Citrus Nutrition and Quality - American Chemical Society

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Downloaded by UCSF LIB CKM RSCS MGMT on December 3, 2014 | http://pubs.acs.org Publication Date: December 15, 1980 | doi: 10.1021/bk-1980-0143.ch008

Relationship of Citrus Enzymes to Juice Quality JOSEPH H. BRUEMMER U.S. Citrus and Subtropical Products Laboratory, U.S. Department of Agriculture, Science and Education Administration, Southern Region, P.O. Box 1909, Winter Haven, FL 33880

The quality of citrus fruit represents the sum total of fruit development--fruit set, growth, tissue differentiation and ripening on the tree. During this development period compounds are formed that are responsible for the color and flavor of the ripe fruit. Many of these compounds have been identified, and research is now directed toward identifying the enzymic reactions that regulate their biosynthesis through metabolic pathways. Stewart (1) recently reviewed the carotenoid pigments identified as present in citrus and assessed their contribution to citrus color. Reported with that review in the same journal was the mode of action of bioregulators in controlling carotenoid biosynthesis through enzyme inhibition (2). Many terpenoids, aliphatic esters and aliphatic aldehydes in citrus fruit have been identified, but the pathways for their biosynthesis have not yet been determined (3). Bruemmer et al. (4) recently reviewed the working hypotheses on the mechanism for regulating the biosynthesis of citrus acids and concluded that supportive data were inadequate to identify the enzyme reactions that regulate acid metabolism in citrus. The q u a l i t y of extracted c i t r u s j u i c e s depends on enzyme r e a c t i o n s that occur not only i n the f r u i t during the development p e r i o d , but a l s o i n the j u i c e during p r o c e s s i n g . When j u i c e i s e x t r a c t e d from c i t r u s f r u i t , enzymes are released from t h e i r normal r e s t r a i n t i n the c e l l . Several of these enzymes c a t a l y z e r e a c t i o n s that adversely a f f e c t t a s t e and appearance of the j u i c e . Unless the r e a c t i o n s are c o n t r o l l e d , the j u i c e products w i l l not meet the standards of q u a l i t y set up by the USDA Food Safety and Quality Service. The two r e a c t i o n s of commercial importance are the h y d r o l y s i s of p e c t i n to p e c t i c a c i d , which c l a r i f i e s j u i c e , and the l a c t o n i z a t i o n of limonoic a c i d Α - r i n g lactone to the b i t t e r compound, l i m o n i n . Research e f f o r t s to i d e n t i f y and c h a r a c t e r i z e the r e a c t i o n s , to i s o l a t e and p u r i f y the enzymes, and to develop methods to c o n t r o l the r e a c t i o n s are described i n t h i s review.

This chapter not subject to U.S. copyright. Published 1980 American Chemical Society In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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3.1.1.11)

E a r l y i n the development of the c i t r u s j u i c e p r o c e s s i n g i n d u s t r y , c l a r i f i c a t i o n of b o t t l e d and canned c i t r u s j u i c e s and c i t r u s beverages was recognized as r e s u l t i n g from the a c t i o n of p e c t i c enzyme(s) that had not been destroyed by heat p a s t e u r i z a t i o n (5). I n v e s t i g a t i o n s of cause and prevention of c l a r i f i c a t i o n and g e l a t i o n i n c i t r u s j u i c e s and t h e i r concentrates were stimulated by the r a p i d expansion of the frozen-orange conc e n t r a t e i n d u s t r y i n the l a t e f o r t i e s . These i n v e s t i g a t i o n s were thoroughly reviewed by J o s l y n and P i l n i k (6). During the past twenty years some progress was made on p u r i f i c a t i o n and mode of a c t i o n of p e c t i c enzymes (7)· More r e c e n t l y , m u l t i p l e forms of p e c t i n e s t e r a s e (PE) that d i f f e r i n k i n e t i c p r o p e r t i e s and temperat u r e s t a b i l i t y have been i s o l a t e d (8). Some of the i r r e g u l a r i t i e s i n heat i n a c t i v a t i o n of PE and j u i c e cloud s t a b i l i t y can be a t t r i b u t e d to the a c t i o n s of the d i f f e r e n t forms of PE. The more recent research on PE and the older work on c i t r u s j u i c e s t a b i l i z a t i o n are reviewed to r e l a t e PE to the problem of j u i c e q u a l i t y . J u i c e C l a r i f y i n g Enzyme. One of the e a r l i e s t reports on a c l a r i f y i n g and c l o t t i n g enzyme i n orange j u i c e was by Cruess (9). He reported that f r e s h orange j u i c e formed a j e l l y - l i k e suspension a few hours a f t e r expression, and that a f t e r a few days the suspended matter coalesced and s e t t l e d , l e a v i n g a c l e a r supernatant l i q u i d . He speculated that heating the j u i c e to 85°C (185°F) destroyed enzyme(s) and prevented the j u i c e from c l e a r i n g . J o s l y n and Sedky (10) showed that c l a r i f i c a t i o n of c i t r u s j u i c e s was always accompanied by decomposition of p e c t i c substances and used the rate of c l a r i f i c a t i o n as a measure of the a c t i v i t y of the p e c t i c enzymes. They (11) found that the rate at which heat p a s t e u r i z e d c i t r u s j u i c e s cleared v a r i e d i n v e r s e l y with both temperature and d u r a t i o n of the heat treatment. They showed that h e a t i n g orange j u i c e to 80°C (176°F) at pH 4 f o r 1 min i n a c t i v a t e d the c l e a r i n g enzyme and that i n a c t i v a t i o n was more r a p i d at pH 2.5 than at pH 4.0. Stevens (12) elaborated on the pH-temperature r e l a t i o n s h i p i n h i s patent f o r s t a b i l i z i n g c i t r u s j u i c e products by s e t t i n g f o r t h four s p e c i f i c ranges of minimum temperatures for short time (0.1 to 3 min) heating of j u i c e s at pH 2.2 to 3.79. A l s o he speculated that c i t r u s j u i c e s contain two cloud-coagulating enzymes of d i f f e r e n t t h e r m o s t a b i l i t i e s . One enzyme, most a c t i v e at low pH and temperature, appeared to be destroyed by h e a t i n g the j u i c e at 65 to 70°C (149 to 158°F). The second enzyme, most a c t i v e at pH 3.0 to 3.3 and about 35°C (95°F), appeared to r e q u i r e h e a t i n g to 88°C (191°F) f o r i n a c t i v a t i o n (12). Stevens (13) d e s c r i b e d a r a p i d t e s t f o r p e c t i c enzymes i n c i t r u s j u i c e . It i n v o l v e d adding p e c t i n under c o n t r o l l e d c o n d i t i o n s of temperature and sample p r e p a r a t i o n , and measuring the time r e q u i r e d f o r flocculation. Stevens, and coworkers (14) f u r t h e r elaborated on the patent work (12, 13). They produced a trend curve of the

In Citrus Nutrition and Quality; Nagy, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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minimum temperatures required to s a t i s f a c t o r i l y i n a c t i v a t e p e c t i c enzymes i n n a t u r a l strength c i t r u s j u i c e s of d i f f e r e n t pHs. Their data i n d i c a t e d that the pulp content of j u i c e was the most important f a c t o r determining the rate and completeness of f l o c c u l a t i o n . Pulpy j u i c e required higher temperature f o r cloud s t a b i l i z a t i o n . They a l s o found that the p a s t e u r i z a t i o n c o n d i t i o n s necessary to s t a b i l i z e n a t u r a l strength or 42°Brix concentrated orange j u i c e f o r frozen storage (-23.3°C; -10°F) was about 65°C (149°F) f o r 1 min. They reported that these c o n d i t i o n s c o i n c i d e d with the minimum c o n d i t i o n s necessary to destroy spoilage organisms. A q u a n t i t a t i v e o b j e c t i v e measurement of c i t r u s j u i c e t u r b i d i t y was used by L o e f f l e r (15, 16) to show that p e c t i c enzyme changes occurred so r a p i d l y a f t e r the j u i c e was reamed from the f r u i t that at l e a s t a p a r t i a l coagulation of the cloud occurred before the j u i c e could be screened, deaerated and heated to a p a s t e u r i z a t i o n temperature. He showed that j u i c e t u r b i d i t y was increased by f l a s h - p a s t e u r i z a t i o n and a l s o by homogenization of the j u i c e before p a s t e u r i z a t i o n . L o e f f l e r (15, 16) presented data on t u r b i d i t y of f l a s h - p a s t e u r i z e d c i t r u s j u i c e s (heat exposure f o r 16 to 18 sec) a f t e r storage at s e v e r a l temperatures. He found that "samples p a s t e u r i z e d at 91°C (196°F) l o s t t h e i r cloud when stored at 35°F (95°F) but others p a s t e u r i z e d at 93-95°C (199-203°F) r e t a i n e d t h e i r cloud almost i n d e f i n i t e l y " . E x t r a c t i o n and I d e n t i f i c a t i o n . I d e n t i f i c a t i o n of PE as the c l e a r i n g enzyme i n c i t r u s j u i c e s progressed r a p i d l y a f t e r MacDonnell et a l . (17) reported on c a t i o n requirement f o r e x t r a c t i o n and s o l u b i l i z a t i o n of the enzyme from various p o r t i o n s of the f r u i t . PE was assayed by the method introduced by Kertesz (18) and modified by Lineweaver and B a l l o u (19). The method involved measuring the rate at which the methyl e s t e r groups i n the p e c t i n molecule are hydrolyzed by t i t r a t i n g the f r e e carboxyl groups with 0.1N NaOH as they are formed. One u n i t of PE was defined as the amount of enzyme which w i l l hydrolyze 1 meq carboxyl groups per min from a 0.5% s o l u t i o n of p e c t i n i n 0.15M NaCl at pH 7.5, 30°C (86°F). McDonnell et a l . (17) showed that the enzyme e x t r a c t e d from orange flavedo had a rather broad pH optimum at about 7.5 i n 0.15M NaCl, but that the enzyme required high i o n i c s t r e n g t h s o l u t i o n s at lower pH to be o p t i m a l l y a c t i v e . For 0.15M CaCl^ the plateau of optimum a c t i v i t y extended from pH 4.0 to 8.5 but a c t i v i t y was only about 50% of the l e v e l of a c t i v i t y found f o r 0.05M CaCl^ at pH 7.5. The increased a c t i v i t y caused by c a t i o n was greater at pH 5 than at pH 7.5 with 0.05M C a C l . 2

Occurrence and D i s t r i b u t i o n . PE was found a s s o c i a t e d with s t r u c t u r a l elements of the orange. McDonnell et a l . (17) reported no a c t i v i t y i n f i l t e r e d orange j u i c e , but found 58, 44 and 28 PE u n i t s per kg wet t i s s u e i n flavedo, albedo and c e l l sacs r e s p e c t i v e l y . Working with four v a r i e t i e s of F l o r i d a oranges and Dancy tangerine, Rouse (20) showed that j u i c e sacs had the highest



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a c t i v i t y and c e n t r i f u g e d (not c l a r i f i e d ) j u i c e the l e a s t . The r a g (segment w a l l t i s s u e e n c l o s i n g the j u i c e sacs) had the next highest a c t i v i t y followed by the peel (flavedo and albedo). In g r a p e f r u i t he found that j u i c e sacs had the highest a c t i v i t y but that albedo and flavedo had more a c t i v i t y than the rag. The d i s t r i b u t i o n of PE i n lemon and lime t i s s u e s was somewhat d i f f e r e n t from that i n orange or g r a p e f r u i t . Rouse and Atkins (21) showed that lemon and lime p e e l (flavedo plus albedo) had the highest a c t i v i t y followed by j u i c e sacs and r a g i n that order. P r e v i o u s l y , Rouse (22) found that j u i c e samples prepared to contain i n c r e a s i n g l y higher amounts of pulp ( j u i c e s a c s ) , a l s o had correspondingly higher PE a c t i v i t i e s . Rouse, Atkins and Huggart (23) showed that PE a c t i v i t y i n orange j u i c e was d i r e c t l y p r o p o r t i o n a l to pulp content. Orange PE was shown to be bound to c e l l w a l l s as an enzymesubstrate complex with p e c t i n (24). The s o l u b i l i z a t i o n of PE (pH 7.5 and 0.15N NaCl) and d e - e s t e r i f i c a t i o n of c e l l w a l l p e c t i n were s i m i l a r l y temperature dependent. A f t e r complete d e - e s t e r i f i c a t i o n of the c e l l w a l l p e c t i n , an e q u i l i b r i u m was e s t a b l i s h e d between bound and f r e e PE i n the e x t r a c t i o n medium. At the pH o f j u i c e (4.5 and below) the enzyme bound to c e l l w a l l s was not s o l u b i l i z e d unless s o l u b l e p e c t i n was added. The bound enzyme was i n a c t i v e at pH 4, whereas the f r e e s o l u b l e enzyme was about 20% more a c t i v e at pH 4 than at the optimum pH (7.5). Assays f o r PE. Because PE hydrolyzes p e c t i n t o p e c t i c a c i d and methanol, the enzyme concentration can be assayed by measuring the r a t e at which f r e e carboxyl groups or methanol i s released from the substrate. Kertesz (18) t i t r a t e d the f r e e carboxyl groups as they were formed by the a c t i o n o f the enzyme on p e c t i n . He used methyl red to i n d i c a t e the pH (6.2) and added 0.1N NaOH at frequent i n t e r v a l s to maintain the pH r e l a t i v e l y constant f o r 30 min. The pH meter replaced the use of i n d i c a t o r s i n subsequent m o d i f i c a t i o n s (17, 19). Current procedures use automatic pH t i t r a t o r s to t i t r a t e a l k a l i at constant pH (25). A blank i s used to c o r r e c t f o r the consumption of a l k a l i due to i t s r e a c t i o n with atmospheric C 0 , or the r e a c t i o n s o l u t i o n i s protected from CO- with a blanket o f N^. The broad pH optimum f o r PE was used By Somogyi and Romani (26) t o devise a r a p i d assay based on the r a t e at which the pH of unbuffered 1% p e c t i n i n 0.2M NaCl changed from 7.0 to 7.5 a f t e r the a d d i t i o n o f the enzyme. At low enzyme concentration the r e a c t i o n r a t e was f i r s t order f o r the_ f i r s t 1-min i n t e r v a l , and r e p r o d u c i b i l i t y was w i t h i n - 5%. Methanol can be determined c o l o r i m e t r i c a l l y but must u s u a l l y be d i s t i l l e d from the r e a c t i o n mixture before being analyzed. Wood and S i d d i q u i (27) described a simple and p r e c i s e s p e c t r o p h o t o m e t r y method f o r measuring methanol i n an assay f o r PE. The method involved permanganate o x i d a t i o n of methanol to formaldehyde and r e a c t i o n o f the formaldehyde with pentane-2,4-dione. The c o l o r r e a c t i o n was developed d i r e c t l y i n the PE r e a c t i o n mixture without i n t e r f e r e n c e from the p e c t i n . However, Termote e t a l . (28) 2


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increased the s e n s i t i v i t y and r e p r o d u c i b i l i t y of the method by developing the c o l o r with methanol d i s t i l l e d from the PE r e a c t i o n mixture. Gas chromatographic (GC) analyses are more s e n s i t i v e and r e p r o d u c i b l e than c o l o r i m e t r i c analyses. Gessner (29) developed a method f o r r e a c t i n g n i t r o u s a c i d with the a l c o h o l s i n e x t r a c t s of t i s s u e homogenates and then analyzing the head space by GC. The d e r i v a t i v e s formed have higher vapor pressures than the a l c o h o l s so that s e n s i t i v i t y of head space analyses i s increased according ly. Gessner claimed good r e p r o d u c i b i l i t y of values with concen t r a t i o n s of methanol at 1 mg/1. The d e r i v a t i z a t i o n method was used to assay PE i n plant t i s s u e (30) . A probable e r r o r of 2% was claimed, and s e n s i t i v i t y was 3 mg/1. The most s e n s i t i v e assay method f o r PE a c t i v i t y i s to use, as s u b s t r a t e , p e c t i n e s t e r i f i e d b i o l o g i c a l l y (31) or chemically (32) with C methanol. In s t u d i e s by Gessner (29) and Bartolome and Hoff (30) the substrate and enzyme were p r e c i p i t a t e d from the reacj^on mixture with a c i d i f i e d ethanol or TCA before a c t i v i t y of the C methanol i n the supernate was determined. The r a d i o a c t i v e assay method was about 100 times as s e n s i t i v e as the t i t r i m e t r i c method f o r PE a c t i v i t y (32). The r a d i o a c t i v e method may be used when the t i t r a t i o n method i s not a p p l i c a b l e , e.g., when the pH of the r e a c t i o n i s near the pK of p e c t i n (about 4.0), or when the r e a c t i o n r a t e i s low because of l i m i t e d amount of enzyme or substrate. However, the automatic t i t r a t i o n method when a p p l i c a b l e , i s advantageous because the r e a c t i o n can be monitored continuously and any d e v i a t i o n s from l i n e a r i t y r e a d i l y recognized. Purification. Orange PE was i s o l a t e d i n i t i a l l y from Navel orange flavedo by e x t r a c t i o n with a borate-acetate b u f f e r at pH 8.2 and then p r e c i p i t a t i o n from the e x t r a c t with (NH,) SO^ (17). L a t e r MacDonnell et a l . (33) used (NH,) S0^ to f r a c t i o n a t e the flavedo e x t r a c t (pH 7) and adsorbed the 40 to 80% s a t u r a t i o n f r a c t i o n on f i l t e r paper pulp (1-2 g/1). PE was extracted from the paper pulp with 0.1M NaCl and t r a n s f e r r e d to a C e l i t e 505 column (0.4U PE/g). The column was f i r s t washed with 0.025M Na HP0^, and the PE was then eluted from the column with 1.0M NaCl c o n t a i n i n g 0.025M Na HP0,. The enzyme was p r e c i p i t a t e d from the e x t r a c t with ( N H ^ S O ^ (90% s a t . , pH 7), s o l u b i l i z e d , d i a l y z e d and then t r a n s f e r r e d to a D u c i l column (0.75U PE/g). The column was washed w i t h 0.025M Na^HPO,, and PE was e l u t e d from the column with 1.0M NaCl c o n t a i n i n g 0.025M Na^PO^. The eluate was d i a l y z e d and f r e e z e - d r i e d ; and i t s powder r e f r i g e r a t e d . This procedure p u r i f i e d orange PE over 100-fold on a t o t a l Ν b a s i s . More r e c e n t l y , Manabe (34) p u r i f i e d PE from C i t r u s n a t s u d a i d a i f r u i t by chromatography on a DEAE-cellulose column followed by Sephadex G-100 column adsorption of the a c t i v e f r a c t i o n . The f i n a l preparation was homogeneous as determined by d i s c e l e c t r o p h o r e s i s and was 460 times as a c t i v e as the o r i g i n a l e x t r a c t on a p r o t e i n b a s i s . 2

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Specificity. Orange PE hydrolyzed methyl and e t h y l e s t e r s of polygalacturonans, but at optimal pH, the r a t e on e t h y l e s t e r s was about 10% f a s t e r than that on the methyl e s t e r s (33, 35). At pH 4 the r a t e s were about equal (35). G l y c y l and g l y c e r y l e s t e r s of polygalacturonans were not hydrolyzed by orange PE (35, 36). Nongalacturonate e s t e r s (over 50 tested) were not hydrolyzed by the p u r i f i e d PE p r e p a r a t i o n of MacDonnell et a l . (33). P u r i f i e d c i t r u s PE d i d not hydrolyze methyl e s t e r s of d i g a l a c t u r o n i c or t r i g a l a c t u r o n a c i d but r e a d i l y hydrolyzed polymeric u n i t s of 10 or more (37). So f a r , the minimum chain length of the o l i g o galacturonan methyl e s t e r s required f o r PE a c t i o n has not been determined. P a r t i a l reduction of the galactopyranosiduronate s t r u c t u r e by NaBH^ decreased PE a c t i o n markedly, i n d i c a t i n g s p e c i f i c i t y of the carboxyl group f o r enzyme r e a c t i o n (38). A c t i v a t i o n - I n h i b i t i o n and Function In Vivo. When 0.15M NaCl was added to an orange PE-pectin r e a c t i o n mixture at pH 7.5, a c t i v i t y was increased 5 - f o l d , and at pH 5 i t was increased 100f o l d (17). As explained by Lineweaver and B a l l o u (19), NaCl caused the apparent a c t i v a t i o n by f r e e i n g the enzyme from the i n a c t i v e i o n i c complex ( p e c t i n - c a r b o x y l ) . They showed that at pH 5.7 p e c t i c a c i d i n h i b i t e d a l f a l f a PE a c t i v i t y 55% i n 0.015M NaCl but only 17% i n 0.2M NaCl. At pH 8.5 p e c t i c a c i d i n h i b i t e d PE a c t i v i t y only 9% i n 0.015 NaCl. They concluded that the s t i m u l a t i o n of a c t i v i t y by c a t i o n s at low pH (17) d i d not show that c a t i o n s were e s s e n t i a l f o r a c t i v i t y , but, r a t h e r , that c a t i o n s f u n c t i o n by preventing product i n h i b i t i o n , which i s greater at low pH. Termote et a l . (28) studied product i n h i b i t i o n of orange PE as an approach to s t a b i l i z i n g cloud of orange j u i c e . Both chemically and enzymically prepared hydrolysates of p e c t i c a c i d with an average degree of polymerization of 8 or higher i n h i b i t e d PE a c t i v i t y at pH 5 i n 0.1M NaCl. Unhydrolyzed p e c t i c a c i d showed the greatest i n h i b i t i o n . No i n h i b i t i o n was observed when the i o n i c s t r e n g t h of the r e a c t i o n mixture was increased to 0.5M NaCl. P e c t i c a c i d hydrolysates with degree of polymerization of 8 to 15 were e f f e c t i v e i n extending the period of cloud s t a b i l i t y i n PE a c t i v e j u i c e s by a f a c t o r of 3 to 5 but d i d not prevent the j u i c e s from c l a r i f y i n g e v e n t u a l l y . Versteeg (8) speculated on the f u n c t i o n o f PE i n v i v o . He noted the high a c t i v i t y of PE i n c i t r u s f r u i t compared to the amount of a v a i l a b l e p e c t i n . The f r u i t contain s u f f i c i e n t a c t i v i t y to d e e s t e r i f y the p e c t i n to low methoxy p e c t i n i n 10 min at optimum pH. He suggested that the methyl t r a n s f e r a s e found by Kauss and Hassid (39) to e s t e r i f y p e c t i c a c i d to p e c t i n i n mung bean shoots and to be l o c a t e d i n a lipid-membrane complex (31) functioned as p e c t i n e s t e r a s e a f t e r the l i p i d membranes were destroyed and the environment changed. However, no d e f i n i t i v e experiments to e s t a b l i s h the r o l e of PE i n f r u i t s were reported.



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Isoenzymes. M u l t i p l e forms of c i t r u s PE were reported by Evans and McHale (40) and Versteeg et a l . (41). PE was p u r i f i e d from West Indian limes and Navel oranges by f r a c t i o n a t i o n of the whole f r u i t e x t r a c t s with (NH.^SO^ (40-65%), adsorption and e l u t i o n from Sephadex G-75 columns (40). The PE a c t i v e f r a c t i o n s were combined and concentrated before separation i n t o two a c t i v e PEs on the b a s i s of t h e i r e l u t i o n volume from a DEAE Sephadex A-50 column. Orange PEI (OPEI) and lime PEI (LPEI) had the same e l u t i o n volume; a l s o OPEII and LPEII had the same e l u t i o n volume. A higher c o n c e n t r a t i o n of NaCl was required at a l l pH values f o r optimum a c t i v i t y of OPEI and LPEI than of OPEII and LPEII. When the component parts of oranges were s e p a r a t e l y analyzed chromâtοgraphi c a l l y with DEAE-Sephadex A-50, OPEI was detected only i n the p e e l , whereas OPEII was i d e n t i f i e d i n j u i c e sacs and s e c t i o n w a l l s (40). Versteeg et a l . (41) p u r i f i e d e x t r a c t s of Navel orange pulp and p e e l by (NH^^SO^ f r a c t i o n a t i o n (30 to 75% s a t u r a t i o n ) and chromatography on Bio-Gel P-100. The enzyme p r e p a r a t i o n e l u t e d from Bio-Gel P-100 was separated i n t o PEI and P E U by chromato graphy on c r o s s - l i n k e d pectate (42) . Each PE was f u r t h e r p u r i f i e d by chromatography on CM Bio-Gel. PEI d i d not bind as s t r o n g l y as PEII to e i t h e r the c r o s s - l i n k e d pectate or CM B i o - G e l . The a c t i v i t y of the p u r i f i e d PEI and PEII combined was about 26% of the a c t i v i t y of the crude e x t r a c t ; and PEI had about twice the a c t i v i t y of PEII. Both enzymes had molecular weight of 36,200 as determined from t h e i r m o b i l i t y based on dodecyl s u l f a t e e l e c t r o p h o r e s i s . PEI had optimum a c t i v i t y at pH 7.6 and PEII at pH 8.0. A l s o , PEI was r e l a t i v e l y more a c t i v e at low pH v a l u e s . PEI had low a f f i n i t y f o r p e c t i n and was weakly i n h i b i t e d by polygalacturonate. PEII had high a f f i n i t y and was s t r o n g l y i n h i b i t e d . Rombouts et a l . (42) synthesized c r o s s - l i n k e d pectate with 0.46 degree of c r o s s - l i n k i n g and was able to get strong b i n d i n g of both PEI and PEII to the pectate. Through PE a c t i v i t y measurements on m e t h y l - e s t e r i f i e d c r o s s - l i n k e d pectate they concluded that b i n d i n g of the enzymes to the pectate matrix involved b i o s p e c i f i c a f f i n i t y as w e l l as i o n exchange e f f e c t s . Versteeg (8) i s o l a t e d a t h i r d isoenzyme of Navel oranges and found that i t was more heat s t a b l e than e i t h e r PEI or PEII and had an i s o e l e c t r i c point s i m i l a r to that of PEI (pH 10.05). The s p e c i f i c a c t i v i t y of the heat s t a b l e f r a c t i o n was low compared to that of PEI or PEII and was not improved much by B i o Gel P-100 chromatography. The molecular weight of t h i s t h i r d PE isoenzyme was estimated to be 54,000 and was c a l l e d high molecular weight (HM-PE) by the author (8). C r o s s - l i n k e d pectate chromato graphy separated HM-PE i n t o three f r a c t i o n s . HM-PEII contained about 85% of the a c t i v i t y of the o r i g i n a l HM-PE and was p u r i f i e d about 20-fold by c r o s s - l i n k e d pectate chromatography (8). Thinl a y e r pH gradient e l e c t r o p h o r e s i s of crude PE e x t r a c t s from d i f f e r e n t c i t r u s f r u i t (oranges, lemon, g r a p e f r u i t and tangerine) gave good separation of PEI, PEII and HM-PEII and revealed a c t i v i t y peaks corresponding to a t o t a l of 12 forms of PE. However PEI and PEII accounted f o r more than 80% of the a c t i v i t y i n oranges (8).



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Mode of A c t i o n of PEI and PEII. Versteeg (8) measured r e a c t i o n rates of PEI and PEII with p e c t i n s p r e s a p o n i f i e d so that degree of e s t e r i f i c a t i o n (DE) ranged from 95.6 to 32%. He found that maximum v e l o c i t i e s (Vmax) f o r the r e a c t i o n s with a l l the substrates were about the same. However, the a f f i n i t y of PEI and PEII to the substrates increased d r a m a t i c a l l y as the DE of the p e c t i n s decreased. A f f i n i t y of PEII f o r p e c t i n was almost 150-fold greater f o r p e c t i n with DE of 32% than of 95.6%. The l o g of a f f i n i t y c o r r e l a t e d with the l o g of percentage free carboxyl groups i n the p e c t i n f o r both PEI and PEII even though a f f i n i t y of PEII was much l a r g e r than that of PEI f o r the p e c t i n s . The slopes of the c o r r e l a t i o n equation were 2.08 f o r PEI and 2.03 f o r PEII, i n d i c a t i n g that doubling the number of f r e e carboxyl groups gave a f o u r - f o l d increase i n a f f i n i t y . Versteeg (8) c a l c u l a t e d the percentage of p o s s i b l e u n i t s that had two carboxyl groups f o r p e c t i n s with DEs of 95 to 32%. He found that l o g % u n i t s w i t h two carboxyl groups i n some f i x e d arrangement c o r r e l a t e d (slope equals 2) w i t h the l o g % f r e e carboxyl groups i n the p e c t i n . Because PE i s i n h i b i t e d by g a l a c t u r o n i c a c i d oligomers with degree of polymerization of 8 or more (28) he concluded that a substrate with two f r e e carboxyl groups separated by s i x monomers was required f o r the optimal enzyme-substrate complex. Versteeg (8) found that PEI and PEII were s i m i l a r i n the products they cleaved from p e c t i n with DE of 95.6%. Separation of r e a c t i o n products a f t e r t h i s substrate was d e - e s t e r i f i e d to DE of 60% showed that about 30% of the substrate was not acted on by the enzyme. In contrast a l l the e s t e r bonds i n p e c t i n with DE of 95.6% could be hydrolyzed by a l k a l i . PEI and PEII d i d not completely d e - e s t e r i f y p e c t i n s with DE ranging from 95.6 to 32%. The lower the i n i t i a l DE of the s u b s t r a t e , the lower was the DE of the f i n a l product of the r e a c t i o n . However, none of the products from PEI and PEII r e a c t i o n s with p e c t i n s (DE 95.6 to 32%) had DEs of l e s s than 11%. Solms and Deuel (38) e a r l i e r noted that 11% DE was the lower l i m i t f o r d e - e s t e r i f i c a t i o n of p e c t i n by PE. S t a b i l i t y of PE Isozymes. The p u r i f i e d forms of PE l o s t only 15% of t h e i r a c t i v i t i e s during 2 years at 4°C (40°F) with 0.1M NaCl i n phosphate b u f f e r (pH 7.5) (8). At 30°C (86°F) PEI was s t a b l e f o r 24 hr at pH 4 and 7, but PEII l o s t a l l a c t i v i t y w i t h i n 6 hr at pH 4. Tested at 30°C (86°F) i n s i n g l e - s t r e n g t h j u i c e r e c o n s t i t u t e d from concentrate, a c t i v i t y of PEI and crude orange PE d e c l i n e d but HM-PE r e t a i n e d i t s a c t i v i t y over the 15 days storage. Versteeg (8) t e s t e d the heat s t a b i l i t y of the p u r i f i e d PEs i n orange j u i c e , pH 4.0, and found that PEII was the l e a s t s t a b l e , being completely i n a c t i v a t e d at 55°C (131 F); PEI was i n a c t i v a t e d at 65°C (149°F), and HM-PE at 85°C (185°F). Heat i n a c t i v a t i o n curves of a crude preparation of orange PE i n orange j u i c e i n d i c a t e d that about 5% of the t o t a l PE a c t i v i t y was due to HM-PE, 60% to PEI and 30% to PEII. e



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Cloud S t a b i l i t y and PE A c t i v i t y . The three forms o f PE were t e s t e d f o r d e s t a b i l i z a t i o n o f orange j u i c e cloud (8). Only PEI and HM-PE were a c t i v e at 5°C (41°F) and 30°C (86°F). PEI was much l e s s a c t i v e than HM-PE a t 5°C (41°F). Versteeg (8) concluded that HM-PE a c t i v i t y was r e s p o n s i b l e f o r d e s t a b i l i z i n g orange j u i c e during storage and that cloud s t a b i l i z a t i o n required heating j u i c e t o 90°C (194°F) because HM-PE r e t a i n e d a c t i v i t y i n j u i c e heated to l e s s than that temperature. The heat s t a b i l i t y of the a c t i v e HM-PE a l s o accounted f o r the observations on i r r e g u l a r heat i n a c t i v a t i o n and cloud s t a b i l i t y patterns (43, 44·, 45^, 46). B i s s e t t e t a l . (45) reported that heating orange j u i c e to 82°C (180°F) i n a c t i v a t e d 94 to 95% o f the PE a c t i v i t y but d i d not g r e a t l y improve cloud s t a b i l i t y o f s i n g l e - s t r e n g t h or concentrated orange j u i c e . A s i m i l a r report by Atkins et a l . (46) showed that 90% i n a c t i v a t i o n of PE a c t i v i t y of g r a p e f r u i t j u i c e heated to 85°C (185°F) d i d not prevent g e l a t i o n and c l a r i f i c a t i o n i n the concentrate. I d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of HM-PE as the heat t o l e r a n t enzyme r e s p o n s i b l e f o r c l a r i f y i n g under-pasteurized c i t r u s j u i c e s represent a major breakthrough i n understanding the r e l a t i o n s h i p between PE and j u i c e q u a l i t y . Progress i n e s t a b l i s h i n g the r o l e of HM-PE i n v i v o , i t s o r i g i n and r e l a t i o n s h i p to PEI and PEII could lead to procedures to c o n t r o l HM-PE formation d u r i n g f r u i t development and maturation. Knowing the d i s t r i b u t i o n of t h i s a c t i v e form of PE i n the s t r u c t u r a l l y defined f r u i t p a r t s could a s s i s t the f r u i t j u i c e t e c h n o l o g i s t to adopt processing c o n d i t i o n s to minimize HM-PE presence i n the product. Limonin D-Ring Lactonase (EC 3.1.1.36) The need to u t i l i z e packinghouse r e j e c t s o f substandardq u a l i t y Navel oranges r e s u l t e d i n i n v e s t i g a t i o n s on the cause and prevention of b i t t e r n e s s i n p a s t e u r i z e d Navel orange j u i c e . Juice from the Navel orange was not b i t t e r when f r e s h l y expressed but became b i t t e r on standing (47). Higby (47) i s o l a t e d s e v e r a l b i t t e r substances from V a l e n c i a and Navel orange pulps, and p u r i f i e d and c h a r a c t e r i z e d one of them as limonin, a substance p r e v i o u s l y i s o l a t e d from seeds of s e v e r a l v a r i e t i e s o f c i t r u s (48). Higby suggested that the precursor of limonin i s limonoic a c i d and that i t i s s t a b l e i n the i n t a c t f r u i t but i s l a c t o n i z e d a t the pH of j u i c e . Emerson (49, 50) compared the r a t e at which b i t t e r n e s s developed i n early-season Navel oranges with the r a t e at which limonin formed from limonoic a c i d i n V a l e n c i a orange j u i c e and i n water at the pH o f the j u i c e and concluded that the precursor i n Navel oranges was probably one of the lactone a c i d s rather than the d i a c i d . He a l s o suggested that i f the d i a c i d were the precursor of limonin, the r e a c t i o n would probably be enzyme catalyzed because a c i d c a t a l y s i s o f the d i a c i d was too slow.



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Maier and Beverly (51) used high voltage e l e c t r o p h o r e s i s to separate limonin, limonoic a c i d and limonoate monolactone i n e x t r a c t s from early-season and l a t e r - s e a s o n Navel oranges and Marsh g r a p e f r u i t . They were able to show that limonoate monolactone was the primary limonoid i n the endocarp and albedo t i s s u e s o f the early-season f r u i t and that f r e e limonin and limonoic a c i d were not n a t u r a l c o n s t i t u e n t s of healthy i n t a c t navel oranges or g r a p e f r u i t . They a l s o showed that the monolactone was not present i n l a t e r season f r u i t . Maier and M a r g i l e t h (52) i d e n t i f i e d the monolactone as limonoic a c i d Α-ring lactone a f t e r separating the A- and D-ring lactones by paper e l e c t r o p h o r e s i s and TLC. They a l s o obtained i n d i c a t i o n s of an enzyme i n c a r p e l l a r y membrane t i s s u e that converted limonoic a c i d Α-ring lactone to limonin. The enzyme was i s o l a t e d and p u r i f i e d from peeled seeds (53) and the r e a c t i o n shown to be s p e c i f i c f o r the D-ring lactone group. The enzyme c a t a l y z e d the l a c t o n i z a t i o n r e a c t i o n at pH 6 and the h y d r o l y t i c r e a c t i o n at pH 8.0. The enzyme was p u r i f i e d about 200-fold and shown to be e s s e n t i a l l y f r e e from contaminating p r o t e i n s by d i s c e l e c t r o p h o r e s i s (53). Hasegawa (54) reported on p r o p e r t i e s of the lactonase. The enzyme p u r i f i e d from g r a p e f r u i t seeds hydrolyzed limonoids that have the D-ring i n t a c t but d i f f e r from limonin i n the v i c i n i t y of the A and A - r i n g s , namely, obacunone, nomilin and ichangin. Limonin D-ring lactonase was shown to be markedly heat r e s i s t a n t , r e t a i n i n g about 30% of i t s a c t i v i t y a f t e r 5 min at 100°C (54). C i t r u s leaves were shown to be the s i t e of limonoic a c i d Ar i n g lactone b i o s y n t h e s i s i n c i t r u s (55). The lactone accumulated to the l e v e l of 2000 ppm i n very small leaves but as the l e a f grew, the lactone content d e c l i n e d . The lactone content o f the f r u i t increased as the l e v e l i n the leaves d e c l i n e d . Hasegawa and Hoagland (55) a l s o showed that limonoic a c i d Α-ring lactone was not synthesized i n the f r u i t but i n the leaves. The r a d i o a c t i v e l a b e l e d lactone was i s o l a t e d from a f r u i t adjacent to a l e a f a c t i v e l y s y n t h e s i z i n g i t from l a b e l e d acetate, i n d i c a t i n g that the lactone was synthesized i n the leaves and transported to the f r u i t (55). Although limonoic a c i d Α-ring lactone has been shown to be a substrate of a lactonase that c a t a l y z e s the formation of limonin i n c i t r u s f r u i t , s e v e r a l other enzymes i n c i t r u s can a l s o use the Ar i n g lactone as substrate, however, the products are not b i t t e r products. These enzymes and t h e i r r e a c t i o n s are reviewed i n the next s e c t i o n . f

Enzymes to Degrade Limonin Precursor Hasegawa et a l . (56) defected the enzymic conversion of 19deoxylimonoic a c i d 3-methyl C e s t e r to the 17-dehydro d e r i v a t i v e by albedo t i s s u e s l i c e s of Navel oranges. They i s o l a t e d the product and i d e n t i f i e d i t by TLC as the r e a c t i o n product formed when the substrate was dehydrogenated by limonoate dehydrogenase (EC 1.1.1)



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i s o l a t e d from c e l l - f r e e e x t r a c t s of Arthrobacter g l o b i f o r m i s (57). They a l s o showed that the 17-dehydro d e r i v a t i v e i s o l a t e d from the r e a c t i o n mixture with the albedo t i s s u e could £g converted back to the substrate, 19-deoxy-limonoic a c i d 3-methyl C e s t e r by b a c t e r i a l limonoate dehydrogenase (56). The c i t r u s and b a c t e r i a l limonoate dehydrogenases both required NAD f o r the dehydrogenase r e a c t i o n (56, 57). Although the c i t r u s enzyme was not i s o l a t e d , i d e n t i f i c a t i o n of the product of the r e a c t i o n as the 17-dehydro d e r i v a t i v e and i s o l a t i o n of 17-dehydrolimonoate Α-ring lactone from Navel orange j u i c e and Navel orange peel (58) i n d i c a t e d that one of the limonoid metabolic pathways i n c i t r u s was the dehydrogenation of carbon 17 i n the l a c t o n e . The 17-dehydrolimonoate Α-ring lactone i s not b i t t e r , so the enzymic d e b i t t e r i n g of f r e s h Navel orange j u i c e with limonoate dehydrogenase from A. g l o b i f o r m i s was suggested (57) and then demonstrated (59). A patent was issued f o r using limonoate dehydrogenase to d e b i t t e r c i t r u s j u i c e s (60). The pH optimum f o r the b a c t e r i a l enzyme a c t i v i t y was much higher (pH 9.5) than j u i c e pH (3.5-4.5), so that the r a t e of d e b i t t e r i n g was very slow (59) i n j u i c e not adjusted to higher pH. Limonoate dehydrogenase i s o l a t e d from a Pseudomonas sp. had a lower pH optimum (pH 8.0) and showed considerable a c t i v i t y even at pH 4 (61). The Pseudomonas enzyme used NADP twice as e f f e c t i v e l y as NAD, and i t s a c t i v i t y was stimulated by ZnCl^. Compared to the A. g l o b i f o r m i s dehydrogenase, the Pseudomonas enzyme was much more s t a b l e at pH 3.5 (62). As l i t t l e as 200 u n i t s of the enzyme decreased the limonin precursor l e v e l i n one l i t e r of f r e s h orange j u i c e to 4 ppm from 21 ppm i n 2 hr at 24°C (75°F). Prospects look good f o r use of the Pseudomonas dehydrogenase to d e b i t t e r orange j u i c e commercially (62). Recently, a n o n s p e c i f i c enzyme capable of degrading the precursor was extracted from Navel orange albedo (63). N i c o l and Chandler (63) used a proximate assay f o r the degrading enzyme; that i s , the substrate was not i d e n t i f i e d except as a limonin precursor. The crude e x t r a c t s from albedo contained degradation a c t i v i t y that was concentrated by 40 to 60% s a t u r a t i o n with (NH^)^SO^. I n s t a b i l i t y of the enzyme prevented f u r t h e r purification. Other Enzymes i n C i t r u s J u i c e s P e c t i n e s t e r a s e and limonin D-ring lactonase are the only enzymes known to c a t a l y z e r e a c t i o n s that adversely a f f e c t the q u a l i t y of c i t r u s j u i c e s . Bruemmer et a l . (64) l i s t e d other enzymes that have been detected i n c i t r u s j u i c e s and described some of the r e a c t i o n s that can occur i n the j u i c e s . None of the r e a c t i o n s appear to n o t i c e a b l y a f f e c t the q u a l i t y of commercial juices. F r e s h l y extracted c i t r u s j u i c e s contain esterase (EC 3.1.1.1) (65^, 66) and phosphatase (EC 3.1.32) (66, 67) a c t i v i t i e s . Native substrates i n orange j u i c e f o r peroxidase


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(EC 1.11.1.7) (68) and diphenol oxidase (EC 1.10.3.1) (69) have been i d e n t i f i e d . The p o t e n t i a l r o l e of p y r u v i c decarboxylase (EC 4 . 1 . 1 . 1 ) c a t a l y z e d r e a c t i o n as a source of acetaldehyde and other aldehydes i n j u i c e was discussed (70). Raymond et a l . (71) i s o l a t e d the decarboxylase from orange j u i c e s e c t i o n s and demonstrated that only 10 to 15% of the enzyme was i n an a c t i v e form. Since the p u r i f i e d enzyme was only a c t i v e w i t h p y r u v i c a c i d and 2 - k e t o b u t y r i c a c i d of the s e r i e s of 2-ketoacids examined, they (71) concluded that the d i r e c t c o n t r i b u t i o n of orange p y r u v i c decarboxylase to the orange v o l a t i l e p r o f i l e was l i m i t e d to acetaldehyde and p o s s i b l y propionaldehyde.

Literature Cited 1. Stewart, I. Citrus color-a review. Proc. Int. Soc. Citriculture, 1977, 1, 308-311. 2. Yokoyama, H.; Hsu, W. J.; Poling, S. M.; Hayman, E.; Debenedict, C. Bioregulators and citrus fruit color. Proc. Int. Soc. Citriculture, 1977, 3, 717-722. 3. Bruemmer, J. H. Aroma substances of citrus fruits and their biogenesis. In "Geruch-und Geschmackstoffe". F. Drawert, ed. Verlag Hans Carl, Nurnberg, Germany, 1975, p 167-176. 4. Bruemmer, J. H.; Buslig, B. S.; Roe, B. Citrus Enzyme Systems: Opportunities for Control of fruit quality. Proc. Int. Soc. Citriculture, 1977, 3, 712-717. 5. Joslyn, Μ. Α.; Marsh, G. L. Some factors involved in the preservation of orange juice by canning. Fruit Prod. J., 1934, 14, 45-50. 6. Joslyn, Μ. Α.; Pilnik, W. Enzymes and enzyme activity. In "The Orange, Its Biochemistry and Physiology". W. B. Sinclair, ed. Univ. of Calif. Press, Berkeley, Calif. 1961, p. 373-435. 7. Rexova-Benkova, L.; Markovic, O. Pectic enzymes. In "Advances in Carbohydrate Chemistry and Biochemistry. R. S. Tipson and D. Horton, eds. Acad. Press, New York, 1976, 33, 323-385. 8. Versteeg, C. Pectinesterases from the orange fruit - their purification general characteristics and juice cloud destabilizing properties. Agric. Res. Rep. 892. 1979, Purdoc, Wageningen, Netherlands. 9. Cruess, W. V. Utilization of waste oranges. Calif. Agr. Exp. Sta. Bull., 1914, 244, 157-170. 10. Joslyn, Μ. Α.; Sedky, A. The relative rates of destruction of pectin in macerates of various citrus fruits. Plant Physiol., 1940, 15, 675-687. 11. Joslyn, Μ. Α.; Sedky, A. Effect of heating on the clearing of citrus juices. Food Res., 1940, 5., 223-232. 12. Stevens, J. W. Method of conserving fruit juices. 1940, U.S. Patent 2,217,261. 13. Stevens, J. W. Method of testing fruit juices. 1941, U.S. Patent 2,267,050.



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Citrus Nutrition and Quality - American Chemical Society

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