Calcium and magnesium interactions in browning of ‘Golden Delicious’ apples with bitter pit

Scientia Horticulturae, 23 (1984) 345--351

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Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

CALC]EUM AND MAGNESIUM INTERACTIONS IN BROWNING OF 'GOLDEN DELICIOUS' APPLES WITH BITTER PIT

J.A. H O P F I N G E R , B.W. P O O V A I A H

and M.E. P A T T E R S O N

Department of Horticultureand Landscape Architecture, Washingto~iState University,Pullman, W A 99164-6414 (U.S.A.) Scientific Paper No. 4902, College of Agriculture Research Center, Washington State University, Pullman, W A , U.S.A., Project 0321 (Accepted for publication 21 February 1984)

ABSTRACT Hopfinger, J.A., Poovaiah, B.W. and Patterson, M.E., 1984. Calcium and magnesium interactions in browning of 'Golden Delicious' apples with bitter pit. Scientia Hortic., 23: 345--351. The role of Ca and Mg in the enzymatic browning of 'Golden Delicious' apples was explored. Enzymatic browning due to polyphenol oxidase (PPO) was stimulated with the addition of 0.8--10 mM MgC12 to the reaction mixture, whereas 0.8--10 mM CaC12 had litl~le effect or decreased the rate of enzymatic browning. Within a week after vacuum infiltration with MgC12, treated fruits exhibited browning symptoms that were similar to bitter pit. Six months after vacuum infiltration, when analyzed for enzyme activity, MgCl~-treated fruits exhibited higher rates of PPO activity, whereas CaCI~treated fruits had lower activity than the MgC12-treated fruits and the controls. A hypothesis is advanced that the initial visible symptoms of bitter pit, i.e. the localized browning, is caused by PPO, its activity being stimulated by the localized Mg/Ca imbalance present in the tissue. The results indicate that Ca is predominant in the prevention of this disorder, whereas a deficiency of Ca is involved in its induction. Keywords: calcium; enzymatic browning; magnesium; polyphenol oxidase.

ABBREVIATION PPO = polyphenol oxidase.

INTRODUCTION

The enzymatic browning reactions in plants can be divided into two distinct classes, referred to as "functional" browning and "adventitious" browning (Mason, 1955). "Functional" browning occurs during the normal development of plant tissues, i.e. browning of seed coats, whereas "adventitious" browning occurs as a result of cellular disruption. Adventitious

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browning is of primary concern to post-harvest physiologists interested in fruit quality. The enzyme responsible for the initiation of this browning reaction is polyphenol oxidase (phenolase, o-diphenol: oxygen oxidoreductase, EC 1.10.3.1). Ponting and Joslyn (1948} demonstrated that the darkening of apple tissue is due to the enzyme PPO, with peroxidases contributing little to the browning reaction. PPO has a copper prosthetic group (Timberlake, 1957), requires oxygen for activity (Joslyn and Ponting, 1951; Hulme, 1958), and has a molecular weight of approximately 34 500 (Kertesz and Zito, 1957). PPO predominantly catalyzes the oxidation of o-dihydroxy phenols; p-dihydroxy phenols are oxidized at a reduced rate and little enzyme activity is found using either monohydroxy or m-dihydroxy phenols as substrates (Ponting and Joslyn, 1948; Reyes and Luh, 1960). These dihydroxy phenolic substrates are initially oxidized to quinones, which then undergo further polymerization and result in the formation of melanin-like brown pigments. It has been shown that enzymatic browning can be decreased with compounds that can either complex with copper (Timberlake, 1957) or complex with the phenolic substrates, thus preventing substrate oxidation, i.e. borates (Bedrosian et al., 1960), sulfates and sulfites (Burton et al., 1963}. Browning has also been reduced by the application of natural fruit acids, i.e. ascorbic, citric and malic acids (Ponting and Joslyn, 1948}. Ca has not been reported to prevent enzymatic browning, but has been used to prevent the nonenzymatic browning of dehydrated potatoes (Simon et al., 1953, 1955). In an earlier study to help apple packers reduce bruising in 'Golden Delicious' apples following controlled atmosphere storage, we observed definite reductions in browning from some treatments involving Ca. These observations established strong presumptive evidence for a Ca interaction in the enzymatic browning of apple fruits. The initial visible symptoms of bitter pit are localized areas of browning beneath the skin, occurring predominantly at the calyx end of the fruit. Associated with these pockets of localized browning is a mineral imbalance between Mg and Ca (Lewis and Martin, 1973; Hopfinger and Poovaiah, 1979). The purpose of the present investigation was to determine the role of Mg and Ca in the enzymatic browning of 'Golden Delicious' apples. MATERIALS

AND METHODS

PPO was isolated and purified by a modification of the method used by Shannon and Pratt (1967). One-hundred grams of ~3olden Delicious' apple tissue was blended with 200 ml of ice¢old 0.1 M glycine buffer pH 10 in a Waring blender. The macerated tissue was initially filtered through several layers of cheese cloth, the filtrate was then filtered through a Biichner funnel under vacuum, using Whatman No. 42 filter paper. The

347 filtrate was poured into 400 ml of - 2 6 ° C acetone and the mixture was allowed to stand for 10 min. The precipitated protein was then filtered in a Btichner funnel, under vacuum, using Whatman No. 42 filter paper. It was washed 3 times with 25 ml of ice-cold 0.1 M glycine buffer pH 10, filtered under vacuum and resuspended in 40 ml of 0.1 M citrate--0.2 M phosphate buffer (dibasic) pH 5.2. The enzyme preparation was centrifuged at 20 000 g for 20 min. The pellet was discarded and the supernatant used as the enzyme source. The enzyme reaction mixture consisted of 4 ml chlorogenic acid (1 mM) and 1 ml of the enzyme preparation. To determine the direct effects of CaC12 and MgC12 in the reaction mixture on the rate of enzymatic browning, an additional 0.1 ml of the appropriate concentration of each salt was added to the enzyme/substrate mixture prepared from untreated fruits. All solutions were buffered in 0.1 M citrate--0.2 M phosphate buffer pH 5.2. To ensure that there were no interactions between the salts and the phosphate buffer, the experiment was repeated using 0.2 M sodium acetate buffer pH 5.2. Enzyme reactions were carried o u t at room temperature (21°C). A Bausch and L o m b Spectronic 20 s p e c t r o p h o t o m e t e r equipped with a digital read-out was used for the enzyme assay; the wavelength was 400 nm. Readings were taken every :minute for 5 min, then the change in optical density (O.D.400) per min was calculated. The amount of protein present in the assay was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). The apples used were harvested from the Washington State University Research Unit at Royal Slope, 135 days after full bloom. The effect of Ca and Mg levels in fruit on browning was determined from fruits infiltrated with each element. Each vacuum infiltration treat~ ment was c o m p o s e d of 40 'Golden Delicious' apples. The apples were submerged in a steel drum with a 10-1 capacity, in either distilled water (control), 2% CaC12(w/v) or 2% MgC12(w/v). The pressure was reduced to 250 mm Hg for 5 min, released slowly, and the apples were allowed to remain in the solution for an additional 3 min. The fruits were then rinsed with distilled water, air-dried, placed in perforated polyethylene bags, and stored at I ° C until removed for enzyme analysis. Atomic absorption analysis for Ca and Mg was carried o u t using an Instrumentation Laboratory aa/ae Spectrophotometer model 151. Freezedried 1;issues were ashed and analysed according to the methods of Chapman and Pratt (1961). RESULTS AND DISCUSSION Treated fruits were continually observed to determine the effects on bitter pit development from the CaC12 or MgC12 that was vacuum-infiltrated into apple fruits. Within 1 week after infiltration, Mg-treated fruits exhibited s y m p t o m s that were morphologically similar to bitter pit (Hopfinger and Poovaiah, 1979), whereas the Ca-treated fruits had no such

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symptoms. Six months after treatment, the Ca-treated fruits remained free from bitter pit, whereas the Mg-treated fruits had 87.5% pit (Table I) and had a 50% increase in bitter pit symptoms over and above the control level. TABLE I Ca and Mg c o n t e n t of infiltrated 'Golden Delicious' apples and its effect on bitter pit symptoms. Means followed by a c o m m o n letter are not significantly different at the 5% level (Student t-test) Treatment Ca (mg 1-z)

Control CaC12 MgCl 2

433.3 a 810.6 b 418.2 a

Mg (rag 1-~)

503.0 a 451.6 a 776.6 b

Mg/Ca

1.16 0.56 1.83

Fruit affected (%). Months after treatment 0.5

6.0

15.0 0.0 50.0

37.5 0.0 87.5

From our previous work (Poovaiah and Leopold, 1973; Hopfinger and Poovaiah, 1979), it was felt that Mg played a more significant role in bitterpit development than is generally indicated in the literature. Using 3H20 efflux as a measure of membrane permeability, we found that Mg, as well as Ca, decreased membrane permeability (J.A. Hopfinger, unpublished results, 1978). Based on our research, the initial effect of Mg is probably not at the membrane, i.e. increasing permeability, although it has been shown that apples with bitter pit do have membranes that are more permeable to solute leakage, see Rousseau et al. (1972), who determined that the increase in solute leakage was due to a decrease in endogenous Ca levels with a concommitent increase in Mg. They showed that when bitter-pitsusceptible fruits were sprayed with Ca, bitter pit was alleviated, membrane permeability was decreased and fruit firmness was increased. These data did not reveal which came first in the ontogeny of this disorder, the loca5D-

,-- 4.0•~. A- 3.0. E

~

C ontrol

0

o)

1.0-

0-4

1;-3

1;-2

1;-1

Salt Concentration(M)

Fig. 1. The effects o f Ca and Mg in the reaction mixture on the rate o f PPO catalyzed browning. Vertical lines indicate SE.

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lized browning or the mineral imbalance. Since the initial visible symptom of bitter pit was the appearance of localized browning associated with the mineral imbalance between Mg and Ca, the effect of these cations upon PPO activity was determined. From Fig. 1, it can be seen that 10 -3 M MgC12 in the assay mixture stimulated the rate of enzyme activity compared to the control, whereas the same concentration of CaC12 had either no effect on the enzymatic rate of browning or slightly inhibited it. To determine the effect of Ca and Mg on PPO activity within the fruit, enzyme activity was determined from fruits that had been infiltrated with H20 (control), 2% CaC12 (w/v), and 2% MgCI2 (w/v) and stored for 6 months. The dal~a from Fig. 2 indicate that the MgC12-treated fruit had higher PPO activity compared to the control, whereas CaC12-treated fruits had lower PPO activity. While the "signal" that initiates or triggers bitter pit remains unknown, the first visible symptom is the darkening of tissue in localized pockets at the calyx end of the fruit. Since this region of the fruit normally contains the highest Mg and lowest Ca levels (Lewis and Martin, 1973), any factor(s) which could increase Mg or decrease Ca in the localized Mg/Ca ratio in the fruit could stimulate the enzymatic browning caused by PPO. This Mg/Ca ratio is in a delicate balance within the fruit. Lewis and Martin (1973) have found that the Mg/Ca ratio increases from the stem to the 7.06.0-

o

Mg

o

5.0.E 4.0-

Control

o~ E 3.0-

ao

1.0.

Ca

1

2

3

4

5

6

Chlorogenic Acid

7

8

9

10

xlO4M

Fig. 2. The effects of post-harvest fruit infiltration of CaC12 and MgCI~ on the rate of

enzymatic browning.

350

calyx end of 'Merton' apple fruits. Our electron probe data (Hopfinger and Poovaiah, 1979) substantiate the Ca trend, i.e. higher in the stem end than in the calyx end, but the Mg levels in Golden Delicious' apples appear to be rather uniform, t h e r e b y resulting in an increase in the Mg/Ca ratio from the stem end to the calyx end. Based on the data from Figs. 1 and 2, it is suggested that the initial visible symptoms of bitter pit, i.e. localized browning, are accentuated by the localized decrease in Ca, which is caused by factor(s) currently unknown. The key to understanding the initiation of bitter pit is to determine what factor(s) initiate the localized mineral imbalance. ACKNOWLEDGEMENT

Supported in part by Columbia River Orchard Foundation and Washington State Tree Fruit Research Commission.

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Simon, M., Wagner, J.R., Silveira, V.G. and Handel, C.E., 1955. Calcium chloride as a non-enzymatic browning retardant for dehydrated white potatoes. F o o d Technol., 9: 271--275. Timberlake, C.F., 1957. Metallic components of fruit juices. II. The nature of some copper complexes in apple juice. J. Sci. F o o d Agric., 8 : 159--168.