Flavor Precursors - American Chemical Society

Chapter 6

Precursor Atmosphere Technology Efficient Aroma Enrichment in Fruit Cells 1

2

2

Ralf G. Berger, Gerd R. Dettweiler, Gabriele M. R. Krempler, and Friedrich Drawert 2

1

Institut für Lebensmittelchemie der Universität Hannover, Wunstorferstrass 14, D-3000 Hannover 91, Germany Institut für Lebensmittelchemie und Analytische Chemie der Technische Universität München, D—8050 Freising 12, Germany

Downloaded by CORNELL UNIV on September 28, 2016 | http://pubs.acs.org Publication Date: May 13, 1992 | doi: 10.1021/bk-1992-0490.ch006

2

Precursor atmosphere (PA)-technology is a short-time storage biotechnology that uses intact, mature fruit cells as a biocatalyst for the production of fruit flavor. Precursor substrate is supplied by exposing fruit tissue to a controlled atmosphere containing vapor of, e.g., volatile alcohols. Increasing the substrate concentration subsequently increased the concentration of certain carboxylic esters in apple cvs.; accumulation factors for ethyl 2-methylbutanoate of 20 in 48 h, and more than 50 in 8 d were recorded. The newly formed esters can be isolated from the PA by adsorption/solvent desorption, or can be transfered into processed products. Analytical and sensory data on pilot scale HTST-juices of PAstored apple demonstrate a significant improvement of the product quality. V o l a t i l e c a r b o x y l i c e s t e r s c o n s t i t u t e an i m p o r t a n t g r o u p o f aroma s u b s t a n c e s i n many f r u i t s , e . g . , i n apple, pear, and b a n a n a . The p r o d u c i n g c e l l s a r e d i s t i n g u i s h e d not o n l y by t h e i r a b i l i t y t o f o r m , a t o r b e y o n d t h e c l i m a c teric maximum, esters from substrates not regularly present in healthy plant c e l l s ; they are a l s o able to t a k e up and m e t a b o l i s e exogenous s u b s t r a t e s when t i s s u e preparations are incubated i n a buffer s o l u t i o n according t o t h e "Aged T i s s u e m o d e l " (1) . Though i n t h e s e t i s s u e p r e p a r a t i o n s an i n d i r e c t s t i m u l a t i o n o f t h e f l a v o r m e t a b o l i s m c a n n o t be r u l e d o u t c o m p l e t e l y , the patterns of d i s t r i b u t i o n o f v o l a t i l e s a f t e r e x p o s u r e , a s compared t o untreated controls, s u g g e s t a d i r e c t b i o c o n v e r s i o n and dynamic i n c o r p o r a t i o n i n t o t h e v o l a t i l e f l a v o r f r a c t i o n ( 2 ) . The o p e r a t i o n o f s e v e r a l p a t h w a y s i n r i p e f r u i t s was

0097-6156/92/0490-0059S06.00/0 © 1992 American Chemical Society

Teranishi et al.; Flavor Precursors ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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FLAVOR PRECURSORS

concluded from t h e s e feeding experiments: β-oxidative s h o r t e n i n g of f a t t y a c i d s ; r e d u c t i o n of c a r b o n y l groups and r e v e r s e ; d e c a r b o x y l a t i o n o f k e t o a c i d s ; d e g r a d a t i o n of u n s a t u r a t e d f a t t y a c i d s a c c o r d i n g t o the alternative pathway ( 3 ) ; f o r m a t i o n o f c a r b o x y l i c e s t e r s from t h e i r single moieties. The t e c h n i c a l a p p l i c a t i o n o f t h i s o b v i o u s p o t e n t i a l to b i o s y n t h e s i s e s e n s o r y k e y compounds was p r e v e n t e d by some drawbacks : A h i g h degree of cleanliness must be m a i n t a i n e d during precursor incubation to avoid microbial i n t e r f e r e n c e , and a large scale production of defined t i s s u e prepara t i o n s f r o m m a t e r i a l s o f v a r i a b l e s i z e and f i r m n e s s appears i m p o s s i b l e . The s o l u t i o n t o t h e p r o b l e m i s s u p p l y i n g i n t a c t m a t u r e f r u i t s w i t h v o l a t i l e p r e c u r s o r s v i a t h e gas phase d u r i n g storage. The e f f i c a c y of the so-called PA-(Precursor Atmosphere) storage i n enhancing the e s t e r content of f r u i t a s been d e m o n s t r a t e d f o r a p p l e ( 4 - 8 ) , p e a r , c h e r r y , strawberry (9) , and banana (20) . F o r t h e p r e s e n t work s e v e r a l a p p l e c v s . were s e l e c t e d b e c a u s e apple i s the major f r u i t i n Europe, v o l a t i l e s and b i o s y n t h e s i s a r e w e l l - k n o w n , as i s t h e l a c k o f aroma o f C A - s t o r e d (11,12) and o f t h e r mally processed products of apple (9). Experimental Samples. A p p l e s were from t h e Fachhochschule Weihenstephan and f r o m l o c a l m a r k e t s and u s e d d i r e c t l y or stored i n cooled c e l l s ( 7 ° C ) before use. I n t a c t , healthy, and color matched fruits were randomly d i v i d e d into c o n t r o l and Ρ Α - s t o r e d samples. Commercial a p p l e juices were f r o m German m a n u f a c t u r e r s . P i l o t s c a l e j u i c e s o f u n t r e a t e d and Ρ Α - s t o r e d a p p l e s were o b t a i n e d by u s i n g a centrifugal press, a p l a t e heat exchanger (Holstein & Kappert), and a two s t a g e t u b u l a r h e a t exchanger for down-cooling. I s o l a t i o n o f Aroma Compounds. 100g p i e c e s were c u t f r o m 6 a p p l e s and immersed i m m e d i a t e l y i n MeOH, i n t e r n a l s t a n d ard was added, and t h e homogenised, centrifuged and c o o l e d ( 4 ° C ) s o l u t i o n a f t e r d i l u t i o n w i t h s a t d . N a C l was extracted with Pentan/Diethylether 1/1. The c o m b i n e d , d r i e d , and c o n c e n t r a t e d e x t r a c t s were u s e d f o r GC and G C MS a n a l y s e s . J u i c e s were t r e a t e d i n t h e same way a s c e n t r i f u g e d s o l u t i o n s (12) . I n s t r u m e n t a l A n a l y s i s . C a p i l l a r y GC ( C a r l o E r b a , V a r i a n , Siemens) and GC-MS ( P e r k i n E l m e r / F i n n i g a n ) using fused silica c a p i l l a r y c o l u m n s c o a t e d w i t h O V - 1 0 1 o r CW20M (G.Leupold, Weihenstephan), l i n e a r temperature programs, and electronic data integration (Merck-Hitachi) was d e s c r i b e d i n d e t a i l elsewhere (23).


6.

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Precursor Atmosphere Technology

61

Sensory A n a l y s i s . 9 randomly s e l e c t e d judges of a group of 14 t r a i n e d persons performed t r i a n g l e tests. All s a m p l e s were a d j u s t e d t o a c o n s t a n t s u g a r a c i d r a t i o by a d d i n g s u c r o s e o r m a l i c a c i d , and were p r e s e n t e d i n n o n transparent glasses.


L o c a l i s a t i o n o f Aroma i n A p p l e . 25 s i z e m a t c h e d a n d P A s t o r e d a p p l e s were c u t i n t o h a l v e s . A c y l i n d r i c a l piece o f t i s s u e ( o . d . 10 mm) was r a d i a l l y e x c i s e d , t h e c y l i n d e r cut into 6 defined zones, the corresponding zones combined, a d j u s t e d t o i d e n t i c a l weight, transfered into MeOH, and t h e v o l a t i l e f r a c t i o n i s o l a t e d a s f o r s e g m e n t s of i n t a c t a p p l e s . T o t a l A c i d . F i l t e r e d homogenate t i t r a t e d t o pH 8 . 1 .

o r j u i c e was d e g a s s e d and

PA-Storaqe. F r u i t s were p l a c e d on a p o r c e l l a n insert i n s i d e a g a s - t i g h t g l a s s j a r (10 L) and k e p t a t a m b i e n t temperature and light conditions, if not otherwise m e n t i o n e d . M i l l i m o l a r amounts o f p r e c u r s o r s were c a l c u l a t e d i n r e l a t i o n t o t h e e x a c t a c t u a l mass o f t h e f r u i t s (ca. 1 kg) and p l a c e d i n a s m a l l g l a s s p l a t e a t the b o t t o m . A f t e r i n d i c a t e d i n c u b a t i o n p e r i o d s t h e j a r was opened t o a l l o w gas exchange w i t h ambient atmosphere. Pressure I n j e c t i o n of Precursor. Butanol soins, in H 0 were a d m i n i s t e r e d u s i n g a D e r m o - J e t m e d i c a l p r e s s u r e i n j e c t o r (Medimex-Holfeld) w i t h a f i x i n j e c t i o n volume o f 90μ1. D i s t i l l e d H2O was i n j e c t e d i n t o c o n t r o l s a m p l e s . A uniform d i s t r i b u t i o n of the i n j e c t e d l i q u i d across the c r o s s s e c t i o n o f t h e f r u i t f l e s h was d e m o n s t r a t e d u s i n g a 0.1% s o i n , o f m e t h y l e n e b l u e . 2

Results and Discussion Application of Precursor.

The b i o s y n t h e s i s o f c a r b o x y l i c esters i n f r u i t i s believed to proceed v i a r e - e s t e r i f i c a t i o n of acyl-CoA intermediates of the β - o x i d a t i o n p a t h way w i t h r e d u c e d and l i b e r a t e d ( a l k y l ) m o i e t i e s o f the same i n t e r m e d i a t e (1,14). According to e a r l i e r results (4-8) a l a c k o f t h e s u b s t r a t e s o f t h e e s t e r i f y i n g enzyme s y s t e m (15) i s the t h e o r e t i c a l b a s i s of the attempts to c r e a t e a r o m a - e n r i c h e d m a t e r i a l s by m a k i n g a v a i l a b l e a d d i t i o n a l p r e c u r s o r . Pressure i n j e c t i o n of precursor s o l u t i o n s i n t o i n t a c t f r u i t would s o l v e the problem o f p r e p a r i n g t i s s u e d i s k s on a t e c h n i c a l s c a l e . n - B u t a n o l was i n j e c t e d i n t o a p p l e s , and t h e a p p l e s were t h e n s t o r e d i n a g a s - t i g h t j a r . Though a s u f f i c i e n t amount o f p r e c u r s o r accumulated i n the t i s s u e ( T a b l e I) , t h e i n c r e a s e s of b u t y l a n d b u t a n o a t e e s t e r s were i n t h e same r a n g e a s f o r t h e c o n t r o l b a t c h (H2O i n j e c t e d ) . O b v i o u s l y , t h e p r e c u s o r did not reach the enzymatic r e a c t i o n s i t e . A previous l o c a l i s a t i o n of v o l a t i l e s across the cross section of a p p l e f r u i t showed t h a t t h e c o n c e n t r a t i o n o f e s t e r s is


62

FLAVOR

Table I . Concentration of V o l a t i l e s a f t e r tion of n-Butanol into Apples


Compound (Mg/100g)

Control 0 d

PRECURSORS

Pressure Injec cv. Jonathan

Precursor I d

injected* 3 d

n-Butanol

186

4030

3610

Et Bu Bu Bu Bu He He He

63 100 55 17 67 148 42 121

85 120 52 24 76 153 53 164

98 103 91 80 193 213 107 330

butanoate acetate butanoate 2-mebutanoate hexanoate acetate butanoate 2-mebutanoate

* 10 χ 500

μg n-Butanol/Fruit

two o r d e r s o f m a g n i t u d e h i g h e r i n t h e p e e l s e c t i o n i n the parenchymatic c e l l s of the f l e s h (26). Previous i n c u b a t i o n experiments using precursors of esters with separated peel or peel d i s c s (22,15,17) a l s o indicated t h a t an a c y l - C o A - a l k y l - t r a n s f e r a s e i s l o c a t e d s u b e p i d e r m a l l y . A d i f f u s i o n a l t r a n s p o r t b r i n g s aroma compounds t o i n n e r p a r t s o f t h e f r u i t . As e x p e c t e d , t h e more p o l a r compounds show a l e s s p r o n o u n c e d c o n c e n t r a t i o n g r a d i e n t i n t h e aqueous m a t r i x o f t h e f r u i t ( 2 6 ) . W i t h r e s p e c t t o the morphological s i t u a t i o n , an a d m i n i s t r a t i o n o f v o l a t i l e p r e c u r s o r s o f aroma e s t e r s v i a t h e s u r r o u n d i n g g a s p h a s e a p p e a r e d more p r o m i s i n g .

Ethanol Atmosphere Storage of cv. Red Delicious.

When Red

D e l i c i o u s a p p l e s were s t o r e d i n g a s - t i g h t j a r s , t h e CO2 c o n c e n t r a t i o n i n c r e a s e d w i t h i n 4 d a y s t o 3.2 % v / v u n d e r t h e above d e s c r i b e d c o n d i t i o n s , and t h e r e was a l s o a s l i g h t increase i n ester concentrations (Figure 1). T h i s i s i n good agreement w i t h o t h e r d a t a on t h e a v e r a g e CO2 p r o d u c t i o n by s t o r e d a p p l e s ( 2 8 ) . I n t h e p r e s e n c e o f e x o g e n o u s e t h a n o l t h e a c c u m u l a t i o n o f CO2 was l o w e r ( c a . 2 % v / v a f t e r 4 days) , but t h e c o n c e n t r a t i o n s o f a l l t h e e t h y l e s t e r s i n the f r u i t increased d r a m a t i c a l l y (Figure 1 ) . The most p r o n o u n c e d e n r i c h m e n t was o b s e r v e d f o r e t h y l 3 - H O - o c t a n o a t e : a more t h a n 2 6 0 f o l d increase within a p e r i o d o f 48 h o u r s a s compared t o t h e c o n t e n t i n t h e u n t r e a t e d c o n t r o l . A p p l e s o f t h e same b a t c h , when s t o r e d a t ambient atmosphere, l o s t d i f f e r e n t p r o p o r t i o n s of the i n t e r n a l e s t e r s and a c c u m u l a t e d a l i p h a t i c a l c o h o l s . The PA i n d u c e d e s t e r a c c u m u l a t i o n , t h o u g h enzyme cata l y s e d , was n o t s i g n i f i c a n t l y a c c e l e r a t e d by e l e v a t e d s t o rage temperatures (Figure 2a/b). While the longer chain f a t t y a c i d e s t e r s showed s l i g h t i n c r e a s e s at 28°C, the c o n c e n t r a t i o n s o f most v o l a t i l e e s t e r s were d e c r e a s e d a s compared t o a c o n t r o l s a m p l e s t o r e d a t a m b i e n t t e m p e r a -


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Precursor Atmosphere Technology C Ethyl 2-methylbutanoate (pg/100g)

I

I PA stored


H I

control

24 48 Incubation time (h) F i g u r e 1. P r e c u r s o r c e n t r a t i o n of e t h y l o l / k g a p p l e c v . Red

atmosphere ( P A ) - s t o r a g e and c o n 2-methylbutanoate (130 mM e t h a n Delicious).

C Ethyl 4-(Z)-decenoate (pg/IOOg)

fl

19 C

a

10 C

8

19 C

B

28 C

C Ethyl 2-methylbutanoate (pg/100g)

fl

19 C

fl

10 C

a

19 C

fl

28 C

F i g u r e 2 a / b . E f f e c t o f t e m p e r a t u r e and Ρ Α - s t o r a g e on concentrations of ethyl esters in apple (100 mM e t h a n o l / k g c v . Red Delicious).


63


64

FLAVOR

PRECURSORS

t u r e ( 1 9 ° C ) . At 1 0 ° C , which i s c l o s e r to the conditions of classical c o n t r o l l e d atmosphere storage, all ethyl esters increased as compared t o an u n t r e a t e d control sample k e p t a t 1 9 ° C . Comparing d i f f e r e n t ethanol concentrations i n the gas p h a s e , s a t u r a t i o n o f t h e e s t e r i f y i n g s y s t e m was r e a c h e d for short chain fatty acids at concentrations < 50 mM EtOH/kg f r u i t . A t higher ethanol c o n c e n t r a t i o n s o n l y the a c y l m o i e t i e s > C5 were a f f e c t e d . P l o t t i n g t h e t o t a l n e o s y n t h e s i s (as a measure o f r e a c t i o n v e l o c i t y ) of ethyl esters vs. ethanol concentration a typical Michaelis c u r v e was o b t a i n e d r e f l e c t i n g t h e k i n e t i c s o f t h e rate l i m i t i n g enzyme ( F i g u r e 3 ) . T h e r e i s a s m a l l n o n - l i n e a r i t y o f pEtOH o v e r t h e H 0 / E t O H s o l u t i o n i n t h e a p p l i e d c o n c e n t r a t i o n r a n g e t h a t was i g n o r e d . While f o r m a l l y not p e r m i s s i b l e , the transformed k i n e t i c d a t a ( F i g u r e 4) were u s e d t o c a l c u l a t e v a n d KM v a l u e s t o compare w i t h known v a l u e s o f p u r e e s t e r a s e s o r l i p a s e s . One c a n c o n c l u d e t h a t t h e a c y l a t i o n o f t h e e x o g e n o u s s u b s t r a t e r u n s v e r y r a p i d l y , and t h a t e t h a n o l c o n c e n t r a t i o n s < 20 mM/kg were q u i t e e f f e c t i v e u n d e r t h e s e c o n d i t i o n s . Aged t i s s u e s o f s t r a w b e r r y c o n v e r t e d e x o g e n o u s 1a l a n i n e e v e n somewhat more r a p i d l y w i t h K M s r a n g i n g f r o m 0.26 t o 1 mM/kg ( 2 9 ) . I n t h e p r e s e n c e o f a s u f f i c i e n t amount o f e t h a n o l t h e i n crease of the concentrations of the e t h y l e s t e r s con t i n u e d f o r one week o r more ( F i g u r e 5) . T h e s u b s e q u e n t d r o p i n t h e c o n c e n t r a t i o n o f e t h y l e s t e r s i s c a u s e d by e x h a u s t i o n o f t h e p r e c u r s o r , and n o t by an i n a c t i v a t i o n o f t h e enzymes i n v o l v e d . P h y s i o l o g i c a l d i s o r d e r s were n o t observed at t h i s p o i n t of time, n e i t h e r v i s u a l l y nor ana lytically. 2

m

a

x

1

ΡΑ-Storage of Apples cv.

Jonathan and Purple Cousinot. B u t y l and h e x y l e s t e r s a r e p r e d o m i n a n t i n t h e s e cult i v a r s . A s was o b s e r v e d f o r t h e i n c u b a t i o n o f c v . .Red Delicious with ethanol, the corresponding esters in creased during Ρ Α - s t o r a g e at i n c r e a s i n g butanol or hexanol concentrations, and a g a i n t h e low m o l e c u l a r w e i g h t e s t e r s were more a f f e c t e d a t l o w e r p r e c u r s o r c o n c e n t r a tions (Figure 6a/b). Regarding the total increase of e s t e r s the e s t e r i f y i n g system i s s a t u r a t e d a t lower c o n c e n t r a t i o n s of p r e c u r s o r with i n c r e a s i n g molecular weight of t h e a l c o h o l . S u r p r i s i n g l y , exogenous a l c o h o l c o n c e n t r a t i o n s > 10 mM/kg r e s u l t i n d e c r e a s e s o f b o t h i n t e r n a l a l c o h o l and e s t e r s c o n t e n t s . A c c o r d i n g t o (20) i t a p p e a r s that the p e r m e a b i l i t y of the peel i s a f u n c t i o n of the e x t e r n a l a l c o h o l c o n c e n t r a t i o n . T h i s may be e x p l a i n e d a s a p r o t e c t i v e mechanism a g a i n s t s t r e s s f u l e x t e r n a l c o n d i t i o n s . The p r e f e r e d f o r m a t i o n o f t h e s y m m e t r i c b u t y l b u t a n o a t e and h e x y l h e x a n o a t e , respectively, points to a concurrent o x i d a t i o n of the precursor a l c o h o l ; the i n t e r m e d i a t e a l d e h y d e s , h o w e v e r , were n o t d e t e c t e d . These findings permit the generation of apples with t a i l o r e d aroma and n o v e l o d o r n o t e s . A p r o b l e m may a r i s e ,


6.

BERGER ET AL.

65


N e o s y n t h e s i s of Et Esters (mM/kg * 24h)

0,2

4

0,1

Of

.


0

.

25

,

1

50 75 100 C E t O H (mM/kg)

F i g u r e 3. I n c r e a s e o f e t h y l a s f ( [ e t h a n o l ] ) ; ( c v . Red

1

ί

125

esters during Delicious).

150

ΡΑ-storage

I: Increase of Ethyl Esters (mM/kg) 0,35-1

0

2

4

6

8 10 l / C * 1000

F i g u r e 4. K i n e t i c s o f e s t e r a p p l e c v . Red Delicious.

12

neosynthesis

14

16

i n PA-stored

C Et Esters (mg/kg)

Time of Incubation (h)

F i g u r e 5. C o n c e n t r a t i o n s o f e t h y l e s t e r s t i m e P A - s t o r a g e (50 mM e t h a n o l / k g c v . Red

upon l o n g Delicious).



66

FLAVOR PRECURSORS

i f t h e r e s i d u a l amounts o f t h e p r e c u r s o r a l c o h o l a d v e r s e ly a f f e c t the sensory q u a l i t y . Experiments with l i m i t e d Ρ Α - s t o r a g e p e r i o d s , f o l l o w e d by a p o s t - i n c u b a t i o n a t am b i e n t c o n d i t i o n s , showed t h a t t h e i n t e r n a l r a t i o s o f p r e c u r s o r a l c o h o l and e s t e r p r o d u c t s may be c o n t r o l l e d t o u l t i m a t e l y achieve well-balanced sensory r e s u l t s (Figure 7) . Due t o t h e c o n t i n u i n g c o n s u m p t i o n o f t h e a l c o h o l , an i m m e d i a t e d r o p i n t h e c o n c e n t r a t i o n o f b u t a n o l was o b s e r v e d upon c h a n g i n g t h e p r e c u r s o r e n v i r o n m e n t ( a f t e r 24 h o u r s ) , but the neosynthesis of e s t e r s proceeded with a d e c l i n e b e g i n n i n g a f t e r 3 days o f s t o r a g e . Thus, making use o f t h e dynamic b i o s y n t h e t i c e v e n t s , a c o n c e r t e d d i l u t i o n o f t h e p r e c u r s o r becomes p o s s i b l e . I n f r u i t s c o n t a i n i n g s e v e r a l s e n s o r y k e y compounds two o r more p r e c u r s o r s w i l l h a v e t o be u s e d ( F i g u r e 8) . E s t e r i f i c a t i o n o f e x o g e n o u s s u b s t r a t e s i n f r u i t a p p e a r s t o be a h i g h l y c o m p e t i t i v e r e a c t i o n , a s an i n t e r n a l e x c e s s o f one a l k y l moiety always r e s u l t e d i n a decrease of e s t e r s not c o n t a i n i n g the r e s p e c t i v e a l k y l moiety. Feeding Jonathan a p p l e s w i t h b u t a n o l o r h e x a n o l c l e a r l y showed t h a t the i n c r e a s e o f t h e r e s p e c t i v e e s t e r s was a t t h e e x p e n s e o f other v o l a t i l e s . The r e s u l t i n g s e n s o r y d e v i a t i o n s from the genuine aroma were compensated by simultaneously f e e d i n g v a r i o u s a l c o h o l s . When compared t o an u n t r e a t e d c o n t r o l , t h e o r i g i n a l m o l a r r a t i o o f e s t e r s was r e s t o r e d , but on a m a r k e d l y h i g h e r l e v e l . T h e s e r a t i o s were c o n s e r v e d f o r s e v e r a l d a y s d u r i n g p r o l o n g e d s t o r a g e a t am b i e n t c o n d i t i o n s . I t i s i n t e r e s t i n g to note that the t o t a l m o l a r amount o f n e w l y f o r m e d e s t e r s i n t h e t h r e e P A e x p e r i m e n t s o f F i g u r e 8 was s i m i l a r (250 t o 270 μ Μ / k g ) . This result underscores the metabolic situation of a l i m i t e d p o o l o f endogenous a c y l m o i e t i e s . More r e f i n e d i n c u b a t i o n p r o t o c o l s c o n s i d e r i n g pROHs, p 0 , and t e m p e r a t u r e w i l l l e a d t o d e s i r e d p a t t e r n s o f aroma compounds. 2

ΡΑ-Effects on Primary Metabolites. The a c y l

moieties r e

q u i r e d f o r e s t e r f o r m a t i o n o r i g i n a t e from a p r i m a r y p a t h way. Therefore, changes of concentrations of other, sensorially or physiologically important constituents were f o l l o w e d d u r i n g Ρ Α - s t o r a g e . The c o n c e n t r a t i o n s of sucrose, fructose, and glucose were not markedly a f f e c t e d . The c o n c e n t r a t i o n o f t o t a l a c i d went t h r o u g h a minimum on d a y 3, but then increased to reach the o r i g i n a l concentration of ca. 10 g / k g a f t e r one week. T h i s t i m e c o u r s e o f a c i d c o n c e n t r a t i o n was s i m i l a r i n P A s t o r e d and u n t r e a t e d f r u i t , and d e p e n d e d l a r g e l y on t h e a c c e s s o f oxygen t o t h e f r u i t . S i m i l a r l y , t h e c o n c e n t r a t i o n o f a s c o r b i c a c i d d r o p p e d f r o m 100 t o 55 m g / k g on t h e s e c o n d d a y o f i n c u b a t i o n , and t h e n r o s e c o n t i n u o u s l y , b u t a g a i n t h e p r e s e n c e o f an a l c o h o l s u b s t r a t e i n the gas p h a s e h a d l i t t l e e f f e c t on t h e a b s o l u t e c o n c e n t r a t i o n s .

Recovery of Volatiles from Apple PA. (21) t h e r e s p i r a t o r y i n c r e a s e measure of the homeostatic

A c c o r d i n g t o Romani of c l i m a c t e r i c f r u i t i s a reaction of a senescing


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BERGER ET AL.


C Internal B u O H (mM/kg) 2i

67

C Bu Esters ( μ Μ / k g ) r 400 300

200

100


0,5

C n-Butanol (mM/kg Apple)

C Internai H e O H (mM/kg)

C He Esters ( μ Μ / k g )

0,7 0,6

400

H

Hexanol He Esters

0,5

300

0,4 200 0,30,2 -

100

0,1 0

2

5

10

20

30

C n-Hexanol (mM/kg Apple)

Figure 6a/b. I n t e r n a i c o n c e n t r a t i o n o f p r e c u r s o r and t o t a l p r o d u c t as f ( [ e x t e r n a l p r e c u r s o r ] ) ; (cv. Jona than, 3d Ρ Α - s t o r e d , 2 3 ° C ) .

C BuOH (μΜ/kg)

C Bu Esters ( μ Μ / k g )

600 -

400 -

200 -

Bu Esters

—·— BuOH 1

1

r» 0

1

2 Days of Incubation

3

F i g u r e 7. L i m i t e d Ρ Α - s t o r a g e and i n t e r n a i t i o n s o f v o l a t i l e s (5 mM b u t a n o l / k g a p p l e not, 2 3 ° C ) .

4

concentra cv. Cousi-



68

FLAVOR

PRECURSORS

t i s s u e . The f o r m a t i o n o f v o l a t i l e f l a v o r s c a n be inte g r a t e d i n t o t h i s view, because the e x c e s s i v e d e g r a d a t i o n o f membrane p o l y m e r s and s t o r a g e compounds i m p l i e s the n e e d t o e l i m i n a t e o s m o t i c a l l y a c t i v e and c y t o t o x i c (free fatty acids) constituents. One means t o m e t a b o l i c r e l i e f i s v o l a t i l i s a t i o n and r e m o v a l f r o m t h e c e l l u s i n g the n a t u r a l c o n c e n t r a t i o n g r a d i e n t . As a r e s u l t , the headspace of ΡΑ-stored fruits contains large amounts of v o l a t i l e e s t e r s . A f l u i d i s e d bed f i l l e d w i t h a polymer adsorbent was developed to isolate volatiles from p r e c u r s o r atmospheres on a l a b s c a l e . I n one t r i a l 15 consecutive isolations at intervals of 32 h o u r s were p e r f o r m e d ( F i g u r e 9) . D u r i n g t h e f i r s t week t h e c o n c e n trations of PA-ennriched esters steadily increased, f o l l o w e d by a s l o w d e c r e a s e d u r i n g t h e following two w e e k s . The l o a d e d p o l y m e r was s o l v e n t d e s o r b e d , a n d t h e flavor concentrates were s e n s o r i c a l l y evaluated using test strips. Even towards the end o f the incubation p e r i o d t y p i c a l sensory r e s u l t s with f r u i t y , apple-like notes were found. A second application of precursor a l c o h o l was n o t u n d e r t a k e n , b u t i s e x p e c t e d t o m a i n t a i n h i g h e s t e r c o n c e n t r a t i o n s i n t h e gas phase.

Juices

from ΡΑ-stored Apples. Aroma e n r i c h e d fruits should represent suitable starting materials for p r o c e s s i n g . A p i l o t s c a l e p l a n t f o r p r o d u c i n g j u i c e from Ρ Α - s t o r e d and u n t r e a t e d a p p l e was u s e d t o d e m o n s t r a t e t h e t r a n s f e r o f v o l a t i l e k e y compounds f r o m t h e f r u i t i n t o a customary p r o d u c t . During these experiments i t turned out t h a t a n a d a p t e d t e c h n o l o g y h a s t o be u s e d : i f an imme diate i n a c t i v a t i o n o f t h e endogenous e s t e r h y d r o l y s i n g activities is not achieved, severe losses of sensory v a l u a b l e compounds w i l l o c c u r . P r o v i d e d t h a t t h i s t e c h n i c a l r e q u i r e m e n t i s met a p r e c e d i n g Ρ Α - s t o r a g e y i e l d s j u i ces t h a t c o n t a i n e l e v a t e d c o n c e n t r a t i o n s of those e s t e r s that determine the fruity character of the over-all f l a v o r . T a b l e I I compares o d o r t h r e s h o l d s and a c t u a l c o n Table I I . V o l a t i l e s of J u i c e of Ρ Α - t r e a t e d Apple Rhein. Bohnapfel (5mM BuOH, 2mM HeOH, l + 2 d S t o r a g e ,

Compound (Mg/100g)

Threshold*

n-Butanol η Hexanol n-Hexanal 2-(E)-Hexenal Butyl acetate E t h y l butanoate Hexyl acetate *

200 200 2 32 8,8 2,7 0,9

cv. 23°C)

Commercial PA-stored ( a v e r a g e o f 3) 1,6 3 17 4 7 < 1 15

87 41 14 < 1 54 41 121

(Mg/100g H 0 ) , Maximum V a l u e a c c . t o L i t e r a t u r e 2


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BERGER ET AL.


Butyl Ester

§11 Hexyl Ester


C Total Esters ( μ Μ / k g )

Control

BuOH

HeOH

Mixture

F i g u r e 8. E f f e c t o f b u t a n o l (5 m M / k g ) , h e x a n o l (2 mM/ k g ) , and o f a m i x t u r e on e s t e r c o n c e n t r a t i o n s (1 + 2d PA, a p p l e c v . Jonathan, 24° C).

F i g u r e 9 . E t h y l e s t e r s i s o l a t e d by a d s o r p t i o n / s o l v e n t d e s o r p t i o n f r o m a p p l e PA (50 mM e t h a n o l / k g , c v . Red Delicious).


69


70

FLAVOR

PRECURSORS

c e n t r a t i o n s o f key v o l a t i l e s i n commercial apple juice and j u i c e made f r o m Ρ Α - s t o r e d a p p l e . B o t h p r e c u r s o r a l c o h o l s show s i g n i f i c a n t l y i n c r e a s e d r e s i d u a l c o n c e n t r a tions, but r e m a i n below t h e odor d e t e c t i o n threshold. While n-hexanal occurs i n a l l j u i c e s in concentrations above i t s t h r e s h o l d , 2 - ( E ) - h e x e n a l remains below. Butyl a c e t a t e and e t h y l b u t a n o a t e were f o u n d i n c o n c e n t r a t i o n s above t h e i r t h r e s h o l d s i n P A - j u i c e s o n l y . The i n c r e a s e d l e v e l o f h e x y l a c e t a t e , f i n a l l y , has a s i g n i f i c a n t impact on t h e f r u i t y o d o r c h a r a c t e r . To s u p p l e m e n t t h e a n a l y t i c a l d a t a j u i c e s f r o m u n t r e a t e d and Ρ Α - s t o r e d a p p l e s were s e n s o r i a l l y e v a l u a t e d . Commer c i a l j u i c e s o f good q u a l i t y s e r v e d as a r e f e r e n c e . At l e a s t 6 o u t o f 9 t e s t p e r s o n s were a b l e t o distinguish t h e P A - j u i c e s i n each of the t r i a n g l e t e s t s . Most persons f o u n d t h a t t h e Ρ Α - j u i c e s were more " a p p l e - l i k e " t h a n t h e c o m m e r c i a l p r o d u c t s . A b o u t one t h i r d o f t h e t e s t p e r s o n s rejected the PA-juice, because, without knowing the o r i g i n s of the samples, they argued the u n u s u a l l y i n t e n s e o d o r i m p r e s s i o n s were c a u s e d by a r t i f i c i a l f l a v o r i n g s . I n summary, t h e b e n e f i c i a l e f f e c t s o f Ρ Α - s t o r a g e o f a p p l e were analytically and sensorically demonstrated. The a p p r o a c h o f Ρ Α - s t o r a g e c a n now be t r a n s f e r e d t o other p r o c e s s e d p r o d u c t s from a p p l e , and, p r o b a b l y , t o other f r u i t s and t h e i r p r o d u c t s a s w e l l .

Acknowledgments.

The DECHEMA, F r a n k f u r t , i s thanked f o r f i n a n c i a l s u p p o r t v i a t h e M i n i s t e r o f R e s e a r c h and T e c h nology, Bonn. H . K o l l m a n n s b e r g e r was involved in some e a r l y Ρ Α - r e s e a r c h and c o n t r i b u t e d e x p e r i m e n t a l a d v i c e and GC-MS a n a l y s e s . F u r t h e r a n a l y t i c a l and t e c h n i c a l s u p p o r t came f r o m A . K e l l e r , G . L e u p o l d , S . N i t z , a n d I . R e i s c h l e .

Literature Cited 1. 2. 3. 4. 5. 6. 7.

Schreier, P. Chromatographic Studies of Biogenesis of Plant Volatiles; Chromatographic Methods; Hüthig: Heidelberg, 1984. Berger, R.G. In Volatile Compounds in Foods and Beverages; Maarse, H . , Ed.; Food Science and Technology no.44; Dekker: New York, 1991, pp 283-304. Berger, R.G.; Dettweiler, G.R.; Kollmannsberger, H.; Drawert, F. Phytochemistry 1990, 29, 20692073. De Pooter, H . L . ; Dirinck, P . J . ; Willaert, G.A.; Schamp, N.M. Phytochemistry 1981, 20, 2135-2138. De Pooter, H . L . ; Montens, J . P . ; Willaert, G.A.; Dirinck, J . P . ; Schamp, N.M. J.Agric.Food Chem. 1983, 31, 813-818. Berger, R.G.; Drawert, F. J.Sci.Food Agric. 1984, 35, 1318-1325. Bartley, I.M.; Stoker, P.G.; Martin, A.D.E.; Hatfield, S.G.S.; Knee, M. J.Sci.Food Agric. 1985, 36, 567-574.


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8.

71

De Pooter, H.L.; van Acker, M.R.; Schamp, N.M. Phytochemistry 1987, 26, 89-92. 9. Drawert, F . ; Berger, R.G. In Lebensmittelqualität; Stute, R., Ed.; VCh: Weinheim, 1988; pp 431-455. 10. Berger, R.G.; Drawert, F . ; Kollmannsberger, H. Ζ.Lebensm.Unters.Forsch. 1986, 183, 169-171. 11. Guadagni, D.G.; Bomben, J . L . ; Harris, J.G. J.Sci. Food Agric. 1971, 22, 110-115. 12. Willaert, G.A.; Dirinck, P . J . ; De Pooter, H . L . ; Schamp, N.M. J.Agric.Food Chem. 1983, 31, 809-813. 13. Dettweiler, G.R. Erzeugung natürlicher Aromen aus geeigneten Vorstufen mit Hilfe von intakten Fruchtgeweben; PhD thesis: TU München, 1989. 14. Tressl, R.; Albrecht, W. In Biogeneration of Aromas; Parliment, T . H . , Croteau, R., Eds.; ACS Symposium Ser. 317; ACS: Washington, DC, 1986; pp 114-133. 15. Knee, M; Hatfield, S.G.S. J.Sci.Food Agric. 1981, 32, 593-600. 16. Berger, R.G. Perf.& Flavorist 1990, 15, 33-39. 17. Paillard, N. In Flavour '81; Schreier, P., Ed.; deGruyter: Berlin, 1981; pp 479-494. 18. Fidler, J . C . ; North, C.J. J.Hort.Sci. 1971, 46, 213-221. 19. Drawert, F . ; Berger, R.G. Ζ.Naturforsch. 1982, 37c, 849-858.19. 20. Banks, N.H. J.Exp.Bot. 1985, 36, 1842-1850. 21. Romani, R.J. J.Food Biochem. 1978, 2, 221-232. RECEIVED December 18, 1991


Flavor Precursors - American Chemical Society

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