Regulation of Acetaldehyde and Ethanol Accumulation in Citrus Fruit

22

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Regulation of Acetaldehyde and Ethanol Accumulation in Citrus Fruit J. H. Bruemmer U.S. Citrus & Subtropical Products Laboratory, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 1909, Winter Haven, FL 33883-1909 Maturation effected the following changes in enzymes and metabolites in orange fruit: ethanol and acetaldehyde accumulated to levels of 10 mM and 0.08 mM, pyruvate decreased about 30%, pyruvate decarboxylase increased over 4 fold, alcohol dehydrogenase increased about 2 fold, the NADH to NAD ratio increased 2 1/2 fold and the terminal oxidase developed CN-insensitivity. The fraction of the total alternative respiratory pathway in actual use increased from 0.46 to 1.08. Induction of the alternative, CN-insensitive oxidase during maturation was interpreted as indicating that membrane function was modified which affected metabolic pathways resulting in the accumulation of ethanol and acetaldehyde. The "essence" of citrus flavor is a complex mixture of volatile alcohols, aldehydes, esters, hydrocarbons, ketones and oxides. Alcohols are the largest class and ethanol is the main organic constituent of the essence. Esters and aldehydes are considered to contribute most to the characteristic flavor and aroma. In these two classes ethyl butyrate and acetaldehyde were shown to be important components of high quality orange juice (_1 ). Ethanol is enzymically related to ethyl butyrate and acetaldehyde through reactions catalyzed by alcohol

This chapter not subject to U.S. copyright. Published 1986, American Chemical Society In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


276

BIOGENERATION O F A R O M A S

a c e t y l t r a n s f e r a s e (AAT) and a l c o h o l dehydrogenase (ADH) respectively. I n j u i c e from immature Hamlin orange e t h a n o l was p r e s e n t at 1 mM but accumulated t o 10 mM at the mature h a r v e s t s t a g e (2^). A c e t a l d e h y d e l e v e l s i n c r e a s e d more s l o w l y than e t h a n o l from 0.03 mM t o a l e v e l of 0.08 mM at m a t u r i t y . Ethanol also accumulated in j u i c e from mature c i t r u s f r u i t d u r i n g hypo-02 o r hyper-CO^ r e f r i g e r a t e d s t o r a g e f o r 6 weeks ( 2 ) . E l e v a t i o n o f the e t h a n o l c o n t e n t o c c u r s d u r i n g the m a t u r a t i o n s t a g e o f f r u i t development when a c i d i t y d e c l i n e s , t o t a l s o l i d s i n c r e a s e , and the f l a v o r compounds accumulate t o c h a r a c t e r i s t i c a l l y r i p e l e v e l s . S i n c e e t h a n o l is a normal p r o d u c t o f a n a e r o b i c r e s p i r a t i o n in p l a n t s , i t s a c c u m u l a t i o n s i g n a l s a change in the pathway o f energy m e t a b o l i s m . T h i s summary of r e s e a r c h on the c o n t r o l of e t h a n o l a c c u m u l a t i o n in c i t r u s f r u i t d e s c r i b e s an approach t o i d e n t i f y the r o l e o f energy m e t a b o l i s m in the b i o r e g u l a t i o n of m a t u r a t i o n . B i o c h e m i c a l changes

during anaerobic metabolism

Immature g r a p e f r u i t responded t o 16-hr. i n c u b a t i o n at 38°C under C0^ by a 1 5 - f o l d i n c r e a s e in j u i c e e t h a n o l and a much s m a l l e r i n c r e a s e in a c e t a l d e h y d e compared t o a i r c o n t r o l s ( 3 ) . M a l a t e , a p r e c u r s o r o f e t h a n o l , was about 25% lower in j u i c e from the a n a e r o b i c a l l y s t o r e d f r u i t , but the d e c l i n e in malate c o u l d account f o r o n l y 7% o f the i n c r e a s e in e t h a n o l . C i t r a t e was a l s o lower and i t s m e t a b o l i s m c o u l d a c c o u n t f o r 15% o f the i n c r e a s e in e t h a n o l . The a l c o h o l dehydrogenase a c t i v i t y was about t w i c e as h i g h in j u i c e from a n a e r o b i c - t r e a t e d f r u i t , but the l e v e l s o f m a l i c enzyme (ME), m a l a t e dehydrogenase (MDH), and p y r u v a t e d e c a r b o x y l a s e (PDC) were not g r e a t l y a f f e c t e d by the t r e a t m e n t . Mature oranges responded t o 6 weeks s t o r a g e at 4 C under 5% 0^ in N2 by a 3- t o 4 - f o l d i n c r e a s e in j u i c e e t h a n o l and a c o n s i s t e n t d e c r e a s e in sugar l e v e l compared t o c o n t r o l s s t o r e d in 21% 0 in N (4_). L e v e l s o f ME, PDC, ADH, c i t r a t e and malate were s i m i l a r in t r e a t e d and c o n t r o l g r o u p s . The r a t i o of o x i d i z e d to reduced forms o f NAD was s e v e r a l f o l d h i g h e r in j u i c e from the low oxygen group than from the 21% 0~ c o n t r o l . E t h y l e n e s t i m u l a t e d r e s p i r a t i o n in c i t r u s (_5,6) and enhanced m a t u r a t i o n o f f r u i t (7). Mature g r a p e f r u i t s t o r e d in a i r c o n t a i n i n g 20 ppm e t h y l e n e c o n t a i n e d 7 t o 10 t i m e s more e t h a n o l a f t e r 4 , 8 o r 12 weeks at 15 C than c o n t r o l f r u i t s t o r e d w i t h o u t e t h y l e n e ( 8 ) . A c e t a l d e h y d e in e t h y l e n e t r e a t e d f r u i t was about 3 times h i g h e r . L e v e l s of ME, PDC, and ADH were s i m i l a r in t r e a t e d and c o n t r o l g r o u p s . E t h y l e n e treatment decreased j u i c e malate about 60% in t h e 12-week p e r i o d compared t o c o n t r o l s . Malate v a l u e s were l o w e s t in samples w i t h h i g h e s t e t h a n o l , which s u g g e s t e d t h a t e t h y l e n e promoted the m e t a b o l i s m o f malate t o e t h a n o l . In pome f r u i t m e t a b o l i s m o f m a l a t e t h r o u g h the d e c a r b o x y l a t i n g system is c o n s i d e r e d p a r t o f the e t h y l e n e - p r o m o t e d r i p e n i n g p r o c e s s (7^. D e c a r b o x y l a t i o n o f malate t o e t h a n o l would e x p l a i n changes in t h e s e m e t a b o l i t e s a f t e r s h o r t term i n c u b a t i o n in CO^ and a f t e r s t o r a g e w i t h 10 ppm e t h y l e n e in a i r . Two o f the enzymes (PDC and ADH) in t h i s d e c a r b o x y l a t i o n pathway ( F i g u r e 1) were i s o l a t e d and p u r i f i e d from oranges and t h e i r r e a c t i o n p r o p e r t i e s examined. 2

2

In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

22.

BRUEM MER

PDC

and ADH

PDC was 65-fold

Acetaldehyde and Ethanol Accumulation in Citrus Fruit

reactions

i s o l a t e d from orange j u i c e s e c t i o n s and p u r i f i e d f o r k i n e t i c measurements of the r e a c t i o n (9):

p y r u v a t e + NADH


277

PDC

• a c e t a l d e h y d e + NAD

+

about

+ CC>

2

The optimum pH was 4.7, which was lower t h a n t h a t (6.1) r e p o r t e d f o r y e a s t PDC ( 1 0 ) . The H i l l p l o t was t y p i c a l of a m o n o - c a t a l y t i c s i t e enzyme and, a l t h o u g h a c t i v i t y was dependent on reduced s u ^ h y d r y l groups, s u b s t r a t e b i n d i n g was n o t . B i n d i n g k i n e t i c s of Mg and t h i a m i n e pyrophosphate (TPP) and f o r m a t i o n of an a c t i v e c y c l i c t e r n a r y complex in the p r e s e n c e of p y r u v a t e a r e p r o p e r t i e s s i m i l a r t o y e a s t PDC. About 15% of orange PDC was in the a c t i v e c y c l i c form w i t h o u t added p y r u v a t e (9). A c t i v a t i o n of PDC by p y r u v a t e was demonstrated in orange j u i c e ( 1 1 ) . A d d i t i o n o f 10 mM p y r u v a t e t o j u i c e i n c r e a s e d 5 - f o l d the r a t e of a c e t a l d e h y d e f o r m a t i o n , s u g g e s t i n g the p r e s e n c e of about 20% in the a c t i v e form. A c t i v a t i o n of PDC by p y r u v a t e c o u l d e x p l a i n the s t i m u l a t i o n of e t h a n o l and a c e t a l d e h y d e a c c u m u l a t i o n in c i t r u s by a n a e r o b i c treatment (3) and by s t o r a g e in hypo-oxygen atmospheres (4) w i t h o u t n o t i c e a b l e i n c r e a s e in PDC l e v e l . A n a e r o b i o s i s in the f r u i t would s u p p r e s s the o x i d a t i v e pathway and i n c r e a s e a v a i l a b i l i t y of p y r u v a t e f o r a c t i v a t i o n of PDC. ADH was i s o l a t e d and p a r t i a l l y p u r i f i e d from orange j u i c e v e s i c l e s and examined f o r s u b s t r a t e s p e c i f i c i t y , maximum r e l a t i v e v e l o c i t y ( V r ) and a f f i n i t y (1/Km) ( 1 2 ) . E t h a n o l is the p r e f e r r e d s a t u r a t e d a l c o h o l f o r r e d u c t i o n t o the a l d e h y d e based on Vr and 1/Km. U n s a t u r a t e d a l c o h o l s , 2 - p r o p e n o l , 2 - b u t e n o l and 2-hexenol, had comparable t o o r h i g h e r V r s and l/Km's than e t h a n o l . ADH had 5- t o 3 0 - f o l d g r e a t e r a f f i n i t y f o r s a t u r a t e d a l d e h y d e s than the c o r r e s p o n d i n g s a t u r a t e d a l c o h o l s , whereas a f f i n i t i e s of the u n s a t u r a t e d a l c o h o l s and a l d e h y d e s were s i m i l a r . The apparent e q u i l i b r i u m c o n s t a n t s (Kapp - 0.003 f o r e t h a n o l - a c e t a l d e h y d e p a i r ) f a v o r a l c o h o l f o r m a t i o n in the s a t u r a t e d s e r i e s . Other a l d e h y d e s compete w i t h a c e t a l d e h y d e f o r the enzyme but the c o n c e n t r a t i o n of a c e t a l d e h y d e is much h i g h e r than o t h e r a l d e h y d e s in j u i c e v e s i c l e s and the 1/Km f o r a c e t a l d e h y d e is 10 X h i g h e r than f o r o t h e r a l d e h y d e s found in the j u i c e v e s i c l e s . f

R e g u l a t i o n of ADH

and malate

dehydrogenase

(MDH)

The redox r a t i o s o f NAD and NADP in mature c i t r u s f r u i t a r e comparable t o r a t i o s r e p o r t e d f o r o t h e r f r u i t ( 1 3 ) . The r a t i o NADH/NAD was more t h a n s i x times h i g h e r in mature, v e r y r i p e oranges ( B r i x - a c i d r a t i o o f 20) than in immature oranges ( B r i x - a c i d r a t i o o f 6 ) . In c o n t r a s t the NADPH-NADP r a t i o f o r immature and mature f r u i t were comparable. T h i s s h i f t i n g toward h i g h e r redox r a t i o o f NAD has been a s s o c i a t e d w i t h m a t u r a t i o n and t r a n s i t i o n toward a n a e r o b i o s i s in p l a n t t i s s u e ( 1 4 ) . When oranges were i n c u b a t e d under N« at 34°C f o r 18 hr the NADH-NAD r a t i o in j u i c e was t w i c e as h i g n as in j u i c e from c o n t r o l f r u i t i n c u b a t e d under a i r ( 1 5 ) . The redox r a t i o o f NAD a f f e c t s MDH and ADH a c t i v i t i e s in e x t r a c t s from orange j u i c e v e s i c l e s ( 1 5 ) .


278

BIOGENERATION OF AROMAS

Dehydrogenase a c t i v i t y of MDH was suppressed by NADH at 5% of the NAD concentration. Oxidative a c t i v i t y of ADH was suppressed when NADH approached the concentration of NAD. The reductase a c t i v i t i e s were not suppressed by NAD at even 10 times the concentration of NADH. The increase in the redox r a t i o in senescing and anaerobic c i t r u s f r u i t could i n h i b i t the oxidation of malate to oxalacetate and increase the flux v i a the decarboxylation pathway through pyruvate to ethanol (Figure 1).


Pyruvate metabolism during

maturation

Juice ethanol and acetaldehyde increased in Hamlin oranges during maturation (Figure 2) (16). The ethanol-acetaldehyde ratios f o r October through February were 44, 55, 100, 109, 145. The series of values showed a noticeable difference before and after December when the f r u i t reached ripe maturity. The monthly series of values for pyruvate also showed a marked decline between November and December (Figure 3), but the other acids exhibited variable trends. PDC and ADH increased in juice v e s i c l e s of Hamlin orange during maturation from October to December (Figure 4). The largest increase occurred from November to January. Pyruvate dehydrogenase (PDH) increased s l i g h t l y from October (2.2 U) to November (2.9 U) in the juice v e s i c l e s and then plateaued through February (2.9 U) (Figure 5). The higher PDC a c t i v i t y would increase competition with PDH f o r pyruvate and thereby increase decarboxylation to acetaldehyde (Figure 1). The higher PDC a c t i v i t y could thus explain the decline in pyruvate and increase in acetaldehyde and ethanol in December. The higher ADH l e v e l in December could explain the higher concentration r a t i o of ethanol to acetaldehyde. ME increased steadily (Figure 5) which probably influenced the equilibrium between malate and pyruvate in the cytoplasm; malate increased from 4.5 mM to 7.0 mM and pyruvate decreased from 68 y M to 48 yM over the season. Phosphoenolpyruvate carboxylase (PEPC) increased during the early part of the season (Figure 5). However, because MDH was much more active than PEPC (100-fold), the increase in PEPC probably had l i t t l e effect on the equilibrium concentration of oxalacetate, which a c t u a l l y declined (Figure 3). Increase in ethanol and decrease in pyruvate l e v e l during ripening could result from stimulation of the pyruvate decarboxylase reaction promoted by the higher enzyme l e v e l . However, a c t i v i t y of the competing enzyme f o r pyruvate, PDH, is controlled by ratios of NADH to NAD and ATP to ADP in plant mitochondria (18). During maturation of Hamlin orange the r a t i o of NADH to NAD in juice v e s i c l e s increased from 0.09 in October to 0.24 in March, while the phosphorylated r a t i o (NADPH/NADP) was constant (17). The PDH from broccoli was very sensitive to increases in the mole f r a c t i o n of NADH (19) . A 10 to 15% increase in r a t i o in whole tissue decreased PDH a c t i v i t y 15 to 25%. The r a t i o of ATP to ADP in juice v e s i c l e s increased i n i t i a l l y from 0.7 in October but plateaued at 1.0 after December. ATP inactivated PDH by enzymic phosphorylation in mitochondria from pea leaf (20). The phosphorylated PDH was activated by a Mg -dependent phosphatase. Both reactions were inhibited by ADP which suggests


BRUEMMER

Acetaldehyde and Ethanol AccumulationinCitrus Fruit


CYTOPLASM

Figure 1.

MITOCHONDRIA

Pyruvate metabolism in c i t r u s f r u i t

(16).


279

280



HOY

DEC

JAN

ADH and PDC during f r u i t maturation (16).


FEB


22. BRUEMMER

281

the potential of the ATP-ADP couple for PDH regulation. The ubiquinol to ubiquinone r a t i o , (UQH/UQ) increased during the season from 0.4 in October to 0.7 in March. As an indicator of the redox state of the respiratory pathway, increase in this r a t i o indicates a change in equilibrium between substrate a v a i l a b i l i t y and oxidative function of the pathway. Thus, increases in UQH/UQ and NADH/NAD ratios suggest that the oxidative capacity of the pathway is inadequate to maintain redox equilibrium. Terminal oxidases


The NADH oxidase of the mitochondrial f r a c t i o n from Hamlin orange juice vesicles became less sensitive to KCN as the f r u i t matured (Table I ) .

Table I.

NADH jçcidation (nmoles 0«, mg protein min , mean ± SE, n=4) {21)

NADH Sept Oct Nov Dec Jan

281 294 303 366 379

± ± ± ± ±

2.8 3.1 4.5 4.7 4.7

NADH + ImM KCN 9 ± 1.4 12 ± 0.9 8 ± 0.3 116 ± 3.1 129 ± 3.2

,

NADH + ImM KCN + ImM SHAM 0 0 6 + 0.7 36 + 0.5 45 + 1.0

Fractions prepared from September, October, and November f r u i t were almost completely inhibited by 1 mM KCN, but preparations from December and January f r u i t were inhibited 65 to 70%. The KCN-insensitive r e s p i r a t i o n was inhibited 65 to 70% by salicylhydroxamic acid (SHAM). The SHAM-sensitive oxidase, or alternative oxidase, accounted for about 22% of the t o t a l , and residual oxidase ( a c t i v i t y in presence of KCN + SHAM) about 10% in mitochondria from mature tissue. Induction of the alternative respiratory pathway has been observed in maturing and aging organs, tissues and c e l l s (22). Aged sweet potato root s l i c e s contained two types of mitochondrial membranes (23). The denser type was deficient in phospholipids and possessed alternative oxidase a c t i v i t y . The l i g h t e r type was i d e n t i c a l to membranes from fresh s l i c e s and had no alternative oxidase. Submitochondrial p a r t i c l e s from aged sweet potato root tissue were low in phospholipid and possessed alternative oxidase a c t i v i t y (24). Addition of phospholipid to the p a r t i c l e s increased the l i p i d content and eliminated KCN-insensitive respiration. Submitochondrial p a r t i c l e s (membranes from washed sonicated mitochondria) prepared from juice vesicles of Hamlin oranges harvested in September contained KCN-insensitive respiratory a c t i v i t y (46% of t o t a l ) using a substrate mixture containing 0.05 M malate, 0.05 M succinate, 0.01 M glutamate and 0.01 M TPP (21).



282

This a c t i v i t y contrasts to the small (3%) KCN-insensitive respiratory a c t i v i t y observed when NADH was used as substrate with the mitochondria (Table I ) . To calculate the contribution of each oxidase to the t o t a l oxygen uptake, the t i t r a t i o n method of Bahr and Bonner (25) was used, (^-uptake of submitochondrial p a r t i c l e s (membranes) prepared from juice vesicles of Hamlin oranges harvested in September and January was measured in the presence and absence of ImM KCN t i t r a t e d with a series of SHAM concentrations using the malate, succinate, glutamate substrate mixture (Table I I ) .


Table I I .

A c t i v i t y of alternative pathway (21)

SHAM mM 0 0.25 0.5 1.0

Sept

Jan

0 0.25 0.5 1.0

0«-uptake ± 1 mM KCN +KCN -KCN % of Control 100 100 83 85 67 77 75 46 100 75 67 63

100 87 78 66

The set of values (as % of control) obtained in the absence of KCN was plotted against the set in the presence of KCN (Figure 6). The direct l i n e a r relationship between the sets of values is described by the equation V

T

= p . g ( i ) + Vcyt. (25)

Where V is the t o t a l r e s p i r a t i o n rate, Vcyt is the CN-sensitive cytochrome mediated r e s p i r a t i o n , and g ( i ) is the maximal contribution of the CN-insensitive alternative r e s p i r a t i o n at given concentrations of the alternative path i n h i b i t o r , SHAM. The slope of the l i n e , p is the f r a c t i o n of the alternative path which is operating or in actual use and p . g ( i ) represents the actual contribution of the alternative path to the t o t a l r e s p i r a t i o n . The slope for the September preparation was 0.46, and the slope for the January preparation was 1.08. The slope of 1.0 indicates that the maximal capacity of the alternative path was in actual operation in the mature January f r u i t . These data suggest that during maturation, membrane function was altered which increased the contribution of the alternative pathway to the t o t a l respiration. T


BRUEMMER



22.

0 -UPTAKE, 2

% OF CONTROL,

g(i)

Figure 6. Estimation of the contribution of the alternative pathway to t o t a l respiration of mitochondrial membranes from September and January f r u i t (21).


283

284



Conclusion Ethanol accumulated in maturing citrus fruit as the end product of pyruvate decarboxylation. Conditions that promote this reaction include low 0^, and high CC^, and ethylene levels. Maturation increased the levels of PDC and ADH and increased the NADH to NAD ratio. The higher redox ratio could slow the PDH reaction which competes with PDC for pyruvate. Development of the alternative oxidase activity when ethanol began to accumulate suggests that membrane function was modified which affected rates of various metabolic pathways. The lower phosphorylation efficiency of the alternative oxidase compared to the cytochrome pathway (22) could affect numerous metabolic activities including decarboxylation of pyruvate. Also, membrane transport of pyruvate and cofactors could be altered in mitochondria containing fewer phosphorylation sites (26). Acknowledgment Mention of a trademark or proprietary product is for identification only and does not imply a warranty or guarantee of the product by the U.S. Department of Agriculture over other products that may also be suitable. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Ahmed, A.M.; Dennison, R. Α.; Shaw, P. E. J. Agric. Food Chem. 1978, 26, 368. Davis, P. L. Proc. Fla. State Hort. Soc. 1970, 83, 294. Bruemmer, J. H.; Roe, B. Proc. Fla. State Hort. Soc. 1970, 83, 290. Davis, P. L.; Roe, B.; Bruemmer, J. H. J. Food Sci. 1973, 38, 225. Eaks, I. L. Plant Physiol. 1970, 45, 334. Reid, M. S.; Pratt, Η. K. Nature 1970, 226, 976. Rhodes, M. J. C.; Wooltorton, L. S. C.; Hulme, A. C. Qual. Plant Mater. Veg. 1969, 19, 167. Davis, P. L.; Roe, B.; Bruemmer, J. H. Proc. Fla. State Hort. Soc. 1970, 87, 222. Raymond, W. R.; Hostettler, J. B.; Assar, K., Varsel, C. J. Food Sci. 1979, 44, 777. Ulbrich, J. In "Methods of Enzymatic Analysis"; Bergmeyer, Η. V., Ed.; Verlay Chemic, Weinheim; Academic Press: New York, 1974; Vol. 4, p. 2186. Roe, B.; Bruemmer, J. H. J. Agric. Food Chem. 1974, 22, 285. Bruemmer, J. H.; Roe. B. J. Agric. Food Chem. 1971, 19, 266. Bruemmer, J. H. J. Agric. Food Chem. 1969, 17, 1312. Yamamato, Y. Plant Physiol. 1963, 38, 45. Bruemmer, J. H.; Roe. B. Phytochem. 1971, 10, 255. Roe, B.; Davis, P. L.; Bruemmer, J. H. Phytochem. 1984, 23, 713. Bruemmer, J. H.; Roe B. Phytochem. 1985, 24, 2105. Randall, D. D.; Rubin, P. M. Plant Physiol. 1977, 59, 1. Rubin, P. M.; Randall, D. D. Arch. Biochem. Biophys. 1977, 178, 342. In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

22. BRUEMMER

Acetaldehyde and Ethanol AccumulationinCitrus Fruit

20.

Randall, D. D.; Williams, M.; Rapp, B. J. Arch. Biochem. Biophys. 1981, 207, 437. 21. Bruemmer, J. H.; Roe, B. Phytochem. 1986, in press. 22. Henry, M.; Nyns, E. Sub-Cell. Biochem. 1975, 4·, 1. 23. Nakamura, K.; Asahi, T. Arch. Biochem. Biophys. 1976, 174, 393. 24. Maeshima, M.; L i , H.; Asahi, T. Plant & Cell Physiol. 1984, 25, 999. 25. Bahr, J. T.; Bonner, W. D., Jr. J. Biol. Chem. 1973, 248, 3441. 26. Robertson, R. N. "The Lively Membranes"; Cambridge University: Cambridge, 1983; p. 136.


RECEIVED February 23, 1986


Regulation of Acetaldehyde and Ethanol Accumulation in Citrus Fruit

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