Limonin and Limonoids - American Chemical Society

4 Limonin and Limonoids

Downloaded by NORTH CAROLINA STATE UNIV on December 29, 2012 | http://pubs.acs.org Publication Date: December 15, 1980 | doi: 10.1021/bk-1980-0143.ch004

Chemistry, Biochemistry, and Juice Bitterness VINCENT P. MAIER, SHIN HASEGAWA, RAYMOND D. BENNETT, and LINDA C. ECHOLS U.S. Department of Agriculture, Science and Education Administration, AR, Fruit and Vegetable Chemistry Laboratory, Pasadena, CA 91106 Limonoids are a group of c h e m i c a l l y r e l a t e d t r i t e r p e n e derivatives found i n the Rutaceae and M e l i a c e a e . L i m o n i n , a b i t t e r member o f the group, occurs widely i n c i t r u s j u i c e s . I t has c o m m e r c i a l s i g n i f i c a n c e because b i t t e r n e s s ( e x c e s s i v e b i t t e r n e s s i n the case of g r a p e f r u i t ) reduces j u i c e q u a l i t y . D r e y e r (1_) a n d C o n n o l l y e t a l . (2) h a v e r e v i e w e d t h e c h e m i s t r y a n d b i o c h e m i s t r y o f l i m o n o i d s . More r e c e n t l y M a i e r e t a l . (3_) p u b l i s h e d a c o m p r e h e n s i v e r e v i e w o f the l i m o n o i d c o n s t i t u e n t s o f C i t r u s and the impact o f l i m o n i n b i t t e r n e s s on j u i c e q u a l i t y . T h i s p a p e r summarizes the c h e m i c a l , b i o c h e m i c a l a n d j u i c e q u a l i t y a s p e c t s o f l i m o n o i d s i n C i t r u s (and r e l a t e d genera) and p r e s e n t s r e l e v a n t advances s i n c e p r e v i o u s r e views . Limonoid S t r u c t u r e s

and R e l e v a n t

Chemistry

L i m o n i n (I) i s a h i g h l y o x y g e n a t e d t r i t e r p e n e d e r i v a t i v e whose s t r u c t u r a l f e a t u r e s i n c l u d e a f u r a n r i n g , two l a c t o n e r i n g s , a f i v e - m e m b e r e d e t h e r r i n g , a n d an e p o x i d e . A l l other c i t r u s l i m o n o i d s a l s o c o n t a i n t h e f u r a n r i n g a n d a t l e a s t one o f t h e l a c tone r i n g s . Compounds I , I I I , V - X I I , a n d X I V - X V I were known as n a t u r a l c i t r u s c o n s t i t u e n t s a t t h e t i m e o f o u r l a s t r e v i e w (_3) · S i n c e t h e n s e v e r a l o t h e r c i t r u s l i m o n o i d s h a v e b e e n i s o l a t e d and t h e i r s t r u c t u r e s d e t e r m i n e d : l i m o n o l ( I I ) , o b a c u n o l ( I V ) , and d e o x y l i m o n o l (XVII) f r o m g r a p e f r u i t s e e d s ( 4 ) , i s o l i m o n i c a c i d (XIII) from s o u r orange and g r a p e f r u i t seeds (5)/ deoxylimonic a c i d ( X V I I I ) f r o m g r a p e f r u i t s e e d s ( 6 ) , and m e t h y l d e a c e t y l n o m i l i n a t e (VIII), calamin (XIX), retrocalamin (XX), cyclocalamin ( X X I ) , a n d m e t h y l i s o o b a c u n o a t e d i o s p h e n o l (XXII) f r o m c a l a m o n d i n s e e d s (7_) . T a b l e I (8) shows t h e c o n c e n t r a t i o n s o f l i m o n o i d s i n s e e d s o f s e v e r a l c i t r u s s p e c i e s , a s w e l l as t h e r e l a t i v e p r o p o r t i o n s o f n e u t r a l and a c i d i c l i m o n o i d s i n e a c h . T a b l e s I I a n d I I I (8) show t h e r e l a t i v e amounts o f t h e m a j o r i n d i v i d u a l n e u t r a l a n d a c i d i c limonoids, respectively, in several species. The c a s e s i n which two d i f f e r e n t s a m p l e s o f t h e same s p e c i e s were a n a l y z e d show t h e

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.


CITRUS NUTRITION AND QUALITY

LIMONIN LIMONOL

X

X=0 Χ = α - Ο Η , β~Η

ISOOBACUNOIC ACID

III IV

XI

OBACUNONE X=0 OBACUNOL Χ=α~ΟΗ,

β-Η

EPIISOOBACUNOIC ACID

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

MAIER ET AL.

Limonin

and

Limonoids


4.




XIX

CALAMIN

XXIII

XX

RETROCALAMIN

19-HYDROXYDEACETYLNOMILINIC ACID


4.

MAIER ET

Table

AL.

Limonin

I. Concentrations

Grapefruit V a l e n c i a Orange Lemon Calamondin Kumquat

67

Limonoids

o f Limonoids i n C i t r u s Seeds

% of t o t a l Neutral

T o t a l , % of f r e s h weight

Seeds


and

limonoids Acidic

77 84 61 83 55

1.5 1.1 1.1 0.75 0.61

(8)

23 16 39 17 45

Journal of Agricultural and Food Chemistry

r a n g e s o f v a r i a t i o n i n c o m p o s i t i o n w h i c h c a n be e x p e c t e d . The c h e m i c a l r e a c t i o n s o f l i m o n i n h a v e b e e n r e v i e w e d p r e v i o u s l y (_1,_3) . From a p r a c t i c a l s t a n d p o i n t t h e m o s t i m p o r t a n t re a c t i o n s a r e t h e o p e n i n g and c l o s i n g o f t h e l a c t o n e r i n g s . In c i t r u s l e a f and f r u i t t i s s u e t h e n a t u r a l l y o c c u r r i n g f o r m i s a s a l t o f l i m o n o i c a c i d Α-ring l a c t o n e (XIV), i n which the A - r i n g i s c l o s e d and t h e D - r i n g i s o p e n . T h i s t a s t e l e s s compound i s o n l y s t a b l e i n the s a l t form. A t a c i d i c pH's, and more r a p i d l y i n t h e presence o f c i t r u s limonoate D-ring lactone hydrolase, the D-ring c l o s e s t o form the b i t t e r substance l i m o n i n (9). The g r a d u a l c o n v e r s i o n o f XIV t o l i m o n i n o c c u r s w h e n e v e r f r u i t t i s s u e s a r e d i s r u p t e d , s u c h a s , t h r o u g h s e v e r e b r u i s i n g , f r e e z e damage o r d u r i n g j u i c e e x p r e s s i o n (10). L i m o n i n o c c u r s as s u c h , w i t h b o t h l a c t o n e r i n g s c l o s e d , i n c i t r u s seeds. The s e e d s a l s o c o n t a i n s m a l l amounts o f t h e s a l t o f XIV (10,11). S t r u c t u r a l r e l a t i o n s h i p s and

pathways

The c i t r u s l i m o n o i d s may be d i v i d e d i n t o g r o u p s on t h e b a s i s of s t r u c t u r a l s i m i l a r i t i e s . One s u c h c r i t e r i o n i s t h e n a t u r e o f C-19, w h i c h may be e i t h e r m e t h y l , a s i n o b a c u n o n e ( I I I ) , o r o x y m e t h y l e n e , as i n l i m o n i n . Another c h a r a c t e r i s t i c f e a t u r e i s the f i v e - m e m b e r e d e t h e r ( A ) r i n g i n compounds s u c h as l i m o n i n and isoobacunoic a c i d (X). The c a l a m o n d i n l i m o n o i d s X I X - X X I I a r e u n i q u e i n two r e s p e c t s : t h e 3 - c a r b o x y l i s m e t h y l a t e d and C-6 i s oxygenated. While these s t r u c t u r a l groupings are h e l p f u l i n sug g e s t i n g p o s s i b l e b i o s y n t h e t i c p a t h w a y s , t h e y do n o t a l l o w a d e c i s i o n t o be made a s t o t h e a c t u a l p a t h w a y by w h i c h l i m o n i n i s s y n thesized. For i n s t a n c e , X c o n t a i n s a l l o f the s t r u c t u r a l f e a t u r e s o f l i m o n i n e x c e p t f o r 1 9 - o x y g e n a t i o n and l a c t o n i z a t i o n and t h u s c o u l d be c o n s i d e r e d t h e i m m e d i a t e p r e c u r s o r . However, i c h a n g i n (XII) i s a l s o a p o s s i b l e i m m e d i a t e p r e c u r s o r , c o n t a i n i n g the A l a c t o n e r i n g but l a c k i n g the A - r i n g . The t r u e pathway w i l l u l t i m a t e l y h a v e t o be d e t e r m i n e d by i n c o r p o r a t i o n s t u d i e s o f p o s s i b l e p r e c u r s o r s i n r a d i o a c t i v e form. N e v e r t h e l e s s , i t seems h i g h l y p r o b a b l e t h a t d e a c e t y l n o m i l i n i c a c i d ( V I I ) i s a key l i m o noid intermediate. Lactone r i n g c l o s u r e and/or a c e t y l a t i o n 1

1



68


Table I I . R e l a t i v e Concentrations of N e u t r a l Limonoids i n C i t r u s S e e d s (8)

Seeds

Limonin

% Distribution Nomilin Obacunone

Deacetylnomilin

Grapefruit

76 83

15 11

1 5

8 1

V a l e n c i a Orange

50 75

34 10

1 1

15 5

N a v e l Orange

87

10

1

2

a

37 39

26 21

1 1

11 17

39

31

29

1

40

40

20

1

70

28

1

1

Tangelo

43

43

13

1

Tangerine

63

16

1

20

60

18

1

21

Sour O r a n g e

Lemon

Lime

a Also Ichangin

25 22

Journal of Agricultural and Food Chemistry



4.

MAIER ET AL.

Limonin

Table I I I . Relative

and

69

Limonoids

Concentrations o f A c i d i c Limonoids i n C i t r u s S e e d s (8)

Nomilinic Acid

Deacetylnomilinic Acid

Isolimonic Acid

Grapefruit

86 85

5 13

8 1

Valencia

83

17

-

6 3

47 64

47 32

95 76 56 33 90

5 15 44 33 10

Seeds

Sour

Orange

Orange

Lemon Lime Tangelo Tangerine N a v e l Orange

9

-

34

a Fruit

tissues Journal of Agricultural and Food Chemistry


70



r e a c t i o n s c o u l d convert i t t o d e a c e t y l n o m i l i n (V), n o m i l i n i c a c i d (IX), o r n o m i l i n ( V I ) , and an e l i m i n a t i o n r e a c t i o n c o u l d then p r o d u c e obacunone ( I I I ) from e i t h e r V o r V I . A 1 , 4 - c y c l i z a t i o n o f V I I w o u l d l e a d t o X, w h i c h c o u l d f o r m l i m o n i n b y 1 9 - h y d r o x y l a t i o n and l a c t o n e r i n g c l o s u r e . 19-Hydroxylation o f V I I would form XXIII, which c o u l d undergo 4 , 1 9 - c y c l i z a t i o n t o produce i s o l i m o n i c a c i d (XIII) o r 1 , 4 - c y c l i z a t i o n , f o l l o w e d by l a c t o n e r i n g c l o s u r e to limonin. A l t e r n a t i v e l y , XXIII c o u l d form X I I by l a c t o n e r i n g c l o s u r e and t h e l a t t e r c o u l d then c y c l i z e t o produce l i m o n i n . The b i o s y n t h e t i c p a t h w a y o f t h e c a l a m o n d i n l i m o n o i d s seems straightforward. M e t h y l d e a c e t y l n o m i l i n a t e (VIII) i s an o b v i o u s p r e c u r s o r o f c a l a m i n ( X I X ) , a n d t h e l a t t e r c o u l d t h e n be c o n v e r t e d t o r e t r o c a l a m i n (XX) b y a r e t r o a l d o l - t y p e r e a c t i o n . 1 , 4 - C y c l i z a t i o n o f XIX would produce c y c l o c a l a m i n (XXI), which c o u l d f o r m m e t h y l i s o o b a c u n o a t e d i o s p h e n o l (XXII) b y o x i d a t i o n o f the 7-hydroxyl t o a ketone, f o l l o w e d by e n o l i z a t i o n . Biosynthesis S i n c e t h i s s u b j e c t was r e v i e w e d i n 1977 (_3) i m p o r t a n t pro g r e s s h a s b e e n made i n s t u d i e s o f t h e b i o s y n t h e s i s o f l i m o n o i d s in Citrus. A n a l y s e s o f C i t r u s f o r XIV c o n t e n t a t v a r i o u s matu r i t y s t a g e s a n d r a d i o a c t i v e t r a c e r work showed t h a t l i m o n o i d s a r e a c t i v e l y s y n t h e s i z e d i n c i t r u s l e a v e s , p a r t i c u l a r l y i n young l e a v e s (12) · Hasegawa e t a l . (13.) a t t e m p t e d t o l o c a t e l i m o n o i d s w i t h i n m e s o p h y l l c e l l s and found t h a t limonoids a r e p r e s e n t i n chloroplasts. F u r t h e r f r a c t i o n a t i o n o f t h i s o r g a n e l l e showed t h e presence o f limonoids i n grana. T h e g r a n a i s o l a t e d f r o m lemon l e a v e s c o n t a i n e d a s much a s 1,000 ppm o f X I V . T h e s e f i n d i n g s s u g g e s t e d t h a t l i m o n o i d s a r e most l i k e l y s y n t h e s i z e d i n c h l o r o p l a s t s , a l t h o u g h f u r t h e r work i s n e e d e d t o c o n f i r m t h i s p o i n t . When 5 - g - s i z e n a v e l o r a n g e s w e r e ^ f e d w i t h 44 χ 10^ cpm o f acetate-2C a t t h e r a t e o f 4.4 χ 10 cpm p e r d a y f o r 10 c o n s e c u t i v e days e i t h e r b y i n j e c t i o n o f t h e f r u i t o r by a p p l i c a t i o n on t h e p e e l , n o l a b e l e d X I V was d e t e c t e d i n t h e f r u i t . A l s o , no C was i n c o r p o r a t e d i n t o X I V when 5 - g - s i z e n a v e l o r a n g e was s i m i l a r l y f e d w i t h 20 χ 10^ cpm o f m e v a l o n a t e - 2 - ^ ^ C . When l a b e l e d a c e t a t e was f e d t o l e a v e s a d j a c e n t t o a 5 - g - n a v e l o r a n g e , r a d i o a c t i v i t y was i n c o r p o r a t e d i n t o X I V i n t h e f r u i t (_12) . T h e s e r e s u l t s s u g g e s t t h a t l i m o n o i d s a r e most l i k e l y n o t s y n t h e s i z e d i n the f r u i t t i s s u e s (12). However, t h e y d o n o t r u l e o u t t h e p o s s i b i l i t y t h a t a p r e c u r s o r ( s ) o f l i m o n o i d s b e y o n d a c e t a t e o r meval o n a t e i s s y n t h e s i z e d i n l e a v e s and t r a n s l o c a t e d t o f r u i t and there converted t o limonoids. C i t r u s t r e e s a r e capable o f t r a n s l o c a t i n g l i m o n o i d s from leaves t o f r u i t t i s s u e s (12). When 20,000 cpm o f X I V , r a n d o m l y l a b e l e d , was f e d t o a l e a f a d j a c e n t t o a 5 - g - s i z e l e m o n , a b o u t 13.3% o f t h e t o t a l a c t i v i t y was t r a n s l o c a t e d t o t h e f r u i t d u r i n g 20 h r s . T h e s e f i n d i n g s o f Hasegawa e t a l . (1^/_13) show t h a t l i monoids i n c i t r u s f r u i t t i s s u e s a r e s y n t h e s i z e d i n leaves and translocated t o the f r u i t . 1 4


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4.

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I t has b e e n m e n t i o n e d i n t h e p r e v i o u s s e c t i o n t h a t c i t r u s seeds c o n t a i n h i g h c o n c e n t r a t i o n s of limonoids. D a t t a and N i c h o l a s (14) showed t h e p r e s e n c e o f l i m o n o i d b i o s y n t h e t i c s y s tems i n g e r m i n a t e d V a l e n c i a o r a n g e s e e d s by d e m o n s t r a t i n g t h e i n c o r p o r a t i o n of mevalonate-2C into I. However, Hasegawa e t a l . (8) c o u l d n o t f i n d s u c h s y s t e m s i n lemon s e e d s , immature o r ma ture. S i n c e l i m o n o i d s a r e a c t i v e l y s y n t h e s i z e d i n y o u n g , imma t u r e l e a v e s (12.) , m o s t l i k e l y t h e r a d i o a c t i v e I f o u n d by D a t t a and N i c h o l a s was s y n t h e s i z e d i n t h e c o l e o p t i l e s o f t h e g e r m i n a t e d orange seeds. The c a p a b i l i t y o f C i t r u s t o t r a n s l o c a t e l i m o n o i d s f r o m t h e f r u i t t i s s u e s t o t h e s e e d s was d e m o n s t r a t e d by a d m i n i s t e r i n g 1 χ 10 cpm o f m e t h y l - " ^ C d e a c e t y l n o m i l i n a t e t o t h e stem end o f detached calamondin f r u i t s . A f t e r 16 h r s o f i n c u b a t i o n , 7,500 cpm o f a c t i v i t y w e r e t r a n s l o c a t e d t o t h e s e e d s , and o v e r 90% o f t h e t o t a l a c t i v i t y i n t h e s e e d e x t r a c t was r e c o v e r e d as t h e o r i g i n a l s u b s t r a t e (8_) . T h e s e r e s u l t s show t h a t l i m o n o i d s p r e s e n t i n c i t r u s seeds are t r a n s l o c a t e d through the f r u i t t i s s u e . I n h i b i t i o n of B i o s y n t h e s i s . T r i e t h y l a m i n e d e r i v a t i v e s such as 2 - ( 4 - e t h y l p h e n o x y ) t r i e t h y l a m i n e and 2 - ( 3 , 4 - d i m e t h y l p h e n o x y ) t r i e t h y l a m i n e markedly i n h i b i t the accumulation of limonoids i n c i t r u s l e a v e s (15J. F o r e x a m p l e , y o u n g lemon l e a v e s s p r a y e d w i t h 500 ppm o f 2 - ( 4 - e t h y l p h e n o x y ) t r i e t h y l a m i n e c o n t a i n e d o n l y 27 ppm o f XIV 8 d a y s a f t e r t h e t r e a t m e n t , w h e r e a s t h e c o n t r o l c o n t a i n e d 344 ppm. S i m i l a r l y , t h o s e s p r a y e d w i t h 300 ppm o f t h e compound c o n t a i n e d 0.3 t i m e s as much XIV as t h e c o n t r o l . Metabolism Metabolism i n B a c t e r i a . Three s p e c i e s of b a c t e r i a , which a r e c a p a b l e o f m e t a b o l i z i n g l i m o n o i d s , have b e e n i s o l a t e d f r o m s o i l b y Hasegawa e t a l . ( 1 6 , 1 7 , 1 8 , 1 9 ) , T a b l e IV.

Table

IV.

Limonoid-metabolizing

Bacteria

Major

B a c t e r i a and

Metabolites

Metabolic

a

Pathways

Pathways

Arthrobacter globiformis

17-dehydrolimonoate

17-dehydrolimonoid

Pseudomonas s p .

deoxylimonate deoxylimonin

deoxylimonoid 17-dehydrolimonoid

17-dehydrolimonoate deoxylimonate deoxylimonin

17-dehydrolimonoid deoxylimonoid

No.

a

321-18

342-152-1

When grown on

limonoate



72


Two m e t a b o l i c p a t h w a y s o f l i m o n o i d s h a v e b e e n e s t a b l i s h e d : one v i a 17-dehydrolimonoids and t h e o t h e r v i a d e o x y l i m o n o i d s . A. g l o b i f o r m i s m e t a b o l i z e s limonoids v i a o n l y the 17-dehydrolimonoid p a t h w a y (17_) . Pseudomonas s p . 321-18, on t h e o t h e r h a n d , p r o d u c e s i n i t s g r o w t h m e d i a o n l y XVI and X V I I I , w h i c h a r e m e t a b o l i t e s o f the d e o x y l i m o n o i d pathway, b u t t h i s organism a l s o p o s sesses the 17-dehydrolimonoid p a t h w a y (18_) . T h i s o r g a n i s m und o u b t e d l y m e t a b o l i z e s l i m o n o i d s p r e f e r e n t i a l l y v i a the deoxyl i m o n o i d pathway. B a c t e r i u m No. 342-152-1 p r o d u c e s m e t a b o l i t e s i n v o l v e d i n b o t h p a t h w a y s (19). The r a t i o o f XV t o X V I I I i s a b o u t 3 t o 1, s u g g e s t i n g t h a t t h i s o r g a n i s m m e t a b o l i z e s l i m o n o i d s p r e f e r e n t i a l l y v i a the 17-dehydrolimonoid pathway. Enzymes i n B a c t e r i a . L i m o n o a t e d e h y d r o g e n a s e s , w h i c h c a t a l y z e t h e c o n v e r s i o n o f XIV t o XV, h a v e b e e n i s o l a t e d f r o m t h r e e s p e c i e s o f b a c t e r i a by Hasegawa e t a l . (17,18^, 19) . E a c h d e h y d r o genase has d i f f e r e n t c h a r a c t e r i s t i c s (Table V ) .

T a b l e V.

Bacterial

Limonoate

Dehydrogenases

Bacteria

Cofactors

Arthrobacter globiformis

NAD

Pseudomonas s p .

NAD

No.

342-152-1

321-18

NAD

Optimum

pH

9.5 o r NADP

8.5 7.5

T h e s e enzymes a t t a c k n o t o n l y XIV, b u t o t h e r l i m o n o i d s w h i c h c o n t a i n a f u r a n r i n g and e p o x i d e , p r o v i d e d t h a t t h e D - r i n g i s o p e n . The enzymes r e q u i r e s u l f h y d r y l g r o u p s and Zn for their catalytic activity.


4.

MAIER ET AL.

Limonin

and

73

Limonoids

Deoxylimonin h y d r o l a s e , which c a t a l y z e s the c o n v e r s i o n o f XVI t o X V I I I , h a s b e e n i s o l a t e d f r o m c e l l - f r e e e x t r a c t s o f Ρ s e u d o m o n a s s p . 321-18 ( 2 0 ) . T h i s enzyme a t t a c k s o n l y t h e c l o s e d D - r i n g o f XVI a n d r e q u i r e s no c o f a c t o r f o r i t s c a t a l y t i c a c t i v ity. U n l i k e o t h e r common h y d r o l a s e s , t h i s enzyme i s i n h i b i t e d by p - c h l o r o m e r c u r i b e n z o a t e and H g C l . 2


L i m o n i n D - r i n g l a c t o n e h y d r o l a s e has been i s o l a t e d

from

Pseudomonas s p . 321-18 (21_) . H y d r o l y z i n g a c t i v i t y i s o p t i m a l a t pH 8.0, w h e r e a s l a c t o n i z i n g a c t i v i t y i s o p t i m a l a t pH 6.0. The enzyme a t t a c k s l i m o n o i d s whose s t r u c t u r e s d i f f e r f r o m I i n t h e v i c i n i t y of the A or A - r i n g . The enzyme d o e s n o t a t t a c k XVI a n d XVIII. 1

Metabolism i n C i t r u s . R e c e n t l y , Hasegawa e t a l . (6) h a v e e s t a b l i s h e d t h e p r e s e n c e o f a d e o x y l i m o n o i d pathway i n C i t r u s . M e t h y l - 1 4 c v i l l was m e t a b o l i z e d i n l e a v e s o f c a l a m o n d i n t o f o r m a deoxy d e r i v a t i v e , showing t h e p r e s e n c e o f e p o x i d a s e a c t i v i t y which i s r e q u i r e d f o r t h e f i r s t s t e p o f t h e d e o x y l i m o n o i d pathway. Compound X V I I I , t h e p r o d u c t o f t h e s e c o n d s t e p , was a l s o i s o l a t e d f r o m g r a p e f r u i t s e e d s (6). Furthermore, deoxylimonate A - r i n g l a c t o n e h y d r o l a s e , which i s i n v o l v e d i n the t h i r d step o f the p a t h w a y , was a l s o d e t e c t e d i n g r a p e f r u i t s e e d s {6). T h e s e f i n d i n g s c l e a r l y show t h a t l i m o n o i d s a r e m e t a b o l i z e d i n C i t r u s n o t o n l y v i a t h e 1 7 - d e h y d r o l i m o n o i d pathway as p r e v i o u s l y e s t a b l i s h e d (22), b u t a l s o v i a t h e d e o x y l i m o n o i d pathway. L i m o n i n D - r i n g l a c t o n e h y d r o l a s e i s t h e o n l y l i m o n o i d enzyme w h i c h h a s b e e n i s o l a t e d f r o m C i t r u s a n d c h a r a c t e r i z e d (11)· I t i s o f i n t e r e s t t o n o t e t h a t t h i s enzyme i s e x t r e m e l y h e a t r e s i s t a n t . I t r e q u i r e s 15 m i n o f h e a t i n g a t 1 0 0 ° C t o i n a c t i v a t e i t c o m p l e t e ly. I t s f u n c t i o n a l c h a r a c t e r i s t i c s are very s i m i l a r t o those o f the b a c t e r i a l h y d r o l a s e mentioned p r e v i o u s l y . A c t i v i t i e s o f limonoate dehydrogenase (2_2) , e p o x i d a s e (6) a n d d e o x y l i m o n a t e Α - r i n g l a c t o n e h y d r o l a s e (6) h a v e b e e n demon s t r a t e d i n C i t r u s , b u t t h e y have n o t been i s o l a t e d y e t .


74



Organoleptic

Aspects

L i m o n i n h a s b e e n known t o be an i n t e n s e l y b i t t e r s u b s t a n c e s i n c e i t was f i r s t i s o l a t e d i n 1841 by B e r n a y (23). Beginning i n 1966 t h e d e v e l o p m e n t o f a n a l y t i c a l m e t h o d s f o r l i m o n i n a l l o w e d c o r r e l a t i o n s of apparent b i t t e r n e s s with j u i c e limonin content to be u n d e r t a k e n . The v a r i o u s r e p o r t s o f r e l a t i v e b i t t e r n e s s o f s u p r a t h r e s h o l d l i m o n i n l e v e l s h a v e b e e n r e v i e w e d by M a i e r e t a l . (3_) . The g e n e r a l c o n c l u s i o n s r e a c h e d i n t h e s e t e s t s were t h a t l i m o n i n l e v e l s l e s s t h a n a b o u t 6 ppm were g e n e r a l l y n o n b i t t e r and t h a t the b i t t e r n e s s p e r c e i v e d a t higher limonin l e v e l s v a r i e d with t h e s w e e t n e s s , a c i d i t y and o i l l e v e l s o f t h e j u i c e . I n d i v i d u a l v s . Group B i t t e r n e s s T h r e s h o l d s . A comprehensive s t u d y o f l i m o n i n t h r e s h o l d s i n m o d e l s y s t e m s and i n o r a n g e j u i c e was r e p o r t e d by G u a d a g n i e t a l . ( 2 4 ) . Limonin t h r e s h o l d s , the minimum c o n c e n t r a t i o n p e r c e i v e d by human s u b j e c t s , were d e t e r m i n e d u n d e r c l o s e l y c o n t r o l l e d c o n d i t i o n s by a s c r e e n e d p a n e l . A panel o f 27 j u d g e s , c h o s e n f r o m a g r o u p o f 60 f o r t h e i r i n d i v i d u a l c o n s i s t e n c y i n d e t e c t i n g b i t t e r n e s s , showed a w i d e r a n g e o f s e n s i t i v ity. The m o s t s e n s i t i v e i n d i v i d u a l h a d a l i m o n i n t h r e s h o l d i n o r a n g e j u i c e o f 0.5 ppm w h i l e t h a t o f t h e l e a s t s e n s i t i v e was 32 ppm (Table V I ) . T h i r t y p e r c e n t o f t h e p a n e l c o u l d d e t e c t 2 ppm l i m o n i n and 62% c o u l d d e t e c t 4 ppm. The g r o u p t h r e s h o l d f o r t h i s t e s t was 6 ppm i n an o r a n g e j u i c e o f pH 3.8, B r i x / a c i d r a t i o (B/A) of 14.8. M o s t t a s t e s t u d i e s c o n c e n t r a t e on t h e g r o u p t h r e s h o l d r a t h e r than the t h r e s h o l d s o f the i n d i v i d u a l s i n the group. The a b o v e study demonstrates t h a t both are important. The g r o u p t h r e s h o l d

Table

VI.

Thresholds

of I n d i v i d u a l s f o r Limonin B i t t e r n e s s i n Orange J u i c e (3)

Limonin Threshold (ppm) 0.5 1.0 2.0 3.0 4.0 5.0 6.0 10.0 32.0 a

pH

3.8,

B/A

14.8

Cumulative

% of

Panel

8 17 30 49 62 70 75 91 99.5 The AVI Publishing Company, Inc.


4.

MAIER ET AL.

Limonin

and

Limonoids

75


i s u s e f u l t o determine the g e n e r a l i z e d e f f e c t o f a p a r t i c u l a r t a s t e f a c t o r . However, i n d i v i d u a l t h r e s h o l d s a r e h e l p f u l i n s t u d i e s aimed a t d e t e r m i n i n g t h e t a s t e q u a l i t y p a r a m e t e r s o f j u i c e a c c e p t a b l e t o a w i d e r segment o f t h e p o p u l a t i o n , e s p e c i a l l y t h o s e i n d i v i d u a l s most s e n s i t i v e t o b i t t e r n e s s . I n f l u e n c e o f J u i c e Components on B i t t e r n e s s . The e f f e c t s o f a number o f j u i c e v a r i a b l e s were a l s o s t u d i e d ( 2 4 ) . A pH optimum f o r t h e l i m o n i n g r o u p t h r e s h o l d was o b s e r v e d a t pH 3.7 t o 3.9 i n r e c o n s t i t u t e d orange j u i c e concentrate systems. T h e t h r e s h o l d was a maximum o f 6.4 ppm a t pH 3.8 w i t h a t h r e s h o l d o f 3.4 ppm a t pH 3.5 a n d pH 4.1. V a r y i n g t h e pH w h i l e k e e p i n g t h e B/A c o n s t a n t a g a i n showed a maximum t h r e s h o l d o f 6.5 ppm a t pH 3.8. A t a c o n s t a n t pH o f 3.65, i n c r e a s i n g t h e B/A f r o m 10 t o 16 i n c r e a s e d t h e l i m o n i n t h r e s h o l d f r o m 6.2 t o 8.5 ppm. A h i g h e r B/A h a d n o g r e a t er e f f e c t . When j u d g e s were p r e s e n t e d j u i c e s c o n t a i n i n g 4 ppm l i m o n i n w i t h d i f f e r e n t pH a n d B/A v a l u e s , t h e y i n d i c a t e d a d e f i n i t e p r e f e r e n c e f o r t h o s e j u i c e s whose pH a n d B/A l e v e l s r e s u l t e d i n t h r e s h o l d s a b o v e 4 ppm a s p r e d i c t e d f r o m t h e a b o v e d a t a . This c o n f i r m e d t h e c o r r e l a t i o n between h i g h l i m o n i n t h r e s h o l d (low l i m o n i n d e t e c t i o n ) and t a s t e p r e f e r e n c e i n orange j u i c e . Another important i n t e r a c t i o n i s that of limonin with the b i t t e r flavanone glycoside naringin. Both o f these b i t t e r subs t a n c e s a r e p r e s e n t i n g r a p e f r u i t j u i c e a n d G u a d a g n i e t a l . (25) found t h a t t h e y i n t e r a c t a t s u b t h r e s h o l d l e v e l s i n an a d d i t i v e way. L e s s t h a n t h r e s h o l d amounts o f l i m o n i n o r n a r i n g i n c o n t r i b u t e t o t h e b i t t e r n e s s o f a m i x t u r e o f t h e two compounds. T h e b i t t e r n e s s o f t h e m i x t u r e c a n be p r e d i c t e d b y a d d i n g t h e t a s t e u n i t c o n t r i b u t i o n o f e a c h component ( t a s t e u n i t = c o n c e n t r a t i o n / threshold). I n f l u e n c e o f Sweeteners on B i t t e r n e s s . I n model system s t u d i e s , n a t u r a l f r u i t j u i c e s u g a r s were o b s e r v e d t o r a i s e t h e l i m o n i n t h r e s h o l d (24). An e x p a n d e d s t u d y o f n a t u r a l a n d a r t i f i c i a l s w e e t e n e r s (26) d e m o n s t r a t e d t h a t s u c r o s e , n e o h e s p e r i d i n dihydroc h a l c o n e (NHD), h e s p e r e t i n d i h y d r o c h a l c o n e g l u c o s i d e (HDG) a n d a s p a r t y l p h e n y l a l a n i n e m e t h y l e s t e r (AP) a l l r a i s e t h e l i m o n i n threshold. A t l o w s w e e t n e s s l e v e l s HDG was t h e m o s t e f f e c t i v e f o l l o w e d b y AP a n d NHD. S u c r o s e was w i t h o u t e f f e c t u p t o t h e 2% level. A t s w e e t n e s s l e v e l s e q u i v a l e n t t o 1% s u c r o s e , HDG, AP a n d NHD r a i s e d t h e l i m o n i n t h r e s h o l d i n w a t e r f r o m 1.0 ppm t o 3.2, 2.5 and 1.3 ppm, r e s p e c t i v e l y . B e c a u s e o f i t s h i g h s w e e t n e s s i n t e n s i t y , t h e c o n c e n t r a t i o n o f NHD (16 ppm) was c o n s i d e r a b l y l o w e r t h a n HDG (80 ppm) a n d AP (90 ppm). A t 3-10% s u c r o s e s w e e t n e s s e q u i v a l e n c y , t h e e f f e c t i v e n e s s o f NHD i n c r e a s e d s u b s t a n t i a l l y , s u c r o s e m o d e r a t e l y a n d HDG s l i g h t l y , w h i l e t h a t o f AP d e c r e a s e d . T h e r e f o r e , t h e s w e e t e n e r s HDG, AP a n d NHD c a n e f f e c t i v e l y s u p p r e s s l i m o n i n b i t t e r n e s s a t low c o n c e n t r a t i o n s . NHD h a s a l s o b e e n f o u n d t o s u p p r e s s n a r i n g i n b i t t e r n e s s ( 2 6 ) . I t was s u g g e s t e d a s b e i n g e s p e c i a l l y u s e f u l i n u p g r a d i n g t h e f l a v o r o f l o w B/A, e a r l y - s e a s o n g r a p e f r u i t j u i c e ( 2 7 ) .



76


B i t t e r n e s s Suppressors. Limonin b i t t e r n e s s s u p p r e s s i o n has a l s o b e e n r e p o r t e d (28) t o o c c u r when t h e c i t r u s c o n s t i t u e n t n e o d i o s m i n (ΝΕΟ), t h e t a s t e l e s s f l a v o n e analog o f t h e b i t t e r f l a v a n none n e o h e s p e r i d i n ( 2 9 ) , i s p r e s e n t i n s o l u t i o n s o r j u i c e s c o n taining limonin. Low l e v e l s o f Ν Ε Ο were e f f e c t i v e i n r a i s i n g t h e l i m o n i n t h r e s h o l d i n orange j u i c e , r e d u c i n g b i t t e r n e s s o f s u p r a t h r e s h o l d l e v e l s o f l i m o n i n , and even r e d u c i n g t h e b i t t e r n e s s o f very high levels of limonin. Ν Ε Ο i s a l s o an e f f e c t i v e suppressor o f n a r i n g i n b i t t e r n e s s a n d w o u l d , t h e r e f o r e , be e s p e c i a l l y u s e f u l i n b i t t e r g r a p e f r u i t j u i c e w h i c h c o n t a i n s b o t h n a r i n g i n and l i m o nin. On t h e b a s i s o f t h e t a s t e t e s t s a p r o c e s s f o r r e d u c i n g t h e b i t t e r n e s s o f c i t r u s j u i c e s was p r o p o s e d w h i c h c o m p r i s e s a d d i n g 50 t o 150 ppm n e o d i o s m i n ( 3 0 ) . A n o t h e r t a s t e l e s s f l a v o n e g l y c o s i d e t h a t i s p r e s e n t i n g r a p e f r u i t and b i t t e r orange, r h o i f o l i n , has the a b i l i t y t o 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 (29). P r e h a r v e s t , P o s t h a r v e s t and P r o c e s s i n g V a r i a b l e s . These f a c t o r s h a v e a s u b s t a n t i a l i n f l u e n c e on t h e l i m o n i n c o n t e n t o f t h e juice. A d e t a i l e d d i s c u s s i o n o f t h e s e i n f l u e n c e s i s g i v e n by M a i e r e t a l . ( 3 ) . P r e h a r v e s t f a c t o r s such as s p e c i e s and c u l t i v a r , r o o t s t o c k , f e r t i l i z a t i o n and i r r i g a t i o n p r a c t i c e s , and time o f h a r v e s t a l l have an i m p o r t a n t b e a r i n g on t h e l i m o n o i d c o n t e n t of the f r u i t . During the postharvest p e r i o d , limonoid metabolism g r a d u a l l y r e d u c e s t h e X I V c o n t e n t o f t h e f r u i t t i s s u e s . The e x t e n t o f XIV l o s s i n c r e a s e s w i t h h o l d i n g time and t e m p e r a t u r e . The m e t a b o l i s m i s a c c e l e r a t e d by b r i e f exposure t o e t h y l e n e gas o r t o a d i l u t e s o l u t i o n o f e t h y l e n e g e n e r a t i n g s u b s t a n c e s such as 2-chloroethylphosphonic acid (31). Commercial n a v e l orange, Shamouti orange and g r a p e f r u i t j u i c e s , and l a b o r a t o r y samples o f M u r c o t t orange and N a t s u d a i d a i j u i c e s f r o m e a r l y s e a s o n f r u i t c o n t a i n h i g h l e v e l s o f l i m o n i n (3_) U n d e r c e r t a i n c o n d i t i o n s l i m o n i n b i t t e r n e s s c a n be f o u n d i n e s s e n t i a l l y a l l types o f c i t r u s j u i c e s . The r o o t s t o c k h a s b e e n shown t o be a n i m p o r t a n t f a c t o r i n d e t e r m i n i n g t h e t i m e r e q u i r e d a f t e r commercial m a t u r i t y i s reached f o r the f r u i t t o y i e l d j u i c e w i t h low l i m o n i n l e v e l s . The r o o t s t o c k s p r o m o t i n g f a s t e s t a t t a i n m e n t o f low l i m o n i n l e v e l s a r e t r i f o l i t a t e orange, t a n g e l o and C l e o p a t r a m a n d a r i n , w i t h s w e e t o r a n g e b e i n g i n t e r m e d i a t e , a n d r o u g h lemon, sweet l i m e , K u s a i e l i m e and E a s t I n d i a l i m e r e t a r d i n g t h e d i s a p p e a r a n c e o f l i m o n i n (32). The amount o f l i m o n i n i n c o r p o r a t e d i n t o t h e j u i c e i s i n f l u e n c e d b y a number o f j u i c e p r o c e s s i n g v a r i a b l e s . F a c t o r s s u c h a s the m a c e r a t i o n o f albedo, c e n t r a l v a s c u l a r bundle and c a r p e l l a r y membranes, t h e t i m e o f c o n t a c t b e t w e e n t h e r a g a n d t h e j u i c e , a n d t h e amount o f p u l p i n c o r p o r a t e d i n t o t h e f i n a l j u i c e p r o d u c t a r e known t o i n f l u e n c e j u i c e l i m o n i n c o n t e n t . The u n e v e n d i s t r i b u t i o n o f XIV i n t h e v a r i o u s t i s s u e s o f t h e f r u i t (Table V I I ) i s l a r g e l y r e s p o n s i b l e f o r the e f f e c t s o f processing v a r i a b l e s (33).


4.

MAIER ET AL.

Limonin

and

77

Limonoids

a Table VII.

Limonoid

Distribution

i n Desert Navel

Orange T i s s u e

Limonoic A c i d A-Ring Lactone Content ppm f r e s h w e i g h t mg p e r f r u i t


A l b e d o and F l a v e d o C a r p e l l a r y Membranes Juice Vesicles

22 10 1.9

415 305 15

H a r v e s t e d November 12 Determined as l i m o n i n

O n l y a b o u t 6% o f t h e t o t a l XIV c o n t e n t o f t h e f r u i t i s p r e s e n t i n the j u i c e v e s i c l e s . Any s t e p i n j u i c e p r e p a r a t i o n t h a t i n c r e a s e s i n c o r p o r a t i o n o f the other t i s s u e s i n t o the j u i c e or t h a t i n c r e a s e s t h e e x t r a c t i o n o f XIV f r o m t h o s e t i s s u e s w i l l i n c r e a s e t h e l i m o n i n content o f the j u i c e . Consequently, techniques that i n c r e a s e j u i c e y i e l d , such as h a r d v e r s u s s o f t e x t r a c t i o n , t e n d t o i n c r e a s e l i m o n i n content (34). Limonin L e v e l s of C i t r u s J u i c e s . A c o m p i l a t i o n of limonin v a l u e s i n v a r i o u s c i t r u s j u i c e s i s g i v e n i n (3_) · L i m o n i n has b e e n found i n e s s e n t i a l l y a l l v a r i e t i e s o f c i t r u s j u i c e s examined. Although t h e r e i s a c o n s i d e r a b l e range of l i m o n i n values r e p o r t e d , c e r t a i n trends are apparent. L i m o n i n l e v e l s g e n e r a l l y d e c r e a s e as t h e s e a s o n p r o g r e s s e s . T h i s d e c r e a s e w i t h m a t u r i t y o f the f r u i t i s s e e n i n most v a r i e t i e s t e s t e d , a l t h o u g h t h e m a g n i t u d e o f t h e c h a n g e and t h e l e v e l a t any one t i m e o f t h e y e a r o r any p a r t o f t h e h a r v e s t i n g s e a s o n i s v a r iable. Two e x t e n s i v e s t u d i e s o f l i m o n i n c o n t e n t t h r o u g h o u t t h e s e a son were r e c e n t l y c o n d u c t e d by A l b a c h e t a l . ( 3 5 ) . Commercial o r a n g e and g r a p e f r u i t j u i c e s f r o m t h r e e c i t r u s p r o c e s s i n g p l a n t s i n s o u t h T e x a s w e r e s a m p l e d t w i c e e a c h day a t t h r e e - w e e k i n t e r v a l s f r o m e a r l y November t o l a t e J u n e f o r two c o n s e c u t i v e y e a r s . The a v e r a g e l i m o n i n c o n t e n t o f t h e s e j u i c e s i s summarized i n T a b l e VIII. The November o r a n g e j u i c e s a m p l e s were a l l a b o v e t h e g r o u p l i m o n i n t h r e s h o l d d e t e r m i n e d by Guadagni (24). Some o f t h e samp l e s o b t a i n e d t h r o u g h F e b r u a r y were a l s o a b o v e t h e 6 ppm l i m o n i n g r o u p t h r e s h o l d . T h r o u g h o u t t h e s e a s o n a s i g n i f i c a n t number o f j u i c e s c o n t a i n e d e n o u g h l i m o n i n f o r b i t t e r n e s s t o be d e t e c t a b l e by 30-50% o f t h e i n d i v i d u a l s o f G u a d a g n i s t a s t e p a n e l . The l i m o n i n c o n t e n t o f t h e s e o r a n g e j u i c e s w o u l d , t h e r e f o r e , be l i k e l y t o l o w er the t a s t e q u a l i t y of the j u i c e f o r a s u b s t a n t i a l p o r t i o n of the population. 1

A n o t h e r s e t o f t e s t s o f two o r a n g e v a r i e t i e s f r o m 5 l o c a t i o n s i n T e x a s were s a m p l e d a t two-week i n t e r v a l s t h r o u g h o u t t h e i r s e a s o n o f m a t u r i t y a n d j u i c e d i n t h e l a b o r a t o r y on a c o m m e r c i a l t e s t


78 Table

CITRUS NUTRITION AND QUALITY V I I I . L i m o n i n C o n t e n t o f C o m m e r c i a l O r a n g e and J u i c e s Throughout the H a r v e s t i n g Season

Limonin Content


Processing Date

Source:

(ppm)

Orange

November December January February March April May June

Grapefruit

Grapefruit

Range

Average

6.2-8.2 4.2-7.8 3.3-5.5 2.3-6.8 2.5-3.9 2.1-3.8 1.7-2.1 1.8-2.4

7.2 5.3 4.5 3.8 3.3 2.7 1.9 2.1

Range

Average

11.2-12.2 7.1-11.6 7.4-16.6 5.3-9.7 4.4-7.1 2.2-4.7 2.4-3.8

11.4 10.0 9.1 7.1 5.1 3.8 3.1 2.1

(35)

e x t r a c t o r (36). A g a i n t h e l i m o n i n l e v e l s d e c r e a s e d as t h e s e a s o n p r o g r e s s e d f o r b o t h H a m l i n and M a r r s e a r l y o r a n g e s on T e x a s s o u r orange r o o t s t o c k . The M a r r s v a r i e t y was s u s p e c t e d o f h a v i n g a b i t t e r n e s s p r o b l e m b e c a u s e i t a r o s e as a bud s p o r t f r o m t h e Washington n a v e l . The d a t a show l i t t l e c o n s i s t e n t d i f f e r e n c e i n t h e l i m o n i n l e v e l s o f t h e two v a r i e t i e s i n any one g r o v e . Much more s i g n i f i c a n t d i f f e r e n c e s w e r e s e e n i n f r u i t o f t h e same v a r i e t y f r o m t h e d i f f e r e n t g r o v e l o c a t i o n s , p r o b a b l y due t o d i f f e r e n c e s i n c u l t u r a l p r a c t i c e s , s o i l t y p e and e n v i r o n m e n t a l f a c t o r s . W h i l e most o f the l i m o n o i d s o f C i t r u s have been i s o l a t e d from s e e d s , s e v e r a l o c c u r i n d e t e c t a b l e amounts i n o t h e r p a r t s o f t h e fruit. M i n o r amounts o f d e a c e t y l n o m i l i n , n o m i l i n , o b a c u n o n e , d e a c e t y l n o m i l i n i c a c i d and n o m i l i n i c a c i d were i d e n t i f i e d i n e x t r a c t s o f n a v e l orange p e e l (37). 17-Dehydrolimonoate A - r i n g l a c t o n e was i s o l a t e d f r o m p e e l and j u i c e o f n a v e l o r a n g e s (38) and n o m i l i n h a s b e e n r e p o r t e d t o o c c u r i n g r a p e f r u i t j u i c e and j u i c e v e s i c l e s (39). The o n l y C i t r u s l i m o n o i d s known t o be b i t t e r a r e l i m o n i n , V I , X I I , o b a c u n o i c a c i d and IX (_3) . I n f l u e n c e o f L i m o n i n C o n t e n t on J u i c e Q u a l i t y . Several studi e s h a v e shown t h a t l i m o n i n b i t t e r n e s s d e t r a c t s f r o m j u i c e q u a l ity. The t a s t e t e s t s r e p o r t e d by G u a d a g n i (24) u s i n g screened l a b o r a t o r y p e r s o n n e l i n d i c a t e d t h a t f a c t o r s which r a i s e the limon i n t h r e s h o l d i n o r a n g e j u i c e h a v e a b e n e f i c i a l e f f e c t on p r e f e r ence. F e l l e r s (40) r e p o r t e d r e s u l t s o f t a s t e t e s t s o f g r a p e f r u i t j u i c e w i t h a d d e d l i m o n i n and n a r i n g i n g i v e n t o a W a s h i n g t o n D. C. c o n s u m e r - t y p e t a s t e p a n e l o f 72 p e r s o n s . T h e s e t e s t s showed a h i g h l y s i g n i f i c a n t l i n e a r drop i n preference r a t i n g s with


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s u c c e s s i v e l y h i g h e r l e v e l s o f b i t t e r n e s s w i t h i n e a c h B/A g r o u p . The i n v e r s e r e l a t i o n s h i p b e t w e e n l i m o n i n c o n t e n t and t a s t e p r e f e r e n c e was c o n f i r m e d i n a n o t h e r s t u d y (41) u s i n g a s t e p w i s e m u l t i p l e r e g r e s s i o n a n a l y s i s o f d a t a f r o m 60 s a m p l e s o f c o m m e r c i a l f r o z e n - c o n c e n t r a t e d o r a n g e j u i c e (FCOJ) p a c k e d d u r i n g two s e a s o n s . T h i s and a l a t t e r r e p o r t (42) c o n c l u d e d t h a t l i m o n i n c o n t e n t was h i g h l y c o r r e l a t e d with the f l a v o r q u a l i t y of the j u i c e . J u i c e Q u a l i t y Standards. In r e c e n t years l i m o n i n content has become w i d e l y r e c o g n i z e d as a q u a l i t y f a c t o r i n m o s t c i t r u s juices. P r e v i o u s l y l i m o n i n h a d b e e n c o n s i d e r e d t o be important i n t h e j u i c e q u a l i t y o f o n l y a few h i g h l i m o n i n c u l t i v a r s . Limon i n i s b e i n g u s e d r o u t i n e l y by some p r o c e s s o r s as a q u a l i t y c o n t r o l element. I n a d d i t i o n , l i m o n i n c o n t e n t i s now i n c l u d e d i n t h e F l o r i d a S t a t e G r a d e s f o r c a n n e d and c h i l l e d g r a p e f r u i t j u i c e and f r o z e n c o n c e n t r a t e d g r a p e f r u i t j u i c e ( £ 3 ) . G r a d e "A" juice p a c k e d d u r i n g t h e p e r i o d f r o m A u g u s t 1 t o December 1 must c o n t a i n l e s s t h a n 5 ppm l i m o n i n o r l e s s t h a n 600 ppm n a r i n g i n ( D a v i s t e s t method). G r a d e "B" j u i c e must c o n t a i n l e s s t h a n 7 ppm l i m o n i n o r l e s s t h a n 750 ppm n a r i n g i n . Determination of Limonin Content. A summary o f t h e methods r e p o r t e d t h r o u g h 1976 f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f l i m o n i n i n c i t r u s j u i c e s was p u b l i s h e d p r e v i o u s l y (3_). S i n c e t h e n i m p r o v e m e n t s h a v e b e e n made i n t h e h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h i c m e t h o d (44, 45) and a d v a n c e s have b e e n made i n t h e d e v e l o p m e n t o f e n z y m a t i c (46) and immunoassay (47, 48) (see C h a p t e r 15) m e t h o d s . The l a t t e r methods h o l d g r e a t p r o m i s e f o r the f u t u r e . However, i n e a c h c a s e t h e r e q u i r e d enzyme o r a n t i body i s n o t y e t c o m m e r c i a l l y a v a i l a b l e . Thus, w h i l e s u b s t a n t i a l p r o g r e s s i s b e i n g made t h e n e e d s t i l l e x i s t s f o r a s i m p l e , s e n s i t i v e , and r a p i d c o m m e r c i a l l y a v a i l a b l e method f o r r o u t i n e i n d u s t r i a l q u a l i t y c o n t r o l purposes. C o n t r o l o f J u i c e B i t t e r n e s s . A number o f a d v a n c e s h a v e b e e n r e p o r t e d i n t h i s f i e l d s i n c e i t was l a s t r e v i e w e d (.3) . A commerc i a l a p p l i c a t i o n o f the c e l l u l o s e a c e t a t e a d s o r p t i o n technique f o r t h e r e m o v a l o f l i m o n i n f r o m c i t r u s j u i c e s was u n d e r t a k e n ( 4 9 ) . New s o r b e n t g e l f o r m s o f c e l l u l o s e e s t e r s f o r a d s o r p t i o n o f l i m o n i n were d e v e l o p e d ( 5 0 ) . Knowledge was g a i n e d t h a t l i m o n o i d s a r e b i o s y n t h e s i z e d i n c i t r u s l e a v e s and t r a n s l o c a t e d t o t h e f r u i t (12) a n d t h a t s p e c i f i c b i o r e g u l a t o r s c a n i n h i b i t a c c u m u l a t i o n o f XIV i n c i t r u s leaves (15). A d d i t i o n a l s t u d i e s were c a r r i e d o u t on t h e u s e o f n e o d i o s m i n t o s u p p r e s s l i m o n i n and o t h e r t y p e s o f b i t t e r n e s s (30,51). The i n f l u e n c e o f e x t r a c t o r and f i n i s h e r p r e s s u r e s on t h e l e v e l o f l i m o n i n and 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 was r e p o r t e d ( 3 4 ) . A l s o , f u r t h e r s t u d i e s were c o n d u c t e d on t h e m i c r o b i a l s o u r c e s and p r o p e r t i e s o f l i m o n o a t e d e h y d r o g e n a s e (52_) , t h e enzyme t h a t c o n v e r t s XIV t o XV and c a n be u s e d t o p r e v e n t l i m o n i n from forming i n f r e s h l y e x p r e s s e d c i t r u s j u i c e s (53).



80


As t h e s e new t e c h n i q u e s f o r r e d u c i n g l i m o n i n b i t t e r n e s s a r e d e v e l o p e d a n d come i n t o u s e , t h e y w i l l a f f o r d a d d i t i o n a l a n d more d i r e c t means o f a c h i e v i n g h i g h e r q u a l i t y c i t r u s j u i c e s . This w i l l be e s p e c i a l l y i m p o r t a n t f o r t h a t s i g n i f i c a n t p o r t i o n o f t h e p o p u l a t i o n t h a t has a l i m o n i n b i t t e r n e s s t h r e s h o l d i n t h e v i c i n i t y o f and b e l o w 2 ppm. U n t i l t h e n , much c a n be done t o r e d u c e b i t t e r n e s s b y c a r e f u l management o f f a c t o r s i n f l u e n c i n g j u i c e l i m o n i n l e v e l s ( c h o i c e o f c u l t i v a r , r o o t s t o c k , r i p e n e s s , p o s t h a r v e s t me tabolism, j u i c e e x t r a c t i o n parameters, pulp contact time, blending of j u i c e , etc.) and f a c t o r s i n f l u e n c i n g the p e r c e p t i o n o f b i t t e r n e s s ( j u i c e pH, a c i d i t y , s w e e t n e s s , e t c ) .

Abstract Limonoids are a group of chemically related triterpene deriv atives found in the Rutaceae and Meliaceae families. Limonin is an intensely bitter limonoid that occurs widely in citrus juices where its presence at above threshold levels generally detracts from juice quality. Studies have shown that the metabolically ac tive form of limonin is limonoate Α-ring lactone (LARL) which is nonbitter. LARL is synthesized in the leaves and translocated to the fruit and seeds. It is slowly degraded in the fruit to non -bitter products by at least two metabolic pathways. LARL under goes acid catalyzed lactonization to limonin when the fruit tis sues are disrupted in juice preparation. Group bitterness thresh olds for limonin in orange juice have been reported to be in the 6 ppm range. On the other hand, individual thresholds as low as 0.5 ppm have been reported. In the latter study, 30% of the panel had thresholds of 2 ppm or below. While limonin content tends to be high in the juice from early-season navel, Shamouti, and Murcott oranges, available data indicate that levels of 2 ppm are not uncommon in commercial orange juice. Bitterness is modulated by juice properties including soluble solids, citric acid content and pH. In addition, several tasteless citrus flavonoids specif ically suppress bitterness and increase juice acceptability, as do the flavanone glycoside derived dihydrochalcone sweeteners. The above areas are reviewed in detail and new developments in limonoid biochemistry are discussed. Acknowledgement The a u t h o r s t h a n k R o g e r F . A l b a c h , USDA-SEA-AR, W e s l a c o , T e x a s ; R u s s e l l L . R o u s e f f , D e p a r t m e n t o f C i t r u s , AREC, L a k e A l f r e d , F l o r i d a ; a n d R. L . M a n s e l l , D e p a r t m e n t o f B i o l o g y , U n i v e r s i t y o f S o u t h F l o r i d a , Tampa, F l o r i d a a n d t h e i r c o a u t h o r s for prepublication copies o f t h e i r respective manuscripts.

Literature Cited 1. Dreyer, D. L. Fortschr. Chem. Org. Naturst., 1968, 26, 190-244.



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2. Connolly, J. D.; Overton, K. H.; Polonsky, J. In "Progress in Phytochemistry"; Reinhold, L.; Liwschitz, Y., Eds.; John Wiley and Sons: New York, 1970; 2. 3. Maier, V. P.; Bennett, R. D.; Hasegawa, S. In "Citrus Science and Technology"; Nagy, S.; Shaw, P. E.; Veldhuis, M. Κ., Eds.; AVI Publishing Co.: Westport, Conn., 1977; 1, 355-396. 4. Bennett, R. D., unpublished data. 5. Bennett, R. D.; Hasegawa, S. Phytochemistry, in press. 6. Hasegawa, S.; Bennett, R. D.; Verdon, C. P. Phytochemistry, in press. 7. Bennett, R. D.; Hasegawa, S. Tetrahedron, in press. 8. Hasegawa, S.; Bennett, R. D.; Verdon, C. P. J. Agric. Food Chem., in press. 9. Maier, V. P.; Margileth, D. A. Phytochemistry, 1968, 8, 243248. 10. Maier, V. P.; Beverly, G. D. J. Food Sci., 1968, 33, 488-492. 11. Maier, V. P.; Hasegawa, S.; Hera, E. Phytochemistry, 1969, 8, 495-407. 12. Hasegawa, S.; Hoagland, J. E. Phytochemistry, 1977, 16, 469471. 13. Hasegawa, S.; Bennett, R. D.; Verdon, C. P. Abstracts Citrus Research Conference, Pasadena, CA, 1979; 3. 14. Datta, S.; Nicholas, H. J. Phytochemistry, 1968, 7, 955-956. 15. Hasegawa, S.; Yokoyama, H.; Hoagland, J. E. Phytochemistry, 1977, 16, 1083-1085. 16. Hasegawa, S.; Bennett, R. D.; Maier, V. P. J. Agric. Food Chem., 1972, 20, 435-437. 17. Hasegawa, S.; Bennett, R. D.; Maier, V. P.; King, A. D., Jr. J. Agric. Food Chem., 1972, 20, 1031-1034. 18. Hasegawa, S.; Maier, V. P.; King, A. D., Jr. J. Agric. Food Chem., 1974, 22, 523-526. 19. Hasegawa, S.; Kim, K. S. Abstracts Citrus Research Conference, Pasadena, CA, 1975; 16-17. 20. Hasegawa, S.; Maier, V. P., Border, S. N.; Bennett, R. D. J. Agric. Food Chem., 1974, 22, 1093-1096. 21. Hasegawa, S. J. Agric. Food Chem., 1976, 24, 24-26. 22. Hasegawa, S.; Maier, V. P.; Bennett, R. D. Phytochemistry, 1974, 13, 103-105. 23. Bernay, S. Annalen, 1841, 40, 317. 24. Guadagni, D. G.; Maier, V. P.; Turnbaugh, J. G. J. Sci. Food Agric., 1973, 24, 1277-1288. 25. Guadagni, D. G.; Maier, V. P.; Turnbaugh, J. G. J. Sci. Food Agric., 1974, 25, 1349-1354. 26. Guadagni, D. G.; Maier, V. P.; Turnbaugh, J. G. J. Sci. Food Agric., 1974, 25, 1199-1205. 27. Fellers, P. J. 27th Annual Citrus Processors Meeting, Lake Alfred, FL, 1976; 13. 28. Guadagni, D. G., Maier, V. P.; Turnbaugh, J. G. J. Food Sci., 1976, 41, 681-684. 29. Horowitz, R. M. In "Biochemistry of Phenolic Compounds",


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32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.


J. B. Harborne, Ed.; Academic Press: New York, 1964; Chap.14; 545. Guadagni, D. G., Horowitz, R. M.; Gentili, B.; Maier, V. P. U.S. Patent 4,031,265, 1977. Maier, V. P.; Brewster, L. C.; Hsu, A. C. J. Agric. Food Chem., 1973, 21, 490-495. Kefford, J. F.; Chandler, Β. V. In "Advances in Food Research, Supplement 2"; Chichester, C. O.; Mrak, Ε. M.; Stewart, G. F., Eds.; Academic Press: New York, 1970; 150-164. Maier, V. P.; Brewster, L. C., unpublished data. Attaway, J. A. Proc. Int. Soc. Citriculture, 1977, 3, 816-820. Albach, R. F.; Redman, G. H.; Cruse, R. R.; Lime, R. J., un published data. Albach, R. F.; Redman, G. H.; Lime, B. J., unpublished data. Bennett, R. D., unpublished data mentioned in (38). Hsu, A. C.; Hasegawa, S.; Maier, V. P.; Bennett, R. D. Phytochemistry, 1973, 12, 563-567. Rouseff, R. 30th Annual Citrus Processors Meeting, Lake Alfred, FL, 1979; 21. Fellers, P. J. 24th Annual Citrus Processors Meeting, Lake Alfred, FL, 1973; 6. Cornell, J. A. 25th Annual Citrus Processors Meeting, Lake Alfred, FL, 1974; 3. Carter, R. D.; Buslig, B. S.; Cornell, J. A. Proc. Fla. State Hortic. Soc., 1975, 88, 358-370. Florida Department of Citrus, Standards for Processed Citrus Products, 1979; Section 20-64.03, 20-64.09. Fisher, J. F. J. Agric. Food Chem., 1978, 26, 497-499. Rouseff, R. L.; Fisher, J. F., unpublished data. Hasegawa, S.; Verdon, C., unpublished data. Mansell, R. L.; Weiler, Ε. W. Phytochemistry, in press. Weiler, E. W.; Mansell, R. L. J. Agric. Food Chem., in press. Kefford, J. F.; Chandler, Β. V. In "Citrus Science and Technology"; Nagy, S.; Shaw, P. E.; Veldhuis, Μ. Κ., Eds.; AVI Publishing Co.: Westport, Conn., 1977; 2, 609-616. Chandler, Β. V.; Johnson, R. L. J. Sci. Food Agric., 1979, 30, 825-832. Guadagni, D. G.; Horowitz, R. M.; Gentili, B.; Maier, V. P. U.S. Patent 4,154,862, 1979. Hasegawa, S.; Verdon, C. P.; Schroeder, M. F., unpublished data. Hasegawa, S.; Brewster, L. C.; Maier, V. P. J. Food Sci., 1973, 38, 1153-1155.

RECEIVED May 22, 1980.


Limonin and Limonoids - American Chemical Society

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