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5 Flavonoids and Citrus Quality RUSSELL L. ROUSEFF
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Florida Department of Citrus, Institute of Food and Agricultural Sciences, Agricultural Research and Education Center, P.O. Box 1088, Lake Alfred, FL 33850
Flavonoids are one of the most widely distributed and di verse chemical groups i n the plant kingdom. While flavonoids can be found i n nature in many organisms, from bacteria to higher plants, they are most prevalent i n the higher plants. They have been found i n the roots, stems, flowers, pollen, fruit, seeds, wood and bark. In terms of chemical structure, flavonoids are C compounds arranged C -C -C with the central group usually linked with oxy gen and numbered as shown below: 15
6
3
6
These compounds are differentiated primarily by the oxida tion state of the central three carbon atom u n i t . Thus, as shown in Figure 1, some compounds are c l a s s i f i e d as flavones, flavanones, flavonols, anthocyanins, etc. (The A and Β rings have been l e f t off for clarity). A secondary means of differentiating flavonoids i s by the position and numbers of attached hydroxy, methoxy or sugar units. In c i t r u s , flavonoids usually occur as glycosides, although the polymethoxylated flavones are a notable exception. Metabolically flavonoids can be thought of as a combination of two d i s t i n c t u n i t s : 1) the C5 fragment o f the A r i n g and 2) the C3-C6 fragment o f the Β r i n g . However, w h i l e s i g n i f i c a n t progress has been made i n the areas o f b i o l o g i c a l o r i g i n s and i n t e r r e l a t i o n s h i p s o f f l a v o n o i d compounds the exact b i o l o g i c a l f u n c t i o n o f these compounds remains a mystery. Q u a l i t y i n any food product i s u s u a l l y defined i n terms o f a 0-8412-0595-7/80/47-143-083$06.50/0 © 1980 American Chemical Society Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
CITRUS NUTRITION AND QUALITY
84
II
FLAVONOLS
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C-OH
CH-
C —
XHOH
C-OH
FLAVANONOLS
LEUCOANTHOCYANINS
Figure 1.
FLAVONES
ANTHOCYANINS
FLAVANONES
Flavonoid structures and nomenclature as determined by the central C group (A and Β rings have been left off) 3
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
5.
ROUSEFF
Flavonoids
and
Citrus
Quality
85
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number of f a c t o r s . These f a c t o r s are u s u a l l y evaluated by the human senses, namely, s i g h t , t a s t e , touch and s m e l l . The primary sense i n e v a l u a t i n g a food item i s t a s t e , although other senses, such as s i g h t and s m e l l , can i n f l u e n c e the perception of t a s t e . Flavonoids play a major r o l e i n the determination o f c i t r u s q u a l i t y p r i m a r i l y due to the t a s t e of flavanone neohesperiodosides. Flavonoids do not play a s i g n i f i c a n t r o l e i n the v i s u a l d e t e r mination o f f r u i t or j u i c e q u a l i t y . Although many f l a v o n o i d s i n the p l a n t kingdom are h i g h l y c o l o r e d , most c i t r u s f l a v o n o i d s are c o l o r l e s s . The compounds r e s p o n s i b l e f o r the deep y e l l o w s and oranges i n c i t r u s are c a r o t e n o i d s . Flavonoids play a s i g n i f i c a n t r o l e i n human n u t r i t i o n but s i n c e t h i s area i s the s u b j e c t of chapter 3, the present d i s c u s s i o n w i l l be l i m i t e d to how f l a vonoids a f f e c t c i t r u s q u a l i t y . P h y s i c a l and Chemical C h a r a c t e r i s t i c s Chemical and p h y s i c a l p r o p e r t i e s of i n d i v i d u a l c i t r u s f l a vonoids and f l a v o n o i d s i n general have been discussed i n other reviews U-5.). In the f l a v o n o i d molecule the A and Β r i n g s are s t a b l e and r e l a t i v e l y u n r e a c t i v e . The c e n t r a l C3 group i s the most r e a c t i v e p o r t i o n o f the molecule, e s p e c i a l l y i f l i n k e d w i t h oxygen to form a h e t e r o c y c l i c r i n g . The dihydropyrone r i n g of flavanones i s very r e a c t i v e and r e a d i l y undergoes r i n g opening r e a c t i o n s w i t h base or a c e t i c anhydride ( 3 ) . However, f l a v o n o i d s are g e n e r a l l y s t a b l e under normal processing or storage c o n d i t i o n s . Flavonoid p h y s i c a l p r o p e r t i e s , such as UV absorption spectrum and s o l u b i l i t y are h i g h l y dependent on the bonding and arrangement of the atoms i n the C group. S i g n i f i c a n t s h i f t s are observed i n the absorption maxima from the UV spectra of f l a v o noids -when r i n g opening o r complexing reagents are i n t r o d u c e d . Thus, much s t r u c t u r a l i n f o r m a t i o n can be i n f e r r e d from the spec t r a l changes of these compounds ( 6 ) . There i s l i t t l e enzymatic a c t i v i t y to a l t e r the f l a v o n o i d composition during storage as the f r e s h l y e x t r a c t e d j u i c e i s p a s t e u r i z e d to i n a c t i v a t e most enzymes. 3
H e s p e r i d i n S o l u b i l i t y . H e s p e r i d i n , a t a s t e l e s s flavanone g l y c o s i d e , i s the l e a s t s o l u b l e of a l l c i t r u s f l a v o n o i d s . It is found i n p r a c t i c a l l y every v a r i e t y of c i t r u s (5) and i s the major f l a v o n o i d i n sweet oranges and lemons. In f r u T t or l e a v e s , h e s p e r i d i n i s found as a s o l u b l e complex which can be e x t r a c t e d w i t h water or a l c o h o l ( 5 ) . During j u i c e e x t r a c t i o n , the complex i s destroyed and hesperTdin s l o w l y p r e c i p i t a t e s as f i n e , w h i t e , needle-shaped c r y s t a l s . Once i n the s o l i d form, h e s p e r i d i n can be r e d i s s o l v e d i n formamide, p y r i d i n e or i n d i l u t e a l k a l i . Hesperidin c r y s t a l s are found i n f r o s t damaged oranges (8) where c e l l s have been d i s r u p t e d due to the formation of i c e c r y s t a l s . I t o c c a s i o n a l l y p r e c i p i t a t e s out o f concentrated orange j u i c e products during storage and i s o f t e n found as a
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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t h i n c r u s t on the surfaces of f a l l i n g f i l m evaporators used i n the production of f r o z e n orange concentrate (9). Hesperidin c r y s t a l s have a l s o been found to coat the surface of j u i c e ex t r a c t o r s . This c r u s t reduces the r a t e o f evaporator heat ex change which adds to the energy costs and slows the r a t e of con c e n t r a t e p r o d u c t i o n . I f the evaporator surfaces are not cleaned p e r i o d i c a l l y , h e s p e r i d i n c r y s t a l s w i l l f l a k e o f f i n t o the con centrated j u i c e . J u i c e c o n t a i n i n g excessive amounts of hesper i d i n c r y s t a l s i s downgraded (10). Shown i n Figure 2 are a c c e p t able and unacceptable amounts of h e s p e r i d i n c r y s t a l s from 710 ml of r e c o n s t i t u t e d orange j u i c e . I t should be pointed out t h a t the presence of h e s p e r i d i n c r y s t a l s i n a c i t r u s product i s a v i s u a l d e f e c t and does not a f f e c t the f l a v o r of the product. Naringin S o l u b i l i t y . N a r i n g i n , the major f l a v o n o i d i n grapef r u i t and pummel ο, has been observed (11) to c r y s t a l i z e i n canned f r u i t or j u i c e during storage. This problem was found p r i m a r i l y when s l i g h t l y immature f r u i t was used. Since n a r i n g i n concen t r a t i o n s are highest i n immature f r u i t and g e n e r a l l y decrease as the f r u i t matures, the e a s i e s t way to prevent t h i s p r e c i p i t a t i o n problem i s to use mature f r u i t . Another means of reducing the n a r i n g i n content i s to reduce or e l i m i n a t e from the f i n a l product those p o r t i o n s of the f r u i t c o n t a i n i n g high n a r i n g i n c o n c e n t r a t i o n s , i . e . , the albedo and segment membrane. Methods of
Analysis
The establishment of p r e c i s e o b j e c t i v e measurements f o r c i t r u s q u a l i t y has been hindered by the l a c k of good a n a l y t i c a l methods and the l a c k of consensus on a common d e f i n i t i o n of q u a l i t y . The e v a l u a t i o n of b i t t e r n e s s i s a prime example. I d e a l l y the method should be r a p i d , s p e c i f i c , accurate and i n expensive. U n f o r t u n a t e l y , no method has been developed t h a t s a t i s f i e s a l l f o u r c r i t e r i a . Compromises and t r a d e - o f f s must be made. One of the e a r l i e s t methods to measure the b i t t e r n a r i n g i n and other flavanones i n g r a p e f r u i t j u i c e was developed by W. B. Davis i n 1947 (12!). This t e s t i s based on the r e a c t i o n of d i l u t e a l k a l i w i t h flavanones to form the corresponding y e l l o w chalcones. The flavanone c o n c e n t r a t i o n i s then determined by measuring the absorbance of the chalcones a t 427 nm. Davis pointed out t h a t the procedure was not s p e c i f i c f o r any flavanone but could be used to determine the p r i n c i p l e flavanones i n c i t r u s j u i c e , i . e . , n a r i n g i n i n g r a p e f r u i t j u i c e and h e s p e r i d i n i n orange j u i c e . He suggested t h a t the method might a l s o be s u i t a b l e f o r the determination of flavones and f l a v o n o l s . This method i s s t i l l widely used to measure n a r i n g i n i n g r a p e f r u i t j u i c e ; a l b e i t i t i s not s p e c i f i c f o r n a r i n g i n , i t i s a s i m p l e , r a p i d and inexpen s i v e method of a n a l y s i s . However, s i n c e g r a p e f r u i t contains both b i t t e r and n o n b i t t e r flavanone g l y c o s i d e s , Davis values are only a crude approximation of b i t t e r n e s s .
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
ROUSEFF
Flavonoids
and Citrus
Quality
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SCORING GUIDE FOR HESPERIDIN FROZEN CONCENTRATED ORANGE JUICE AND CONCENTRATED ORANGE FOR MFG.
UNACCEPTABLE Figure 2.
USD A
ACCEPTABLE
visual aids used ίο determine acceptable and unacceptable amounts of hesperidin crystals in orange juice
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Other c o l o r i m e t r i c methods have been developed to determine f l a v o n o i d b i t t e r n e s s i n c i t r u s products (1^3, 1^, 15^ jJ6). The b a s i c approach i n v o l v e s the a d d i t i o n of a reducing agent, such as sodium borohydride or magnesium, to j u i c e f o l l o w e d by the a d d i t i o n of HC1 to form a c h a r a c t e r i s t i c c o l o r . A g a i n , the c o l o r i s due to both b i t t e r and n o n b i t t e r flavanone g l y c o s i d e s i n a d d i t i o n to the t a s t e l e s s flavanone aglycones. An o l d e r and even l e s s s p e c i f i c method i s the f e r r i c c h l o r i d e method (17^, 18) f o r n a r i n g i n . U n f o r t u n a t e l y , t h i s method r e a c t s w i t h many f l a v o n o i d s , both b i t t e r and n o n b i t t e r , as w e l l as c i t r i c a c i d and other hydroxy compounds present i n the j u i c e . D i r e c t u l t r a v i o l e t s p e c t r o p h o t o m e t r y methods have been developed to measure n a r i n g i n i n g r a p e f r u i t (19) and h e s p e r i d i n i n orange j u i c e (20, 21). While these methods are r a p i d , they are a l s o n o n s p e c i f i c f o r f l a v o n o i d b i t t e r n e s s . Chromatographic methods were developed to separate a few of the c i t r u s f l a v o n o i d s from the complex mixture of c i t r u s f l a v o n o i d s . The e a r l y paper chromatographic methods f o r flavanones (22, 23) were d i f f i c u l t to quanti t a t e because of band broadening and uneven s o l v e n t development. Several t h i n l a y e r chromatographic (TLC) methods were developed to separate the b i t t e r from the n o n b i t t e r flavanone g l y c o s i d e s (24^, 2!5, 26, 27). G a s - l i q u i d chromatography (GLC) has not been employed f o r the a n a l y s i s of flavanone g l y c o s i d e s because they are n o n - v o l a t i l e and t h e r m a l l y u n s t a b l e . One GLC method (28) has been developed f o r the a n a l y s i s of the flavanone aglycones. However, the method i s extremely time consuming i n t h a t the samples must be e x t r a c t e d , hydrolyzed and d e r i v a t i z e d before a n a l y s i s . Furthermore, the procedure cannot d i s t i n g u i s h between b i t t e r and nonbitter flavonoids. S w i f t (28) developed a TLC-spectrophotometric a n a l y s i s of f i v e methoxylated flavones from orange p e e l . Since the a n a l y s i s c o n s i s t e d of two s t a g e s , one f o r s e p a r a t i o n and the l a s t f o r q u a n t i t a t i o n , the a n a l y s i s was r a t h e r time consuming and a l s o s u b j e c t to some p o s i t i v e e r r o r s due to incomplete sample sepa r a t i o n . Tatum et a K (29) developed several d i r e c t TLC methods f o r methoxylated and hydroxylated f l a v o n e s , coumarins and psoralens i n c i t r u s . Maier and M e t z l e r (30) developed a semiq u a n t i t a t i v e , two-dimensional TLC procedure to determine a number of c i t r u s f l a v a n o n e s , f l a v a n o l s , f l a v o n e s , coumarins and psoralen aglycones. High performance l i q u i d chromatographic methods (HPLC) have been developed f o r the separation of n a r i n g i n from n a r i r u t i n (3l_, 32) i n c i t r u s j u i c e s . A method to determine the major methoxylated flavones i n c i t r u s has been developed (33). Gradient e l u t i o n HPLC has been employed to separate a wide v a r i e t y of c i t r u s flavanone g l y c o s i d e s , coumarins and psoralens (34). Thus, w i t h the recent improvements i n methodology, i t i s now p o s s i b l e to evaluate the r e l a t i v e c o n t r i b u t i o n of i n d i v i d u a l c i t r u s f l a v o n o i d s because the c o n c e n t r a t i o n of each can now be r a p i d l y and a c c u r a t e l y measured.
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
5.
ROUSEFF
Flavonoids
and
Citrus
Quality
89
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C i t r u s Flavonoids as Q u a l i t y Factors Flavonoids have no odor or mouth f e e l and, i n g e n e r a l , do not c o n t r i b u t e s i g n i f i c a n t l y to the c o l o r of most c i t r u s j u i c e s . T h e i r primary e f f e c t on c i t r u s q u a l i t y i s due to the b i t t e r t a s t e of c e r t a i n flavanone g l y c o s i d e s . Thus, q u a n t i t a t i v e d e s c r i p t i o n s of d e s i r a b l e c i t r u s q u a l i t i e s are u s u a l l y based on the absence or maximum c o n c e n t r a t i o n l i m i t s f o r these compounds. B i t t e r n e s s i s a g e n e r a l l y u n d e s i r a b l e f l a v o r component and i s u s u a l l y d e t r i m e n t a l to the q u a l i t y of c i t r u s products. Any b i t t e r n e s s i n orange or tangerine products reduces t h e i r q u a l i t y , whereas, a l i t t l e b i t t e r n e s s i s a c t u a l l y d e s i r a b l e f o r g r a p e f r u i t products. However, i t has been shown (35) t h a t as the concent r a t i o n of b i t t e r m a t e r i a l s i n c r e a s e , f l a v o r scores and o v e r a l l product a c c e p t a b i l i t y decrease. Thus, excessive b i t t e r n e s s i s u s u a l l y considered o b j e c t i o n a b l e and the F l o r i d a Department of C i t r u s has enacted r e g u l a t i o n s which l i m i t the amount of n a r i n g i n which may be present during the e a r l y weeks of the season (36). I t i s d i f f i c u l t to q u a n t i t a t i v e l y d e f i n e g r a p e f r u i t q u a l i t y i n terms of b i t t e r n e s s because i n d i v i d u a l t a s t e thresholds and b i t t e r n e s s preferences vary markedly. Guadagni et^ al_. (37) found t h a t 7% of a 27 member t a s t e panel could d e t e c t as l i t t l e as 1.5 ppm n a r i n g i n i n water. Y e t , another 7% of t h a t same panel could not d i s t i n g u i s h a 50 ppm n a r i n g i n s o l u t i o n from water alone. This represents over a 30-Fold d i f f e r e n c e i n t a s t e t h r e s h o l d s . F e l l e r s (38) used a l a r g e population of g r a p e f r u i t j u i c e users to evaluate n a r i n g i n b i t t e r n e s s preference l e v e l s . Some of h i s data i s shown i n Table I. Using a low n a r i n g i n j u i c e he added various amounts of n a r i n g i n to d i f f e r e n t l o t s of the same j u i c e . The t a s t e r s were not t o l d the j u i c e s contained d i f f e r e n t b i t t e r n e s s l e v e l s but were asked to evaluate each j u i c e on a s i x p o i n t s c a l e from poor = 1 to e x c e l l e n t = 6 . As expected f l a v o r scores f e l l as the n a r i n g i n content of the j u i c e i n c r e a s e d . Correspondingly, the percentage of the panel t h a t thought the j u i c e was too b i t t e r increased w i t h i n c r e a s i n g n a r i n g i n concent r a t i o n . However, the s u r p r i s i n g r e s u l t o f t h i s study was t h a t at 1900 ppm n a r i n g i n (an e x c e s s i v e l y b i t t e r j u i c e ) , 12% of the t a s t e r s d i d not t h i n k the j u i c e was b i t t e r enough. These r e s u l t s show the tremendous range i n i n d i v i d u a l b i t t e r n e s s response, as w e l l as i n s e n s i t i v i t y . Taste and S t r u c t u r e of C i t r u s
Flavonoids
I n t e r e s t i n g l y , flavanone g l y c o s i d e s e x i s t as s t r u c t u r a l isomers of which one w i l l be i n t e n s e l y b i t t e r w h i l e the other i s t a s t e l e s s . The flavanone p o r t i o n of the b i t t e r molecule i s t a s t e l e s s , w h i l e the g l y c o s i d e p o r t i o n i s t a s t e l e s s or s l i g h t l y sweet ( 1 ) . B i t t e r n e s s i s observed only when the sugars and the
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Table I. E v a l u a t i o n of n a r i n g i n b i t t e r n e s s as a f a c t o r i n preference of F l o r i d a g r a p e f r u i t j u i c e Nan* ngin cone, Mean r a t i n g
(ppm)
300
700
1100
1500
1900
3.7
3.6
3.4
3.4
3.3
17 59
31 55
33 55
46 44
51 37
24
14
17
10
12
(201)
(198)
(198)
(197)
(197)
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Bitterness Too b i t t e r Just right Not enough bitterness Don't know (Number of respondents)
a
Percentage of t a s t e r s Source: (38) α
flavanone aglycone are l i n k e d i n a s p e c i f i c f a s h i o n . These com pounds have been thoroughly studied to determine the r e l a t i o n s h i p between t a s t e and s t r u c t u r e and has been thoroughly reviewed by Horowitz ( U 37). While no s i n g l e s t r u c t u r a l f e a t u r e has been a s s o c i a t e d witiï b i t t e r n e s s , the l i n k a g e of the sugars rhamnose and glucose i s very important. Linked from C - l i n the rhamnose to the C-2 i n g l u c o s e , the r e s u l t i n g d i s a c c h a r i d e i s c a l l e d neohesperidose (2-0-(rhamnopyranosyl) glucopyranose). I f the e s s e n t i a l l y t a s t e l e s s neohesperidose (40) i s l i n k e d to a f l a vanone through the 7-hydroxy p o s i t i o n , the r e s u l t i n g flavanone g l y c o s i d e w i l l be i n t e n s e l y b i t t e r . However, i f the same sugars are l i n k e d C - l to C-6 to form 6-0-(rhamnopyranosyl) glucopyranose ( r u t i n o s e ) and l i n k e d i n the same way to the i d e n t i c a l flavanone aglycone, the r e s u l t i n g molecule i s t a s t e l e s s . The d i f f e r e n t rhamnose-glucose l i n k a g e s are i l l u s t r a t e d i n Figure 3 f o r the b i t t e r n a r i n g i n and i t s n o n b i t t e r isomer, n a r i r u t i n . A l l c i t r u s flavanone neohesperidosides are b i t t e r and a l l flavanone r u t i n o s i d e s are t a s t e l e s s Since the neohesperiodose sugar plays such an important r o l e i n flavanone b i t t e r n e s s one might expect other f l a v o n o i d s w i t h a neohesperiodose attached at the 7 p o s i t i o n to be e q u a l l y b i t t e r . F o r t u n a t e l y , t h i s i s not the case, f o r even s u b t l e changes i n the flavanone molecule can destroy i t s b i t t e r n e s s . As shown i n Figure 4 i f the b i t t e r neohesperidin i s converted to neodiosmin (the corresponding f l a v o n e ) the r e s u l t i n g compound i s t a s t e l e s s (41). Similar r e s u l t s have been observed f o r other flavanone neohesperidosides ( 1 ) . No c i t r u s f l a v o n e g l y c o s i d e has been found to be b i t t e r . Furthermore, i f the rhamnose p o r t i o n of the neohesperidoside i s removed from n a r i n g i n , the r e s u l t i n g g l u c o s i d e (prunin) i s s t i l l b i t t e r , but a t a much reduced i n t e n s i t y (See Table I I ) . While
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
5.
ROUSEFF
Flavonoids
and Citrus
NARIRUTIN
(naringenin
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OH
Quality
OH
91
0 7-β-rutinoside)
Figure 3. Structural isomers of naringenin illustrating the two possible configura tions of the sugars attached at the 7 position. Naringin is bitter whereas narirutin is tasteless.
OH Neodiosmin
0 (tasteless)
Figure 4. Minor structural changes to the aglycone portion of the molecule can destroy bitterness. Adding a single double bond between Carbons 2 and 3 will convert bitter flavanone neohesperidosides to tasteless flavone neohesperidosides.
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a l l flavanone neohesperidosides are b i t t e r , some are more b i t t e r than o t h e r s . Thus, as shown i n Table I I , n a r i n g i n and p o n c i r i n are two of the most b i t t e r flavone g l y c o s i d e s found i n c i t r u s . Each i s about o n e - f i f t h as b i t t e r as quinine d i h y d r o c h l o r i d e . Neohesperidin and n e o e r i o c i t r i n are only one-tenth as b i t t e r as the other two flavanone g l y c o s i d e s . Thus, i t appears the number and p o s i t i o n of r i n g s u b s t i t u t i o n s a f f e c t s the degree of b i t t e r ness of the o v e r a l l molecule. Table II. R e l a t i v e B i t t e r n e s s of C i t r u s Flavonoid and Glycosides
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Compound
Taste
Hesperetin Eriodictoyl Naringenin? Hesperidin Neohesperidin Narirutin Naringin Didymin Poncirin . Eriocitrin Neoeriocitrin Prunin a
3
0
0
b
0
SI. sweet No t a s t e No t a s t e No t a s t e Bitter No t a s t e Bitter No t a s t e Bitter No t a s t e Bitter Bitter
Aglycones
Relative Bitterness
*
— — —
2 —
20 —
20 —
2 6
On a molar b a s i s w i t h q u i n i n e d i h y d r o c h l o r i d e taken as 100 Aglycones Rutinosides . Neohesperidosides Glucoside Source: ( 1 , 39) b
Hagen e t a K (42) determined the r e l a t i v e amounts of a l l the flavanone g l y c o s i d e s i n Texas g r a p e f r u i t . T h e i r r e s u l t s are shown i n Table I I I . Naringin i s the dominant f l a v o n o i d i n grapef r u i t and i s p r i m a r i l y r e s p o n s i b l e f o r the immediate b i t t e r t a s t e i n g r a p e f r u i t . The e q u a l l y b i t t e r p o n c i r i n and the much l e s s b i t t e r neohesperidin are present i n r e l a t i v e l y small amounts and do not c o n t r i b u t e s i g n i f i c a n t l y to o v e r a l l b i t t e r n e s s . While there are no b i t t e r flavone g l y c o s i d e s i n c i t r u s , there are some h i g h l y methoxylated f l a v o n e aglycones t h a t are r e p o r t e d l y b i t t e r . S w i f t (28) i d e n t i f i e d s i n e n s e t i n , n o b i l e t i n , t e t r a - O - m e t h y s c u t e l l e r e i n , 3, 5, 6, 7, 8, 3 ' , 4 heptamethoxyflavone and t a n g e r e t i n from the b i t t e r f r a c t i o n of orange peel j u i c e . He l a t e r determined the i n d i v i d u a l and c o l l e c t i v e t a s t e thresholds of these flavones and compared ,them to the c o n c e n t r a t i o n s found i n commercial orange j u i c e (43). Orange j u i c e w i t h an added t o t a l of 24 ppm of these flavones could not be d i f f e r e n t i a t e d from the orange j u i c e alone. Since the maximum t o t a l 1
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
5.
ROUSEFF
Flavonoids
and Citrus
93
Quality
c o n c e n t r a t i o n found i n orange j u i c e over several years was 7 ppm, he concluded t h a t these flavones were not important c o n t r i b u t o r s to the f l a v o r o f orange j u i c e . Table I I I .
Flavanone Glycosides i n Texas Canned G r a p e f r u i t J u i c e
Compound
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Naringin Naringin r u t i n o s i d e Neohesperidin Hesperidin Poncirin Isosakuranetin r u t i n o s i d e Source:
Concentration (ug/ml) 306 124 10.5 9.9 17.0 5.3
(42)
Flavonoid B i t t e r n e s s Suppressors A l l known c i t r u s f l a v o n e g l y c o s i d e s are odorless and gene r a l l y t a s t e l e s s y e t they appear to have an important f u n c t i o n i n reducing the perceived b i t t e r n e s s of flavanone neohesperidosides and l i m o n o i d s . Horowitz (1_) was the f i r s t to i d e n t i f y the b i t t e r ness suppressing f u n c t i o n of these compounds. He found t h a t r h o i f o l i n , the f l a v o n e e q u i v a l e n t o f the b i t t e r n a r i n g i n , could p a r t i a l l y suppress the b i t t e r n e s s o f n a r i n g i n . Thus, higher n a r i n g i n concentrations were necessary before b i t t e r n e s s could be d e t e c t e d , i f the s o l u t i o n contained l a r g e amounts o f r h o i f o l i n . Guadagni e t al_. (41) found neodiosmin, the t a s t e l e s s f l a v o n e analog o f b i t t e r neohesperidin, to be a very e f f e c t i v e b i t t e r n e s s suppressor. As l i t t l e as 10 ppm i n water increased the b i t t e r ness t h r e s h o l d o f n a r i n g i n and l i m o n i n , a nonflavonoid b i t t e r compound, 3.5-and 4 . 0 - f o l d , r e s p e c t i v e l y . Neodiosmin was a l s o e f f e c t i v e i n reducing preceived b i t t e r n e s s o f l i m o n i n i n orange juice. I t was speculated t h a t these f l a v o n e neohesperidosides are so s i m i l a r i n s t r u c t u r e t h a t they compete w i t h flavanone neohesperidosides f o r s i t e s on the b i t t e r n e s s receptors i n the mouth. This e x p l a n a t i o n assumed t h a t these flavones could be adsorbed a t t a s t e s i t e s without producing an a p p r e c i a b l e t a s t e response of t h e i r own. U n f o r t u n a t e l y , n e i t h e r o f these b i t t e r n e s s suppressors has been found to occur n a t u r a l l y i n the sweet orange (C_. s i n e n s i s ) although Nakabayashi (44) found r h o i f o l i n i n the sour orange "(CT aurantium). Dunlap and Wender (45) reported f i n d i n g r h o i T o l i n i n g r a p e f r u i t e x t r a c t s but no attempt was made to d e t e r mine i t s c o n c e n t r a t i o n i n j u i c e . Neodiosmin has y e t to be found i n g r a p e f r u i t . Thus, i t remains to be shown i f these compounds play any r o l e i n the natural r e d u c t i o n o f n a r i n g i n b i t t e r n e s s as g r a p e f r u i t matures.
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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94
Chalcones and Dihydrochalcones. Chalcones and d i h y d r o chalcones are i n t e n s e l y sweet compounds (39) t h a t are e f f e c t i v e i n r a i s i n g the t h r e s h o l d a t which the b i t t e r n e s s of n a r i n g i n and limonin i s perceived (46). As i l l u s t r a t e d i n Figure 5, chalcones are e a s i l y formed from flavanone g l y c o s i d e s by the a d d i t i o n of a l k a l i and dihydrochalcones are formed from hydrogenated c h a l cones. L i k e the flavanone neohesperidosides, the chalcones and dihydrochalcones vary i n the i n t e n s i t y of t h e i r t a s t e response. Dihydrochalcones are sweeter than chalcones and of the d i h y d r o chalcones, neohesperidin dihydrochalcone (NHD) i s the sweetest (39). I t has been estimated t h a t NHD i s 1,500 times sweeter than sucrose on an equal weight b a s i s . Thus, i t has been proposed t h a t NHD be added to e x c e s s i v e l y b i t t e r j u i c e to reduce the perceived b i t t e r n e s s and i n c r e a s e the q u a l i t y of the j u i c e (47). A 1969 F l o r i d a Department of C i t r u s Market Research Report i n d i c a t e d f a v o r a b l e s a l e s and consumer acceptance of a r t i f i c i a l l y sweetened g r a p e f r u i t j u i c e . F e l l e r s (47) found the average t a s t e t h r e s h o l d of NHD i n s i n g l e - s t r e n g t h g r a p e f r u i t j u i c e to be about 8 ppm. Neohesperidin dihydrochalcone l e v e l s between 8-12 ppm produced a s i g n i f i c a n t sweetening of the g r a p e f r u i t j u i c e w i t h only a s l i g h t but acceptable a f t e r t a s t e . Higher l e v e l s of neohesperidin d i hydrochal cone produced j u i c e s t h a t were e x c e s s i v e l y and unacceptably sweet. Thus, NHD employed i n the proper amounts could be used to upgrade the f l a v o r of e a r l y season g r a p e f r u i t j u i c e or r e p l a c e a s i g n i f i c a n t amount of sucrose used i n covering syrups i n canned or f r o z e n g r a p e f r u i t s e c t i o n s . Chalcones have been proposed (48) as a precursor t h a t i s e n z y m a t i c a l l y c y c l i z e d to form flavanone g l y c o s i d e s . The enzyme r e s p o n s i b l e f o r t h i s s t e r e o s p e c i f i c r i n g c l o s u r e disappears o r i s i n a c t i v a t e d as the f r u i t matures ( 5 ) . However, no chalcones have been i s o l a t e d from c i t r u s . T h e r e f o r e , i f chalcones are a p a r t of the metabolic pathway t h a t leads to the formation of flavanone g l y c o s i d e s they probably e x i s t as short l i v e d , unstable i n t e r m e d i a t e s . I t appears u n l i k e l y t h a t these compounds play a s i g n i f i c a n t r o l e in m i t i g a t i n g bitterness at t h e i r natural l e v e l s . Taxonomic S i g n i f i c a n c e of Flavonoids In terms of i t s f l a v o n o i d c o m p o s i t i o n , c i t r u s i s unique w i t h i n the p l a n t kingdom. Some c i t r u s f l a v a n o i d s are found nowhere e l s e . Furthermore, the r e l a t i v e f l a v o n o i d composition v a r i e s w i t h each v a r i e t y . Thus, the f l a v o n o i d composition of c i t r u s j u i c e s has been proposed (2) as a measure of j u i c e authenticity. In most of the p l a n t kingdom, flavanones occur only i n small amounts compared to other f l a v o n o i d s , y e t they are the predominant f l a v o n o i d i n c i t r u s . C i t r u s flavanones u s u a l l y occur as g l y c o s i d e s , whereas i n other p l a n t s , flavanones are seldom found i n the g l y c o s i d e form (1). Four types of glycosides have been found i n c i t r u s . They are O-glucosides, C - g l y c o s i d e s , r u t i n o s i d e s and neohesperidosides. Kefford (2) was one of the f i r s t i n v e s t i gators to recognize t h a t flavanone composition could be used to
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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5.
ROUSEFF
Flavonoids
and Citrus
Quality
DIHY PR 0 CHALCONE
Figure 5.
Conversion of bitter flavanone neohesperidosides to the corresponding intensely sweet chalcones and dihydrochalcones
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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CITRUS NUTRITION AND QUALITY
d i s t i n g u i s h d i f f e r e n t v a r i e t i e s of c i t r u s . He c l a s s i f i e d sweet oranges, mandarins, lemons and c i t r o n s together because hesper i d i n was the predominant flavanone i n each. Since the p r i n c i p a l f l a v o n o i d i n g r a p e f r u i t and pummelo was n a r i n g i n , Kefford put these c u l t i v a r s i n a separate group. Horowitz [1) observed t h a t the f l a v o n o i d g l y c o s i d e s of many v a r i e t i e s of c i t r u s occurred e i t h e r a l l i n the r u t i n o s i d e or neohesperidoside forms. He thus proposed t h a t a l l c i t r u s could be d i v i d e d according to t h e i r g l y c o s i d e form. Albach and Redman (49) confirmed t h i s method of c l a s s i f i c a t i o n w i t h t h e i r flavanone survey of 41 c i t r u s c u l t i v a r s and 49 hybrids from Texas. They found t h a t the r e l a t i v e amounts of flavanones w i t h i n d i f f e r e n t c u l t i v a r s of the same species were f a i r l y constant. Some of t h e i r r e s u l t s are shown i n Table IV. Most of the commercial c i t r u s c u l t i v a r s c o n t a i n only the non b i t t e r r u t i n o s i d e s , whereas the sour orange and pummelo (C. grandis) have only b i t t e r flavanone neohesperidosides. Working w i t h hybrids of known parentage, they found i f one v a r i e t y con t a i n i n g only flavanone r u t i n o s i d e s i s crossed w i t h another con t a i n i n g only neohesperidosides, the r e s u l t i n g h y b r i d contained both types of flavanone g l y c o s i d e s . An example of t h i s i s shown i n Table IV f o r the cross between s i n e n s i s χ Poncirus t r i f o l i a t a . Thus, i t i s suspected t h a t the Meyers lemon (C. limon) and the g r a p e f r u i t are hybrids r a t h e r than pure v a r i e t i e s because they c o n t a i n both flavanone r u t i n o s i d e s and neohesperidosides. Kamiya e t al^. (50) d i v i d e d c i t r u s i n t o 12 groups based on the number and kind of flavanone g l y c o s i d e s found. With t h i s c l a s s i f i c a t i o n system, they were able to d i s t i n g u i s h between n u c e l l a r and z y g o t i c seedlings using both leaves and f r u i t . Of the 94 hybrids examined, 53 c u l t i v a r s were judged as t r u e hy b r i d s . The remaining c u l t i v a r s were not c l a s s i f i e d as t r u e hy b r i d s because t h e i r flavanone g l y c o s i d e p a t t e r n was not s u f f i c i e n t l y d i f f e r e n t from the female parent. Ta turn e t al_. (29) used the presence of v a r i o u s methoxylated flavones from l e a f e x t r a c t s to d i s t i n g u i s h between n u c e l l a r and z y g o t i c s e e d l i n g s . They a l s o i d e n t i f i e d the predominant f l a vanone g l y c o s i d e and three u n i d e n t i f i e d coumarins i n t h e i r samples. Ting e t ^ a l . (51) showed t h a t there were q u a n t i t a t i v e as w e l l as q u a l i t a t i v e cfifferences i n the methoxylated f l a v o n e content from the j u i c e s of d i f f e r e n t c i t r u s v a r i e t i e s . They f u r t h e r showed t h a t c e r t a i n blends of j u i c e s could be d i s t i n guished based on the amounts of methoxylated flavones found. Both d i r e c t l y and i n d i r e c t l y , f l a v o n o i d composition a f f e c t s the q u a l i t y of i n d i v i d u a l c i t r u s c u l t i v a r s and h y b r i d s . Some c u l t i v a r s , such as the sour orange, are d i r e c t l y a f f e c t e d by the presence of b i t t e r neohesperidosides to the p o i n t they are un p a l a t a b l e . The q u a l i t y of other c u l t i v a r s may be i n d i r e c t l y a f f e c t e d due to the presence of b i t t e r n e s s suppressing f l a v o n e neohesperidosides. C e r t a i n h y b r i d s , such as the Κ e a r l y , a l s o c o n t a i n b i t t e r flavanone g l y c o s i d e s a t l e v e l s which reduce i t s a c c e p t a b i l i t y . T h e r e f o r e , the knowledge of the r e l a t i v e type and
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
5.
ROUSEFF
Flavonoids
and Citrus
97
Quality
Table IV. Thin Layer Chromatographic Survey of Flavanones i n C i t r u s Taxa Relative fluorescence i n t e n s i t y 7-Rutinosides o f : 7-Neohesperidosides 3
3
_l. CO
-s —1.
CO
&
—
-s
np 1IC
ca Φ 3 —1. 3
ο
-s ο QL
_l. ο ,c+
naringenin + glucose. Naringinase s o l u t i o n s were found to r a p i d l y convert n a r i n g i n to n a r i n g e n i n . The same process d i d not occur when naringinase was added to j u i c e . Due to the n a t u r a l l e v e l s of glucose i n j u i c e , the r e a c t i o n r a t e of the second step was decreased by over 60% (68). Thus, prunin accumulated as an i n t e r m e d i a t e product and was s l o w l y converted to n a r i n g e n i n . The i n h i b i t i o n of glucose on the second r e a c t i o n r a t e could be e l i m i n a t e d i f the enzyme, e m u l s i n , was a l s o added to n a r i n g i n a s e . However, s a t i s f a c t o r y d e b i t t e r i n g was achieved w i t h n a r i n g i n a s e a l o n e , and complete h y d r o l y s i s to naringenin was not r e q u i r e d (68). I n v e s t i g a t i o n s on p o s s i b l e commercial use of enzymes to r e move n a r i n g i n b i t t e r n e s s were c a r r i e d out f o r j u i c e (70, 7 1 ) , con c e n t r a t e C71), and f r u i t s e c t i o n s (72). Those i n v e s t i g a t i o n s showed t h a t i t was t e c h n i c a l l y p o s s i b l e to use enzymes to reduce n a r i n g i n b i t t e r n e s s on a commercial s c a l e . However, due to e n zyme expense, most of the i n d u s t r y has found i t more c o s t e f f e c t i v e to use more mature ( l e s s b i t t e r ) f r u i t r a t h e r than employ d e b i t t e r i n g enzymes. The Japanese have been very a c t i v e i n the use of enzymes to reduce n a r i n g i n b i t t e r n e s s . This i s due i n p a r t to the presence of n a r i n g i n i n Natsudaidai orange, an important c u l t i v a r i n Japan. They were one of the f i r s t to i n v e s t i g a t e the commercial
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1000.
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Ε
χ I
ι 15
SEPTEMBER
ι I
ι 15
OCTOBER
ι I
ι
ι
15
I
NOVEMBER
1
1
15
I
DECEMBER
lJ 15
JANUARY
Figure 8. Effect of extractor pressure on totalflavanoneglycoside concentration for Florida grapefruit at different stages of maturity (35) ((Φ) hard squeeze; (O) soft squeeze)
Table V. E f f e c t o f Enzyme Treatment on N a r i n g i n Content and B i t terness P e r c e p t i o n i n G r a p e f r u i t Juice.(79) Sample
Juice volume (mL)
Circulation time (min)
Control l 2a 3 4
— 1500 1500 500 500
— 100-30 180-134 120 125-150
a
a
a
Naringin content (ug/mL)
Bitterness rating*
285 240 195 122 95
3
3.8 3.6 3.3 3.2 2.9
a = blended samples of 2-6 batches o f j u i c e . b = average b i t t e r n e s s scores from 20 member t a s t e p a n e l , where 1 = no b i t t e r n e s s , 2 = j u s t p e r c e p t i b l e , 3 = d e f i n i t e l y p e r c e p t i b l e , 4 = moderately i n t e n s e , 5 = very intense and 6 = extremely i n t e n s e . Journal of Food Science
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a p p l i c a t i o n o f naringinase (73) and have done e x t e n s i v e s t u d i e s to f i n d inexpensive sources f o r t h i s enzyme (74·, 75). The most s u c c e s s f u l approaches have been to soak the peeled f r u i t i n enzyme s o l u t i o n s f o r up to 16 hour before processing (76) o r adding naringinase preparations w i t h low pectinase a c t i v i t y along w i t h the f r u i t as i t i s being canned (77). Roe and Brummer (78) s u c c e s s f u l l y i n f u s e d naringinase i n t o whole shaved g r a p e f r u i t (flavedo removed). This reduced the b i t t e r n e s s o f the albedo to the p o i n t t h a t i t was as e q u a l l y e d i b l e as the f r u i t segments. In a d d i t i o n , the process increased the n u t r i t i o n a l and d i e t a r y f i b e r content o f the f i n a l product. Cost has not been the only problem i n employing enzymes t o reduce n a r i n g i n b i t t e r n e s s . The other problem was the u n d e s i r able l o s s o f cloud due to pectinase i m p u r i t i e s i n the naringinases employed (70). However, both problems have been reduced i n a promising new procedure. Olson e t a K (79) have s u c c e s s f u l l y immobilized naringinase i n a hollow f i b e r r e a c t o r to reduce n a r i n g i n b i t t e r n e s s . Enzyme a c t i v i t y was not a f f e c t e d even a f t e r g r a p e f r u i t j u i c e was pumped through the hollow f i b e r r e a c t o r f o r up to three days. Thus, much more j u i c e can be t r e a t e d w i t h the same amount o f enzyme and the cost per u n i t o f j u i c e was reduced. Pectinase i m p u r i t i e s d i d not cause a l o s s o f j u i c e cloud because the high molecular weight p e c t i n s could not d i f f u s e through the hollow f i b e r membrane t o come i n c o n t a c t w i t h the enzyme. As i l l u s t r a t e d i n Table V, they could reduce j u i c e n a r i n g i n l e v e l s by c o n t r o l l i n g the amount and the c i r c u l a t i o n time o f the j u i c e . N a r i n g i n c o n c e n t r a t i o n was reduced from 285 to 95 ppm w i t h a corresponding r e d u c t i o n i n the b i t t e r n e s s r a t i n g . This i s a promising approach to the problem o f j u i c e n a r i n g i n b i t t e r n e s s , however, the process s t i l l remains t o be evaluated under p i l o t plant conditions. Enzymes have a l s o been developed to reduce o r e l i m i n a t e problems a s s o c i a t e d w i t h h e s p e r i d i n s i n s o l u b i l i t y . Since hesp e r i d i n problems are v i s u a l problems, enzymes have been used t o convert the i n s o l u b l e h e s p e r i d i n t o the more s o l u b l e g l y c o s i d e . The a d d i t i o n o f these enzymes has g r e a t l y reduced the t u r b i d i t y i n canned mandarin s e c t i o n s due to h e s p e r i d i n c r y s t a l formation during storage (80). 1
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RECEIVED July
7, 1980.
Nagy and Attaway; Citrus Nutrition and Quality ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
