Chemistry and Function of Pectins - American Chemical Society

17 Pectin M e t h y l a t i o n Changes a n d C a l c i u m I o n Effects o n the Texture o f F r e s h , Fermented, a n d A c i d i f i e d

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Cucumbers Roger F. McFeeters Agricultural Research Service, U.S. Department of Agriculture, and Department of Food Science, North Carolina Agricultural Research Service, North Carolina State University, Raleigh, NC 27695 The commercial use of calcium ion in cucumber pickle products has stimulated efforts to better understand the mechanisms by which calcium affects cucumber texture. Recent results suggest that a high level of pectin methylation has little effect upon the ability of calcium to maintain the firmness of acidified cucumber tissue, while in fermented cucumbers maintenance of a minimum degree of methylation may be important to firmness retention. Efforts are being made to measure calcium binding characteristics in cucumber tissue and to obtain estimates of the effectiveness of calcium ion in inhibiting tissue degradation by pectolytic enzymes. The ability of calcium ions to act as a firming agent in processed fruits and vegetables has been the subject of many studies over the years (1). Among the commodities in which calcium has been observed to cause firming are: snap beans (2), tomatoes (3), apples (4), carrots (5), apricots (6), and jalapeno peppers (7). Cucumbers are commercially preserved by fermentation (processed pickles), acidification and pasteurization (fresh-pack pickles) and refrigeration of mildly acidified fruit (refrigerated dills). Calcium ion has been found to be effective as a firming agent in all three types of products. It is now being used in most commercial cucumber pickle products. Investigations of the structure of cucumber cell walls and the interaction of calcium with the cell walls have followed the practical application of calcium. There have been major advances in our understanding of the structure of plant cell walls over the past 15 years (8). However, it has not proven to be an easy task to explain specific textural changes which occur during ripening or processing of fruits and vegetables in terms of changes in the structures of cell wall polymers (9). This is perhaps not surprising since the detailed structures of cell wall polymers are proving to be very complex This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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( 8 ) . T h e r e a l s o appears t o be g r e a t v a r i a b i l i t y i n the d i s t r i b u t i o n o f p o l y s a c c h a r i d e s i n d i f f e r e n t f r u i t s and v e g e t a b l e s (10). R e c e n t l y , e f f o r t s have been d i r e c t e d toward t r y i n g t o e x p l a i n and u l t i m a t e l y c o n t r o l the t e x t u r a l changes t h a t o c c u r i n cucumbers d u r i n g p r o c e s s i n g and s t o r a g e . Cucumbers have not been an e x c e p t i o n i n t h a t i t i s d i f f i c u l t t o see c l e a r r e l a t i o n s h i p s between s t r u c t u r a l changes i n the c e l l w a l l and t e x t u r e e f f e c t s i n the t i s s u e . D e s p i t e the d i f f i c u l t i e s which a r e e n c o u n t e r e d , the cucumber has s e v e r a l c h a r a c t e r i s t i c s which make i t a good model t o i n v e s t i g a t e t e x t u r e / c e l l w a l l s t r u c t u r a l r e l a t i o n s h i p s . The cucumber mesocarp i s a l a r g e p r o p o r t i o n o f the t o t a l f r u i t t i s s u e , and i t can be i s o l a t e d i n q u a n t i t y w i t h o u t g r e a t d i f f i c u l t y . The mesocarp t i s s u e i s r e l a t i v e l y u n i f o r m i n s t r u c t u r e and c o n t a i n s l i t t l e s t a r c h t o i n t e r f e r e w i t h the a n a l y s i s of c e l l w a l l p o l y s a c c h a r i d e s (11). A s i m p l e t e x t u r e t e s t has been d e v i s e d which can be used w i t h s m a l l p i e c e s o f cucumber t i s s u e and which r e l a t e s w e l l w i t h human p e r c e p t i o n o f f i r m n e s s (12). F i n a l l y , cucumbers can be o b t a i n e d f o r e x p e r i m e n t a l purposes throughout most of the y e a r i n a wide range o f s i z e s . T h i s paper w i l l g i v e a b r i e f background on t e x t u r a l i n v e s t i g a t i o n s o f cucumbers and cucumber p r o d u c t s . Recent work on the s t r u c t u r e o f cucumber c e l l w a l l s and i n i t i a l e f f o r t s t o d e t e r m i n e t e x t u r e / s t r u c t u r e r e l a t i o n s h i p s w i l l then be r e v i e w e d . Cucumbers can be s o f t e n e d e n z y m a t i c a l l y by f u n g a l p o l y g a l a c t u r o n a s e s . They a l s o c o n t a i n n a t u r a l p e c t o l y t i c enzymes, though the c o n d i t i o n s i n which t h e s e enzymes c o n t r i b u t e t o f r u i t s o f t e n i n g have not been d e t e r m i n e d . F i n a l l y , s o f t e n i n g o c c u r s s l o w l y d u r i n g s t o r a g e o f b o t h p a s t e u r i z e d and fermented cucumbers, where p o l y g a l a c t u r o n a s e s have been i n a c t i v a t e d t o n o n d e t e c t a b l e l e v e l s . The mechanism(s) o f t h i s p o s t - p r o c e s s i n g s o f t e n i n g has not been d e t e r m i n e d . B e l l and coworkers i n the 1950's i n v e s t i g a t e d the s o f t e n i n g of cucumbers i n commercial f e r m e n t a t i o n s (13, 1_4, 15, 16) . They found t h a t s o f t e n i n g o f s m a l l s i z e cucumbers was caused p r i m a r i l y by the p r e s e n c e o f p o l y g a l a c t u r o n a s e s i n the f e r m e n t a t i o n b r i n e s , which degraded p e c t i c s u b s t a n c e s i n the f r u i t . These s o f t e n i n g enzymes were p r i m a r i l y o f f u n g a l o r i g i n and were p r e s e n t on the cucumber f r u i t s and f l o w e r s when they were put i n t o f e r m e n t a t i o n t a n k s . Lampi e t a l . (17) attempted t o measure changes i n the p e c t i c s u b s t a n c e s of cucumbers d u r i n g f e r m e n t a t i o n , but were unable t o show any c o n s i s t e n t p a t t e r n o f changes w i t h the t e c h n i q u e s a v a i l a b l e t o them. The use o f c a l c i u m as a f i r m i n g agent f o r cucumber p r o d u c t s began i n the 1960's w i t h commercial t r i a l s conducted o v e r s e v e r a l y e a r s . I t was found t h a t a d d i t i o n o f 0.1% C a C l t o p a s t e u r i z e d cucumber p r o d u c t s r e s u l t e d i n a s i g n i f i c a n t improvement i n the r e t e n t i o n o f a f i r m t e x t u r e d u r i n g s t o r a g e (18). T h i s l e d t o commercial use o f C a C l i n t h e s e p i c k l e p r o d u c t s . F l e m i n g e t a l . (19) found t h a t 0.1% C a C l a l s o h e l p e d p r e v e n t f i r m n e s s l o s s e s i n fermented cucumber s l i c e s and i n s m a l l whole cucumbers a t low NaCl c o n c e n t r a t i o n s . P a r t i c u l a r l y i m p o r t a n t t o the use o f c a l c i u m i n commercial cucumber f e r m e n t a t i o n s was the f i n d i n g by Buescher and coworkers (20, 21) t h a t a t C a C l c o n c e n t r a t i o n s up t o 1%, whole cucumbers remained f i r m even when f u n g a l p o l y g a l a c t u r o n a s e s were i n t e n t i o n a l l y added t o the f e r m e n t i n g cucumbers (Table 1 ) . 2

2

2

2



17.

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Methylation Changes and Calcium Ion Effects

219

T a b l e 1. Cucumber F i r m n e s s 30 Days A f t e r the B e g i n n i n g o f F e r m e n t a t i o n as I n f l u e n c e d by N a C l , C a C l and P o l y g a l a c t u r o n a s e . From Buescher e t a l . (20) 2

Treatment

P i c k l e Firmness (kg)

2% NaCl Control 0.1 M C a C l Polygalacturonase 0.1 M C a C l + P o l y g a l a c t u r o n a s e 2

2

7.9C 9.6AB 2.3E 8.9BC

5% NaCl Control 0.1 M C a C l Polygalacturonase 0.1 M C a C l + Polygalacturonase 2

Mean f i r m n e s s o f 18 p i c k l e s . V a l u e s w i t h the not s i g n i f i c a n t l y d i f f e r e n t .

8.0C 10.OA 5.8D 10.OA

same l e t t e r s


are


220


H i g h c a l c i u m c o n c e n t r a t i o n s were a l s o found t o p r e v e n t breakdown o f the l o c u l a r t i s s u e o f l a r g e cucumbers d u r i n g f e r m e n t a t i o n (22), presumably by p r e v e n t i n g the d e g r a d a t i o n o f p e c t i n by cucumber p o l y g a l a c t u r o n a s e (23). These o b s e r v a t i o n s have l e d t o e f f o r t s t o d e v e l o p a b e t t e r u n d e r s t a n d i n g o f the s t r u c t u r e o f cucumber c e l l w a l l s and the r o l e t h a t c a l c i u m i o n s p l a y i n i m p r o v i n g t e x t u r e . The cucumber c e l l w a l l c o n t a i n s about 30% c e l l u l o s e (10), 15% p e c t i n (24), and n o n c e l l u l o s i c n e u t r a l s u g a r s (10, 25, 2 6 ) . O t h e r t h a n g l u c o s e , which i s p r e s e n t p r i m a r i l y i n c e l l u l o s e , g a l a c t o s e and x y l o s e a r e the most abundant n e u t r a l s u g a r s i n the c e l l w a l l . The degree o f p e c t i n m e t h y l a t i o n i n cucumbers has been r e p o r t e d t o be 65% i n a Japanese f r e s h market c u l t i v a r (27) and 57% i n 4 cm d i a m e t e r p i c k l i n g cucumbers (28). P r e l i m i n a r y e v i d e n c e from t h i s l a b o r a t o r y i n d i c a t e s t h a t p e c t i n m e t h y l a t i o n may i n c r e a s e d u r i n g development o f the cucumber f r u i t . D e m e t h y l a t i o n o f p e c t i n has been t h e most o b v i o u s change o b s e r v e d i n c e l l w a l l s t r u c t u r e , b o t h i n the c h i l l i n g i n j u r y o f f r e s h cucumbers (_27, 29) and d u r i n g f e r m e n t a t i o n (25) . B e l l e t al. (30) showed t h a t cucumber p l a n t s c o n t a i n p e c t i n e s t e r a s e i n a l l p a r t s o f t h e p l a n t , i n c l u d i n g the f r u i t . As p a r t o f a s e r i e s o f s t u d i e s o f c h i l l i n g i n j u r y i n cucumber f r u i t s , Fukushima and Yamazaki (29) found t h a t a d e c r e a s e i n h o t , w a t e r - s o l u b l e , h i g h m e t h y l a t i o n p e c t i n and an i n c r e a s e i n h o t , w a t e r - i n s o l u b l e , low m e t h y l a t i o n p e c t i n o c c u r r e d when f r u i t were s t o r e d a t 0 o r 5 C . They s u g g e s t e d t h a t d e m e t h y l a t i o n o f the p e c t i n by p e c t i n m e t h y l e s t e r a s e r e s u l t e d i n a more r i g i d c e l l w a l l s t r u c t u r e , and t h a t t h i s d e e s t e r i f i c a t i o n may be a common c h a r a c t e r i s t i c o f c h i l l i n g s e n s i t i v e p l a n t s (29). Tang and M c F e e t e r s (25) i n v e s t i g a t e d changes i n the c e l l w a l l s o f cucumber mesocarp t i s s u e when cucumbers were fermented i n 6% N a C l , a p r o c e d u r e s i m i l a r t o commercial f e r m e n t a t i o n s . F i g u r e 1 shows t h a t o n l y s m a l l changes i n the n o n c e l l u l o s i c n e u t r a l sugars o c c u r r e d d u r i n g the e x p e r i m e n t . The t o t a l p e c t i c s u b s t a n c e s showed l i t t l e change d u r i n g f e r m e n t a t i o n and s t o r a g e . The s i z e o f t h e p e c t i n m o l e c u l e s i n t h e major p e c t i n f r a c t i o n s a l s o showed almost no change. The average degree o f p o l y m e r i z a t i o n o f the major p e c t i n f r a c t i o n ( a c i d - s o l u b l e p e c t i n ) i s o l a t e d from f r e s h cucumbers was e s t i m a t e d t o be 402 r e s i d u e s . A f t e r f e r m e n t a t i o n , the degree o f p o l y m e r i z a t i o n o f the E D T A - s o l u b l e f r a c t i o n , which was the major p e c t i n f r a c t i o n a f t e r f e r m e n t a t i o n , was 403 a f t e r 3 months and d e c l i n e d s l i g h t l y t o 365 a f t e r 6 months. The major change o b s e r v e d was a l a r g e d e c r e a s e i n t h e a c i d - s o l u b l e p e c t i n f r a c t i o n d u r i n g the f e r m e n t a t i o n p e r i o d and an i n c r e a s e i n the E D T A - s o l u b l e p e c t i n d u r i n g the same p e r i o d ( F i g u r e 2 ) . The a c i d - s o l u b l e p e c t i n had a degree o f e s t e r i f i c a t i o n o f 62%, w h i l e t h e E D T A - s o l u b l e m a t e r i a l had a degree o f e s t e r i f i c a t i o n t o o low t o measure. These r e s u l t s i n d i c a t e d t h a t p e c t i n d e e s t e r i f i c a t i o n was t h e major change t o o c c u r i n t h e c e l l w a l l d u r i n g f e r m e n t a t i o n . There were no s u b s t a n t i a l changes i n the c e l l w a l l c o m p o s i t i o n from 1 t o 6 months a f t e r b r i n i n g , even though t h e r e was n e a r l y a 30% d e c l i n e i n t i s s u e f i r m n e s s when C a C l ^ was not added t o the f r u i t . T h u s , a s i t u a t i o n was o b s e r v e d i n which a s u b s t a n t i a l change i n t e x t u r e o c c u r r e d i n cucumber f r u i t w i t h o u t an o b v i o u s change i n the s t r u c t u r e o f the c e l l w a l l .



McFEETERS


F i g u r e 1. Changes i n c e l l w a l l n e u t r a l sugar c o n t e n t b r i n i n g and s t o r a g e o f cucumbers.

during




TIME,

MONTHS

F i g u r e 2. Changes i n p e c t i n f r a c t i o n s and s t o r a g e o f cucumbers.

during

fermentation



17.

McFEETERS


223

M c F e e t e r s e t a l . (28) r e c e n t l y i n v e s t i g a t e d t h e e f f e c t s o f p e c t i n m e t h y l a t i o n i n nonfermented cucumber t i s s u e when the degree o f m e t h y l a t i o n was v a r i e d between 12 and 50%. Hudson and Buescher (31) have s t u d i e d the e f f e c t o f m e t h y l a t i o n on t e x t u r e when the degree o f e s t e r i f i c a t i o n was i n the range o f 6 t o 18%. M c F e e t e r s e t a l . (28) were a b l e t o v a r y the degree o f m e t h y l a t i o n by b l a n c h i n g cucumber s l i c e s a t d i f f e r e n t t e m p e r a t u r e s . H e a t i n g the s l i c e s a t 66 C o r l e s s caused l i t t l e o r no i n a c t i v a t i o n o f p e c t i n e s t e r a s e p r e s e n t i n the t i s s u e (30) . D u r i n g s t o r a g e i n pH 3 . 7 , 2.0% N a C l b r i n e , the p e c t i n was e x t e n s i v e l y demethylated i n t h e s e t i s s u e s ( F i g u r e 3 ) . When s l i c e s were h e a t e d a t 81 C , p e c t i n e s t e r a s e was i n a c t i v a t e d . However, d u r i n g s t o r a g e , p a r t i a l r e a c t i v a t i o n o f the enzyme o c c u r r e d . The r e s u l t was an i n t e r m e d i a t e l e v e l o f m e t h y l a t i o n . F i n a l l y , i f cucumber t i s s u e was h e a t e d i n b o i l i n g water, only s l i g h t r e a c t i v a t i o n of p e c t i n e s t e r a s e a c t i v i t y o c c u r r e d and the degree o f m e t h y l a t i o n remained n e a r 50%. The e f f e c t o f changing the degree o f p e c t i n m e t h y l a t i o n on the f i r m n e s s o f cucumber mesocarp t i s s u e was d e t e r m i n e d o v e r a 6-month s t o r a g e p e r i o d . When t h e s l i c e s were s t o r e d a f t e r b l a n c h i n g i n a b r i n e which c o n t a i n e d 10 mM c a l c i u m i o n , t h e r e were some t e x t u r e d i f f e r e n c e s , but the d i f f e r e n c e s o b s e r v e d were not v e r y l a r g e . The f i r m e s t t e x t u r e was o b t a i n e d when the t i s s u e was b l a n c h e d a t i n t e r m e d i a t e t e m p e r a t u r e s , i . e . 66 and 81 C . S l i c e s b l a n c h e d a t 99 C were l e s s f i r m t h a n the o t h e r t r e a t m e n t s . However, much o f the o b s e r v e d d i f f e r e n c e c o u l d be a t t r i b u t e d t o d i f f e r e n c e s i n the t i s s u e f i r m n e s s i m m e d i a t e l y a f t e r b l a n c h i n g , b e f o r e the t i s s u e was exposed t o b r i n e s o l u t i o n s . The r a t e s o f f i r m n e s s l o s s d u r i n g the 6-month s t o r a g e p e r i o d were s i m i l a r , r e g a r d l e s s o f the degree o f p e c t i n m e t h y l a t i o n . When the c a l c i u m c o n c e n t r a t i o n was v a r i e d i n s l i c e s b l a n c h e d a t 54, 66, and 81 C , t h e r e was a v e r y c l e a r i n c r e a s e i n f i r m n e s s r e t e n t i o n as the c a l c i u m c o n c e n t r a t i o n i n c r e a s e d ( F i g u r e 4 ) . However, the p a t t e r n o f f i r m n e s s changes was t h e same, r e g a r d l e s s o f b l a n c h t e m p e r a t u r e . T h u s , the r e s u l t s o f t h e s e s t u d i e s d i d not show any d i r e c t r e l a t i o n s h i p between p e c t i n m e t h y l a t i o n and f i r m n e s s changes. C a l c i u m i o n was e f f e c t i v e i n p r e v e n t i n g f i r m n e s s l o s s d u r i n g s t o r a g e , r e g a r d l e s s o f t h e degree o f p e c t i n m e t h y l a t i o n . S t u d i e s o f c a l c i u m i o n b i n d i n g by p o l y p e c t a t e have shown t h a t b l o c k s o f a t l e a s t 14 c o n s e c u t i v e d e m e t h y l a t e d c a r b o x y l groups on a d j a c e n t p o l y g a l a c t u r o n a n m o l e c u l e s a r e r e q u i r e d f o r c o o p e r a t i v e c r o s s - l i n k i n g t o form an "eggbox" t y p e s t r u c t u r e (32, 3 3 ) . The f a c t t h a t c a l c i u m i s e f f e c t i v e i n p r e v e n t i n g s o f t e n i n g , even a t h i g h d e g r e e s o f p e c t i n m e t h y l a t i o n , s u g g e s t s t h a t o t h e r t y p e s o f p o l y s a c c h a r i d e / c a l c i u m i n t e r a c t i o n s may be i n v o l v e d i n the cucumber t i s s u e . C a l c i u m i o n has been shown t o form c r y s t a l l i z a b l e c o o r d i n a t i o n complexes w i t h many mono- and d i s a c c h a r i d e s (34, _35) . Cook and Bugg (35^) have s p e c u l a t e d upon the p o s s i b l e importance o f c a l c i u m / g a l a c t o s e i n t e r a c t i o n s i n bone t i s s u e . I t may be u s e f u l t o c o n s i d e r whether such i n t e r a c t i o n s occur i n p l a n t c e l l w a l l s . Hudson and Buescher (31) have found a r e l a t i o n s h i p between cucumber t i s s u e f i r m n e s s and the degree o f m e t h y l a t i o n o f the mesocarp t i s s u e . When t h e p e c t i n m e t h y l a t i o n was l e s s than 13%, the f i r m n e s s o f the t i s s u e d e c l i n e d as the degree o f m e t h y l a t i o n




2 STORAGE

4 TIME,

6

MO.

F i g u r e 3. E f f e c t o f b l a n c h temperature on changes i n m e t h y l a t i o n o f the cucumber c e l l w a l l d u r i n g s t o r a g e .

pectin




MCFEETERS



226


d e c r e a s e d . They s u g g e s t t h a t t h i s may be caused by a change i n the c o n f o r m a t i o n o f the p e c t i n m o l e c u l e s a t v e r y low e s t e r i f i c a t i o n s i m i l a r t o t h a t r e p o r t e d by L e e p e r and D u l l (36) f o r p e c t i n s o l u t i o n s . However, because i t was n e c e s s a r y t o compare t r e a t m e n t s w i t h and w i t h o u t c a l c i u m i o n s p r e s e n t , the t e x t u r e d i f f e r e n c e s o b s e r v e d may have been caused by f a c t o r s o t h e r t h a n d i f f e r e n c e s i n the degree o f m e t h y l a t i o n . S i n c e we a r e i n t e r e s t e d i n t r y i n g t o b e t t e r u n d e r s t a n d the i n t e r a c t i o n o f m e t a l i o n s w i t h the cucumber c e l l w a l l , i t was o f i n t e r e s t t o d e v e l o p a t e c h n i q u e t o determine t h e e x t e n t and a f f i n i t y o f i o n b i n d i n g i n cucumber t i s s u e . S i n c e i t i s g e n e r a l l y thought t h a t c a l c i u m i s bound t o p l a n t t i s s u e s by i n t e r a c t i n g w i t h the f r e e c a r b o x y l groups p r e s e n t i n p e c t i n , an e f f o r t has been made t o r e l a t e c a l c i u m bound i n cucumber mesocarp t i s s u e t o the c o n c e n t r a t i o n o f the f r e e c a r b o x y l groups o f p e c t i n . P i e c e s o f mesocarp t i s s u e were i s o l a t e d from cucumbers and p l a c e d i n a b r i n e t o g i v e an e q u i l i b r a t e d c o n c e n t r a t i o n o f 0.6% a c e t i c a c i d , 200 ppm SO^ ( f o r p r e s e r v a t i o n ) , and v a r y i n g c o n c e n t r a t i o n s o f c a l c i u m i o n from 1 t o 16 mM. A f t e r e q u i l i b r a t i o n , the degree o f p e c t i n m e t h y l a t i o n i n the mesocarp t i s s u e was measured (24) and the c o n c e n t r a t i o n o f f r e e c a r b o x y l groups i n the t i s s u e c a l c u l a t e d . The c o n c e n t r a t i o n o f c a l c i u m bound by the t i s s u e was c a l c u l a t e d as the d i f f e r e n c e i n c a l c i u m c o n c e n t r a t i o n between the mesocarp t i s s u e and b r i n e s o l u t i o n as d e t e r m i n e d by a c o l o r i m e t r i c p r o c e d u r e (37, 3 8 ) . These d a t a were used t o c o n s t r u c t a S c a t c h a r d p l o t t o a n a l y z e b o t h the moles o f c a l c i u m bound per c a r b o x y l group i n the t i s s u e and the a f f i n i t y o f c a l c i u m binding. F i g u r e 5 shows an example o f the a n a l y s i s o f such a b i n d i n g e x p e r i m e n t . The i n t e r c e p t on the X - a x i s gave a r a t i o o f 0.43 c a l c i u m i o n s bound p e r f r e e c a r b o x y l group i n the t i s s u e . I f each d i v a l e n t c a l c i u m i o n were b i n d i n g t o two p e c t i n c a r b o x y l g r o u p s , a r a t i o o f 0.5 would be e x p e c t e d . A n a l y s i s o f the s l o p e o f the S c a t c h a r d c u r v e , gave an a f f i n i t y c o n s t a n t o f 888 f o r c a l c i u m b i n d i n g . Kohn (32) d e t e r m i n e d c a l c i u m b i n d i n g c o n s t a n t s as a f u n c t i o n o f the degree o f p e c t i n e s t e r i f i c a t i o n f o r p e c t i n s o l u t i o n s a t n e u t r a l pH. S t a b i l i t y c o n s t a n t s v a r i e d from

Chemistry and Function of Pectins - American Chemical Society

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