extract was the highest when compared to that of extract from the peeled and whole parsnips. 32 re 33 Water extracts of parsnips kept at 4°C and 22°C for 24 hours showed no difference in activity against DOPA as substrate. EDTA, 0.03% in the reaction mixture, slightly enhanced the oxidation of DOPA by the parsnip extract. Optimum pH for the browning of 0.16 x lO-ZM catechol solution was 8.5. Michaelis constant for the browning reaction Of catechol was found to be 1.7 x 10-2M by three graphical methods. Parsnip phenoloxidase showed strong heat resistance. At 100°C the activity declined rapidly during the first 10 minutes of heating, but decreased at a markedly slower rate upon further heating. Com- plete inactivation of the enzyme was observed after 120 minutes at pH 8.0. Gel electrOphoresis confirmed the enzymic nature of the browning agent in parsnips. Bands in the polyacrylamide gel which resulted in browning re- actions with DOPA, catechol, and chlorogenic acid were also stained with protein stain. Tyrosine failed to react with any of the gel bands. 10. 34 Preliminary storage studies indicated that dipping in NaHSO3 solution of concentrations up to 0.81% for 10 minutes did not stop the browning of parsnips on subsequent storage in air. Keeping the parsnips immersed in 0.48% solution of NaHSO3 resulted in prevention of browning for 4 months of observation. 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