Natural Flavors Produced by Biotechnological Processing - American

Chapter 9

Natural Flavors Produced by Biotechnological Processing 1

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David W. Armstrong , Bruce Gillies , and Hiroshi Yamazaki Downloaded by UNIV LAVAL on April 27, 2016 | http://pubs.acs.org Publication Date: February 21, 1989 | doi: 10.1021/bk-1989-0388.ch009

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Division of Biological Sciences, National Research Council of Canada, Ottawa K1A 0R6, Canada Institute of Biochemistry, Carleton University, Ottawa K1S 5B6, Canada

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The world market for flavors is growing steadily with a distinct trend towards 'natural' compounds. Biotechnology offers many advantages over traditional extraction of botanical materials for flavors production. These include highly specific end product generation (e.g. optically-active compounds), high yields and purity, along with guaranteed supply. Commercial exploitation of biotechnology in this area not only relies upon technical advances but as well on satisfy ing certain regulatory considerations. These aspects are highlighted using examples of whole c e l l microbial culture and isolated enzyme systems. A r e c e n t J a p a n e s e r e p o r t p r e d i c t s t h a t p r o d u c t s from the f o o d and b e v e r a g e i n d u s t r y w i l l top the l i s t o f b i o t e c h n o l o g y p r o d u c t s i n s a l e s by the y e a r 2000 (J_) . W h i l e demand f o r f o o d p r o d u c t s can f l u c t u a t e , p a r t i c u l a r l y at the b u l k end o f the m a r k e t , c e r t a i n h i g h p r i c e d p r o d u c t s such as f l a v o r / a r o m a compounds a r e e x p e r i e n c i n g a c o n s t a n t i n c r e a s e i n demand ( 2 ) . F l a v o r i n g compounds, s u b s t a n c e s g r a t i f y i n g t a s t e and s m e l l , r e p r e s e n t 10-15$ by weight of w o r l d - u s e f o o d a d d i t i v e s , which amounts to 25$ o f the v a l u e o f the t o t a l food a d d i t i v e s market ( 3 . ) · R e c e n t l y , i t has been e s t i m a t e d t h a t 5 , 0 0 0 - 1 0 , 0 0 0 n a t u r a l f l a v o r compounds e x i s t (JO w i t h 4,300 h a v i n g been i d e n t i f i e d ( 5.) . The market f o r f l a v o r s i s a n t i c i p a t e d to grow at an a n n u a l r a t e o f about 30$ (6_) . An a g i n g p o p u l a t i o n and the a s s o c i a t e d d i m i n u a t i o n o f t a s t e a c u i t y among o l d e r p e r s o n s has r e s u l t e d i n a need f o r p r o d u c t s w i t h more intense flavors ( χ ) . O t h e r market demands f o r a g r e a t e r

0097-6156/89yO388-0105$06.00A) Published 1989 American Chemical Society Teranishi et al.; Flavor Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Downloaded by UNIV LAVAL on April 27, 2016 | http://pubs.acs.org Publication Date: February 21, 1989 | doi: 10.1021/bk-1989-0388.ch009

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v a r i e t y of f l a v o r e d products has a l s o i n c r e a s e d flavor material consumption. A recent 'sensory a u d i t ' conducted by a l e a d i n g f o o d p r o c e s s o r r e v e a l e d t h a t c o n s u m e r s w e r e most c o n c e r n e d a b o u t ' f r e s h n e s s ' and 'naturalness' in products t h e y buy C7 ) . This trend c a n be a s c r i b e d t o i n c r e a s i n g h e a l t h and n u t r i t i o n c o n s c i o u s life-styles which has e n c o u r a g e d the d e v e l o p m e n t of n a t u r a l f o o d products (8) . One way food p r o c e s s o r s have r e s p o n d e d t o t h i s t r e n d t o w a r d s ' n a t u r a l n e s s ' i s an i n c r e a s e d u s e of n a t u r a l food f l a v o r a n t s found i n p l a n t s (9^) . U n f o r t u n a t e l y , t h e s e raw m a t e r i a l s a r e s u b j e c t t o v a r i o u s p r o b l e m s (_8 ) . Often, the d e s i r e d f l a v o r c o m p o n e n t i s i n low c o n c e n t r a t i o n and therefore e x t r a c t i o n from b o t a n i c a l m a t e r i a l i s expensive. As w e l l , the s u p p l y o f the m a t e r i a l s i s s u b j e c t to seasonal v a r i a t i o n and t o t h e v a g a r i e s o f t h e w e a t h e r , w h i c h s i g n i f i c a n t l y a f f e c t s the y i e l d and q u a l i t y o f the flavor. The s u p p l y i s a l s o i n f l u e n c e d by the s o c i o - p o l i t i c a l s t a b i l i t y of the p r o d u c i n g regions. The i n d u s t r y , i f i t i s t o meet c o n s u m e r demand e f f e c t i v e l y , n e e d s c o m p e t i t i v e new sources of n a t u r a l f l a v o r s , and biotechnology has become a n e c e s s a r y solution. NATURAL F L A V O R S . The m e a n i n g o f t h e t e r m ' n a t u r a l ' may vary between d i f f e r e n t s o c i a l communities. In the U n i t e d S t a t e s the Code of F e d e r a l R e g u l a t i o n s (CFR 101.22.a.3) d e f i n e s the term ' n a t u r a l f l a v o r ' to i n c l u d e not o n l y animal or p l a n t d e r i v a t i v e s but a l s o i n c l u d e s e n z y m i c and f e r m e n t a t i v e p r o c e s s e s . A natural flavor, a c c o r d i n g t o t h e U.S. FDA g u i d e l i n e s s e t down i n 1958, m u s t be p r o d u c e d f r o m n a t u r a l s t a r t i n g m a t e r i a l s and t h a t t h e e n d - p r o d u c t m u s t be i d e n t i c a l t o s o m e t h i n g a l r e a d y known t o e x i s t i n n a t u r e . Thus, biocatalytic, but not c h e m i c a l , t r a n s f o r m a t i o n of n a t u r a l substances c a n be l e g a l l y l a b e l l e d a s n a t u r a l . A p p l i c a t i o n of B i o t e c h n o l o g i c a l Flavors-Product ion

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B i o t e c h n o l o g i c a l approaches f o r p r o d u c t i o n of flavors have the p o t e n t i a l to a l l e v i a t e the problems o u t l i n e d with t r a d i t i o n a l b o t a n i c a l - d e r i v e d processing routes. A d d i t i o n a l l y , o t h e r b e n e f i t s c a n be e x p e c t e d which s h o u l d s e r v e t o c o n t r o l c o s t s , s u p p l y and consistency. F o o d t e c h n o l o g i s t s h a v e h a d much e x p e r i e n c e with t h e use o f i n d u s t r i a l m i c r o o r g a n i s m s and enzymes i n t r a d i t i o n a l processes ( b r e a d and c h e e s e m a k i n g , a l c o h o l fermentation). N u m e r o u s e a r l y r e p o r t s , some d a t i n g b a c k to the e a r l y 1900s, r e a l i z e d the p o t e n t i a l of microorganisms for flavour/aroma production. However a l i m i t e d knowledge base prevented t h e a p p l i c a t i o n o f many biotechnological routes for flavor production. More r e c e n t l y , the l e v e l of knowledge p e r t a i n i n g to metabolic p a t h w a y s and t h e i r r e g u l a t o r y m e c h a n i s m s has a l l o w e d for

Teranishi et al.; Flavor Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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Natural Flavors by Biotechnological Processing

more c o n t r o l t h r o u g h p h y s i o l o g i c a l and/or g e n e t i c manipulations. With r a p i d advances o c c u r r i n g i n g e n e t i c e n g i n e e r i n g and f e r m e n t a t i o n technology, the area of b i o t e c h n o l o g i c a l f l a v o r s p r o d u c t i o n i s sure t o b e n e f i t . T h e r e h a s b e e n much work c o n d u c t e d recently i nthe area of plant c e l l culture, or phytoproduction, e s p e c i a l l y where t h e p r o d u c t i s a plant-unique mixture of i n d i v i d u a l f l a v o r substances such as v a n i l l a e x t r a c t of which v a n i l l i n i s t h e major component. As w e l l , t h e possibility of genetically engineering improved v a r i e t i e s o f p l a n t s f o r high y i e l d and c o n s i s t e n t quality products i s of considerable interest especially f o r more c o m p l e x p l a n t - u n i q u e flavors. Many f l a v o r compounds a r e s e c o n d a r y m e t a b o l i t e s f o r which a d e t a i l e d understanding of their production i s not well understood. P r e s e n t l y more k n o w l e d g e e x i s t s i n microbial metabolism r e l a t i v e to plant b i o s y n t h e t i c p a t h w a y s a n d t h e r e f o r e h a s r e s u l t e d i n more s u c c e s s f u l development of m i c r o b i a l - b a s e d f l a v o r b i o p r o c e s s i n g . As w e l l , s c a l e - u p o f m i c r o b i a l c u l t u r e s and i s o l a t e d e n z y m e s h a s b e c o m e r e l a t i v e l y common p r a c t i c e w h i l e t h e t r a n s l a t i o n o f plant c e l l c u l t u r e to large commercial scales i s not yet well e s t a b l i s h e d . This review will f o c u s on t h e m i c r o b i a l whole c e l l and i s o l a t e d enzyme systems f o r f l a v o r production. M I C R O B I A L WHOLE C E L L AND E N Z Y M I C S Y S T E M S : A number o f single flavour substances have been i d e n t i f i e d which a r e associated with p a r t i c u l a r f l a v o r s ( 1 0 ) : 2 - i s o b u t y 1 t h i a z o l e with tomato f l a v o r ; methyl- and ethyl-cinnamates with strawberry flavor; methyl a n t h r a n i l a t e with grape f l a v o r ; and benzaldehyde with cherry flavor. S i n g l e f l a v o r components such as these s h o u l d be a m e n a b l e t o m i c r o b i a l p r o d u c t i o n from n u t r i e n t s v i a multienzyme steps or enzymic production from a p p r o p r i a t e p r e c u r s o r s . An i m p o r t a n t a t t r i b u t e o f m i c r o b i a l whole cell b i o c a t a l y s t s i s t h e a b i l i t y t o s y n t h e s i z e p r o d u c t s de novo from r e l a t i v e l y i n e x p e n s i v e n u t r i e n t s such a s s i m p l e s u g a r s . C i t r i c a c i d , p r o d u c e d by t h e f u n g u s A s p e r g i l l u s n i g e r , i s p e r h a p s t h e b e s t known f l a v o r compound. Another fungus Trichoderma r e e s e i can produce a s t r o n g c o c o n u t - l i k e impression which i s the r e s u l t o f the f o r m a t i o n o f 6-pentyl-α-pyrone ( c h e m i c a l synthesis r e q u i r e s seven s t e p s ) . The y e a s t Sporobolomyces odorus can p r o d u c e t h e l a c t o n e s 4 - d e c a n o l i d e and cis-6-dodencen-H-olide , responsible f o r a peach-like impression (JJ_) . Apart from ' f r u i t y flavors there seems t o be a g r o w i n g i n t e r e s t i n t h e p r o d u c t i o n o f n a t u r a l f l a v o r chemicals r e l a t e d t o " t o a s t e d " or cooked meat f l a v o r s f o r a p p l i c a t i o n t o m i c r o w a v e p r o d u c t s ( 1 2 ) . I t h a s b e e n shown t h a t a m i x t u r e o f c e r t a i n s u g a r s a n d dihydroxyacetone ( d e r i v e d from a f e r m e n t a t i o n ) g i v e an 1


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i m p r o v e m e n t i n b r o w n i n g and t a s t e due t o t h e d e v e l o p m e n t of r o a s t i n g and a r o m a t i c compounds (13)» R e c e n t l y we h a v e f o u n d t h a t t h e y e a s t Candida u t i l i s c a n c o n v e r t g l u c o s e , anu o t h e r fermentable s u g a r s , t o e t h y l a c e t a t e (1k ) . E t h a n o l c a n a l s o be c o n v e r t e d d i r e c t l y t o e t h y l a c e t a t e o r a c e t a l d e h y d e by t h i s yeast (15) depending upon t h e c o n c e n t r a t i o n o f e t h a n o l p r o v i d e d i n t h e medium ( F i g u r e 1 ) . Ethyl acetate i s important f o r i t suse i n ' f r u i t y * flavors which a c e t a l d e h y d e has an i m p o r t a n t a p p l i c a t i o n i n providing ' f r e s h n e s s ' t o many p r o d u c t s including f r u i t - b a s e d d r i n k s (JJO . B o t h c o n v e r s i o n s o p e r a t e when i r o n l i m i t i n g c o n d i t i o n s a r e imposed and they demonstrate that r e l a t i v e l y simple physiological m a n i p u l a t i o n s can r e s u l t i n marked changes i n metabolism. This yeast i s capable of 'aerobic' f e r m e n t a t i o n and r e a d i l y forms e t h a n o l from fermentable sugars. Under i r o n - s u f f i c i e n t a e r o b i c c o n d i t i o n s u t i l i s produces biomass and i s c u r r e n t l y used commercially f o r t h i s purpose (single cell protein). The imposition of i r o n - l i m i t e d c o n d i t i o n s r e s u l t s i n the t r i c a r b o x y l i c a c i d (TCA) c y c l e b e i n g s e v e r e l y r a t e limited ( F i g u r e 2 ) . C e r t a i n i r o n - r e q u i r i n g enzymes i n t h e TCA c y c l e i n c l u d i n g s u c c i n a t e d e h y d r o g e n a s e a n d a c o n i t a s e become a c t i v i t y l i m i t e d . Under i r o n - l i m i t a t i o n the i n t r a c e l l u l a r l e v e l of acetyl-CoA accumulates which i n the presence o f e t h a n o l a l l o w s f o r i n c r e a s e d e t h y l a c e t a t e f o r m a t i o n v i a an a l c o l o l t r a n s f e r a s e mechanism. T h r o u g h t h e same m e t a b o l i c s c h e m e , when t h e l e v e l o f e t h a n o l i s e l e v a t e d a b o v e approximately 3·5ί (w/v), a c e t a l d e h y d e a c c u m u l a t e s . As the r a t e o f o x i d a t i o n o f e t h a n o l to a c e t a l d e h y d e exceeds that of the o x i d a t i o n o f a c e t a l d e h y d e to a c e t i c acid, the aldehyde begins t o accumulate. This intracellular accumulation forward i n h i b i t s acetyl-CoA synthetase which r e s u l t s i n a r e d u c t i o n i n e t h y l a c e t a t e formation and a change i n p r o d u c t d i s t r i b u t i o n t o a c e t a l d e h y d e . A p a r t f r o m p r o d u c i n g two i m p o r t a n t f l a v o r substances, the spent y e a s t i t s e l f c a n be u s e d p o s t - p r o c e s s i n g i n natural flavour applications. C a n d i d a u t i l i s c a n be used f o r i m p a r t i n g ' s a v o r y ' o r 'meaty' f l a v o r s as i t i s c l a s s i f i e d by t h e U . S . FDA a s GRAS ( G e n e r a l l y - R e c o g n i z e d - A s - S a f e ) and i s one o f o n l y t h r e e yeasts a l l o w a b l e f o r t h i s purpose i n human f o o d p r o d u c t s

(17). Another i m p o r t a n t a t t r i b u t e o f whole cell/enzyme systems i s f o r the p r o d u c t i o n of o p t i c a l l y - a c t i v e compounds. As e a r l y a s t h e 1 9 6 0 s , t h e s y n t h e s i s o f o p t i c a l l y a c t i v e gamma- a n d d e l t a - l a c t o n e s by m i c r o b i o l o g i c a l r e d u c t i o n was d e m o n s t r a t e d ( 1 8 ) . Production of o p t i c a l l y - p u r e L-glutamate f o r use i n the f l a v o r e n h a n c e r MSG by m i c r o b i a l m e a n s i s a n o t h e r testimonial to this potential of biotechnology.



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Fig. 1. Change i n p r o d u c t d i s t r i b u t i o n from ethyl a c e t a t e t o a c e t a l d e h y d e o f C a n d i d a u t i l i s due t o increasing l e v e l s o f added e t h a n o l .

Cell

Environment

Fig. 2. Pathway o f e t h a n o l u t i l i z a t i o n and e t h y l a c e t a t e o r a c e t a l d e h y d e p r o d u c t i o n by C a n d i d a utilis TCA c y c l e a c t i v i t y i n h i b i t e d u n d e r iron-limited conditions. Acetyl-CoA synthetase forward inhibited by a c e t a l d e h y d e a c c u m u l a t e d when e l e v a t e d l e v e l s o f ethanol 3-5$ w/v) p r e s e n t i n t h e m e d i u m .



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T r a n s f o r m a t i o n o f p r e c u r s o r compounds to f l a v o r s can a l s o r e a d i l y take a d v a n t a g e o f b i o t e c h n o l o g i c a l processing. For example, the s e s q u i t e r p e n e valencene, r e a d i l y a v a i a b l e from o r a n g e o i l b u t o f low commercial v a l u e , c a n be t r a n s f o r m e d by c e r t a i n b a c t e r i a ( 1 8 ) t o the h i g h e r v a l u e s e s q u i t e r p e n e , nootkatone (grapefruit c i t r u s f l a v o r ) c u r r e n t l y v a l u e d at about $4200/kg (19) up f r o m $ 1 3 0 0 - 1 6 0 0 a y e a r e a r l i e r ( s y n t h e t i c $700/kg). Another b i o t r a n s f o r m a t i o n having s i g n i f i c a n t impact on t h e c i t r u s j u i c e i n d u s t r y i s t h e a b i l i t y o f A^. n i g e r t o c o n v e r t the b i t t e r component n a r i n g i n to the n o n - b i t t e r f o r m n a r i n g e n i n (_2£) . R e c e n t c o n s u m e r demand f o r g r a p e f r u i t - b a s e d f l a v o r s i s d r i v i n g t h e p r i c e up f o r n a r i n g i n ( c u r r e n t l y p r i c e d a t $55/kg and r i s i n g ) which c o u l d be c o n v e r t e d b i o l o g i c a l l y t o an e v e n h i g h e r priced n o n - b i t t e r form. An a r e a w h e r e b i o t r a n s f o r m a t i o n s w o u l d i m p r o v e p r o d u c t y i e l d s , a n d s i m u l t a n e o u s l y r e s u l t i n an o p t i c a l l y - a c t i v e end p r o d u c t , i n v o l v e s L - m e n t h o l production. C u r r e n t l y the market f o r t h i s t e r p e n e i s approximately 3000 t o n s / y and r e l i e s upon t r a d i t i o n a l e x t r a c t i o n of p l a n t m a t e r i a l s . In the peppermint p l a n t , oil 'maturation o c c u r s between the i n i t i a l flowering and f u l l b l o o m , d u r i n g w h i c h t i m e o n l y a s m a l l p o r t i o n (40$) of L-menthone i s c o n v e r t e d to L - m e n t h o l . I t has been proposed t h a t L - m e n t h o n e c o u l d be e x t r a c t e d a t p e a k l e v e l s from the p l a n t and s u b s e q u e n t l y c o n v e r t e d v i a dehydrogenase a c t i v i t y of v a r i o u s microorganisms. Various systems employing the b a c t e r i u m Pseudomonas p u t i d a and the y e a s t R h o d o t o r u l a m i n u t a have r e s u l t e d i n L - m e n t h o l w i t h 100$ optical activity (20) . The p r o d u c t i o n o f f l a v o r c o m p l e x e s where p o s s i b l y hundreds of i n d i v i d u a l f l a v o r s u b s t a n c e s c o n t r i b u t e t o an overall f l a v o r i m p r e s s i o n i s h i g h l y s u i t e d to b i o t r a n s f o r m a t i o n . T o c r e a t e t h e same c o m p l e x f l a v o r by t h e a d d i t i o n o f i n d i v i d u a l f l a v o r c o m p o n e n t s w o u l d be v e r y d i f f i c u l t i f not i m p o s s i b l e . As a r e s u l t , t h e e x o g e n o u s a d d i t i o n o f whole c e l l s and/or enzymes to a c c e l e r a t e the flavor m a t u r a t i o n p r o c e s s o f a number o f f o o d p r o d u c t s i s r a p i d l y a t t a i n i n g commercial r e a l i t y . Cheese r i p e n i n g o r a g i n g i s an a r e a o f i n t e n s e r e s e a r c h a n d s h o u l d lead to s i g n i f i c a n t c o s t r e d u c t i o n s . I n t h e U.S. alone over 1 b i l l i o n kilograms of Cheddar-type cheese i s produced annually. Normally, the a g i n g p r o c e s s t a k e s p l a c e over a p e r i o d o f b e t w e e n 3-9 months at a c o s t o f 1-2 cents/kg.month. The a g i n g p r o c e s s i n v o l v e s p r i m a r i l y the a c t i o n o f endogenous b i o c a t a l y s t s which t r a n s f o r m s a r e l a t i v e l y b l a n d e l a s t i c mass t o a w e l l - b o d i e d c h e d d a r cheese. Research h a s s h o w n t h a t two k e y enzymic m e c h a n i s m s i n v o l v i n g l i p a s e and p r o t e a s e a r e c r i t i c a l to c h e e s e a g i n g (j21_) . The l i p a s e p r e f e r e n t i a l l y hydrolyses triglycerides, yielding C _C free fatty acids while the p r o t e a s e p r o v i d e s a b a l a n c e d f l a v o r development through p r o t e o l y t i c cleavage. A number o f p r o d u c t s are now available commercially f o r a c c e l e r a t e d cheese 1

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9.

A R M S T R O N G ET AL.


ripening including a 1ipase/protease preparation derived from A s p e r g i l l u s o r y z a e . L i p a s e s f r o m C a n d i da cylindracae (rugosa) and Mucor m i e h e i show d i f f e r e n t s p e c i f i c i t y f o r f a t t y a c i d c h a i n l e n g t h s and have b e e n used to generate d i f f e r e n t cheese f l a v o r complexes. Apart from producing flavor substances or complexes, b i o c a t a l y s t s could f a c i l i t a t e current extraction processes. A Japanese process uses fungal c e l l u l a s e enzymes to enhance j u i c e e x t r a c t i o n from oranges without d i s r u p t i n g the r i n d o i l g l a n d s which contain 'off f l a v o r s and b i t t e r n e s s . C e l l u l a s e s and p e c t i n a s e s have a l s o been used to degrade p l a n t cell w a l l s t o r e l e a s e more o i l f r o m o i l s e e d s d u r i n g processing. The u s e o f 5 0 0 - 1 0 0 0 g o f enzyme p e r t o n o f seeds at t e m p e r a t u r e s of 30-50°C can produce a 2-6$ increase in o i l yield (22).


1

NOVEL FLAVOR B I 0 P R 0 C E S S I N G S Y S T E M S . Typically the systems d e s c r i b e d above are o p e r a t e d in aqueous environments. I t h a s r e c e n t l y b e c o m e e v i d e n t t h a t many enzymes (23) and e v e n c e r t a i n w h o l e c e l l s (24) can f u n c t i o n i n a p o l a r or o r g a n i c s o l v e n t s s u c h as h e x a n e . Although the above s y s t e m s a r e r e f e r r e d to as ' n o n - a q u e o u s ' , t e c h n i c a l l y , t h e e n z y m e s h a v e t o be i n a b i p h a s i c system (organic solvent/water) with the aqueous component b e i n g p r e s e n t i n l o w a m o u n t s , e v e n down t o a m o n o m o l e c u l a r l a y e r on an e n z y m e . T h i s knowledge will c e r t a i n l y b e n e f i t b i o t e c h n o l o g i c a l p r o d u c t i o n of flavor p r o d u c t i o n f o r a number o f r e a s o n s . S i n c e many f l a v o r compounds or t h e i r p r e c u r s o r s have l i m i t e d water s o l u b i l i t y , the use o f a p o l a r s o l v e n t s r a t h e r than water c o u l d a l l o w f o r more e f f i c i e n t c o n v e r s i o n s due to b e t t e r enzyme/substrate i n t e r a c t i o n . I n a d d i t i o n , some r e a c t i o n s may o n l y occur to a s i g n i f i c a n t degree i n a more a p o l a r e n v i r o n m e n t s u c h as es t e r i f i c a t i o n with l i p a s e e n z y m e s (25_ ) . N o r m a l l y , i n the case o f a l i p a s e - b a s e d conversion, e s t e r s are hydrolytically converted t o t h e a c i d and a l c o h o l m o i e t i e s . In certain a p o l a r s o l v e n t s t h i s r e a c t i o n c a n be r e v e r s e d o w i n g t o t h e p r e f e r r e d p a r t i t i o n i n g o f t h e e s t e r away f r o m the enzyme t h e r e b y ' p u l l i n g ' the r e a c t i o n ( F i g u r e 3 ) . More d e t a i l e d d i s c u s s i o n s on t h e u s e o f o r g a n i c m e d i a f o r b i o c a t a l y s i s c a n be f o u n d e l s e w h e r e (26). We h a v e c o n d u c t e d s t u d i e s r e l a t e d t o t h e production of f l a v o r e s t e r s using immobilized l i p a s e from the yeast C . c y l i n d r a c a e i n a n o n - a q u e o u s s y s t e m (.25^, 2J) . The l i p a s e f r o m t h i s m i c r o o r g a n i s m was found to have a broad r a n g e s p e c i f i c i t y a n d was u s e f u l t o p r o d u c e a number o f commercially important esters including ethyl butyrate, i s o b u t y l a c e t a t e and isoamyl acetate (Table I ) . The s t u d i e s f o c u s s e d on t h e p r o d u c t i o n o f e t h y l b u t y r a t e , u s e f u l i n p i n e a p p l e - b a n a n a f l a v o r s , owing to i t s l a r g e m a r k e t d e m a n d o f o v e r 1 4 0 , 0 0 0 kg a n n u a l l y (28) and s e l l i n g p r i c e f o r the n a t u r a l e s t e r r a n g i n g upwards of


111

112


$150/kg. The s y s t e m was found to a m o u n t s o f t h i s e s t e r and as w e l l operational stability (upwards of

DEVELOPMENTS

produce significant had an excellent a month operation).

Table I. Ester production by i m m o b i l i z e d £. cylindracea l i p a s e u s i n g a range of s u b s t r a t e s . The molar concentration i s t h a t of e s t e r i n the heptane phase a f t e r 24 h . P e r c e n t m o l a r c o n v e r s i o n i s t h a t of initial a c i d t o e s t e r a f t e r 24 h


ESTER

CONVERSION

(M)

Ethyl propionate Ethyl butyrate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl laurate Ethyl isobutyrte Ethyl isovalerate Isobutyl acetate Isoamyl acetate Isoamyl butyrate

0.19 0. 25 0.09 0.21 0.26 0.13 0.18 0.01 0.06 0.06 0.22

SOURCE: R e p r i n t e d - w i t h p e r m i s s i o n S c i e n c e and T e c h n o l o g y L e t t e r s .

from r e f . 27.

76 100 44 84 100 52 72 3 25 24 91 Copyright

1987

The k e y f i n d i n g was the r e q u i r e m e n t f o r i n t e r m i t t e n t hydration of the i m m o b i l i z e d enzyme f o r l o n g term operational stability (Figure 4). Without this hydration p r o t o c o l , u p w a r d s o f 90% of the initial a c t i v i t y was l o s t a f t e r o n l y 2 c y c l e s ( c a . 48 h). A n o t h e r e v e n more r e c e n t a d v a n c e f o r f l a v o r processing i n v o l v e s the use of s u p e r c r i t i c a l fluids. Supercritical fluids, including supercritical C0 , c r e a t e d under e l e v a t e d p r e s s u r e s , e x i s t in a nebulous r e g i o n b e t w e e n t h a t o f a l i q u i d and that of a gas. The a b i l i t y to c o n t i n u o u s l y a d j u s t the s o l u b i l i t y of the d e s i r e d c o m p o n e n t t o be e x t r a c t e d by m o d i f i c a t i o n of pressure a n d / o r t e m p e r a t u r e has l e d t o t h i s technology being q u i c k l y a d o p t e d by f o o d a n d flavor/fragrance industries (decaffeination, o i l extraction, etc.). As w e l l , the c o n c e r n over the t o x i c i t y of c e r t a i n s o l v e n t s , has e n c o u r a g e d the s e a r c h f o r an a l t e r n a t i v e e x t r a c t i v e process. S u p e r c r i t i c a l C0 has been the 'solvent' of choice by m o s t f l a v o r i n d u s t r i e s e m p l o y i n g this technology since i t i s i n e r t , nonflammable, nonexplosive and l e a v e s no r e s i d u e i n the f i n a l p r o d u c t . Moreover C0 i s r e a d i l y a v a i l a b l e i n l a r g e q u a n t i t i e s and high p u r i t y and o f g r e a t i m p o r t a n c e i t i s p r o b a b l y t h e next c h e a p e s t s o l v e n t t o w a t e r ( u n d e r 10 cents/kg). A d d i t i o n a l l y , as the c r i t i c a l temperature of C0 i s only 2

2

2

2


9.


ARMSTRONG ET A L

Non-polor

/

ζ Water /

So 1 v e n t

/ /

/

Ester / / / /

/

//\

/

PiC i • P.I coho'l / / /


/

///

\\ •^v

V / V / // ΛA V/// ]/////]

/ / Λ / ^7~7///////l ////j (//// / / / / / /

Y//// /

/ L /pase ///

/

Ζ /pass Ester + hUO^filcohol + Rcid

F i g . 3. system. Reversal esters.

P r o d u c t i o n o f e s t e r s i n a non-aqueous lipase Water p r e s e n t i n l o w l e v e l s a r o u n d enzyme. o f equilibrium occurs t o favor formation o f A c t i v e s i t e = AS.

3.2 3.0 2.8 2.6

I *·< CO lu 2.0 1.8 1.6 1.4 j

2' — ι ι •—ι I 1 1 I I I I I I I I • I I I I I I I 1 ' 0 I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 r e p e t i t i o n s w i t h washing 1

F i g . 4. Repeated use of immobilized l i p a s e i n non-aqueous system. Between each r e p e t i t i o n t h e i m m o b i l i z e d enzyme a d s o r b e d t o a s i l i c a g e l s u p p o r t m a t r i x was w a s h e d w i t h w a t e r . Fresh substrate ( e t h a n o l , b u t y r i c a c i d ) was a d d e d i n h e x a n e . One c y c l e r e p r e s e n t s c a . 24 h .


113

114

FLAVOR CHEMISTRY:

TRENDS AND

DEVELOPMENTS

3 1 ° C a t 74 b a r p r e s s u r e , t h e r m a l l y l a b i l e flavor c o m p o n e n t s s u c h a s j a s m i n e c a n be r e a d i l y e x t r a c t e d f r o m the b o t a n i c a l s o u r c e . F o r t h e most p a r t , i t i s t h e s e a t t r i b u t e s d e s c r i b e d above t h a t the f l a v o r i n d u s t r y has f o u n d a t t r a c t i v e f o r f l a v o r compound e x t r a c t i o n that hasled b i o t e c h n o l o g i s t s to adapt t h i s technology t o biocatalysis. P i o n e e r i n g s t u d i e s i n v o l v i n g the use of lipase i n supercritical C0 have demonstrated, as d e t a i l e d above f o r s o l v e n t - b a s e d non-aqueous systems, t h a t h y d r o l y t i c r e a c t i o n s c a n be s u p p r e s s e d a n d s y n t h e s i s c a n be s i g n i f i c a n t l y e n h a n c e d ( 2 8 , 2 9 ) . One g r o u p (29_) f o u n d t h a t t h e r e a c t i o n r a t e i n t h i s t y p e o f s y s t e m was u p w a r d s o f 270 t i m e s t h a t ( m e a s u r e d i n m o r e product/mole e n z y m e . s e c o n d ) c o m p a r e d w i t h an a q u e o u s system. Undoubtedly, more n o v e l biotechnological p r o c e s s r o u t e s w i l l become a v a i l a b l e and a s i l l u s t r a t e d by t h e s e e x a m p l e s , e v e n o n c e c o n s i d e r e d ' e x t r e m e * environments could f a c i l i t a t e natural flavor production.


2

REGULATORY

CONSIDERATIONS-AN

UPDATE

I n t h e U . S . , FEMA ( F l a v o r a n d E x t r a c t Manufacturers' A s s o c i a t i o n ) i s i n the process of d r a f t i n g new g u i d e l i n e s f o r n a t u r a l f l a v o r / a r o m a c h e m i c a l s and a c c e p t e d p r o c e s s i n g m e t h o d s (Y2) . Apart from the a c c e p t e d use o f f e r m e n t a t i o n , heat r e a c t i o n s and ^enzymes, i t i s e x p e c t e d t h a t i n o r g a n i c a c i d s and b a s e s ' w i l l be a l l o w a b l e f o r u s e a s c a t a l y s t s . Obviously this could allow f o r production of certain natural flavors, i n t h e absence o f enzymes, under a p p r o p r i a t e c o n d i t i o n s , however the r e q u i r e m e n t for natural substrates will rema i n . A l t h o u g h r e g u l a t o r y a g e n c i e s , s u c h a s t h e FDA, a r e t r y i n g t o keep pace with t h i s r a p i d l y advancing t e c h n o l o g y many p o t e n t i a l b o t t l e n e c k s a n d ' g r e y ' areas p e r s i s t p r i m a r i l y i n i n t e r p r e t a t i o n of the r e g u l a t o r y requirements. T h e FDA s t a t e s t h a t i t s p o l i c i e s a r e s u f f i c i e n t l y comprehensive to apply to those involving biotechnology. Although, statements (J30.) s u c h a s " . . . t h e u s e o f new m i c r o o r g a n i s m s found i n a food such a s y o g u r t c o u l d be c o n s i d e r e d a f o o d a d d i t i v e . Such s i t u a t i o n s w i l l be e v a l u a t e d c a s e - b y - c a s e . . . " c o u l d have significant ramifications. F o r e x a m p l e , i f a new microorganism, possibly manipulated u s i n g rDNA t e c h n o l o g y , i s added t o a food f o r f l a v o r improvement, a f o o d a d d i t i v e p e t i t i o n ( F A P ) c o u l d be n e c e s s a r y . This w o u l d a d d much t o p r o d u c t d e v e l o p m e n t c o s t s a n d p o s s i b l y delay e f f e c t i v e e a r l y market p e n e t r a t i o n . Normally a GRAS ( G e n e r a l l y - R e c o g n i z e d - A s - S a f e ) s t a t u s f o r a food s u b s t r a t e a l l o w s f o r immediate use i n food products without regulatory clearance. A major concern i s that t h e u s e o f rDNA t e c h n i q u e s c o u l d c a u s e a s u b s t r a t e , t h a t was n o r m a l l y c l a s s i f i e d a s GRAS, t o b e s e e n a s a f o o d


9.

ARMSTRONG ET AL.



additive thereby n e c e s s i t a t i n g regulatory clearance b e f o r e u s e (3±) . T h e FDA h a s i n d i c a t e d t h a t t h e u s e o f an a n i m a l f o o d s u b s t a n c e p r e p a r e d by rDNA t e c h n o l o g y must be a p p r o v e d by a n FAP (.32). Although, i f the substance produced t h r o u g h t h i s new t e c h n o l o g y c a n be s h o w n t o b e i d e n t i c a l t o a GRAS s u b s t a n c e , i t p r o b a b l y w o u l d be c l e a r e d f o r u s e w i t h o u t b e i n g l a b e l l e d a s a food a d d i t i v e . N o r m a l l y a n FDA GRAS s t a t u s i s i d e n t i f i e d with the substance i t s e l f and i t s intended l i m i t a t i o n s on u s e , w i t h o u t m e n t i o n a s t o i t s method o f manufacture. I t a p p e a r s t h a t t h e FDA w i l l , however, r e q u i r e c l e a r a n c e by e i t h e r a f o o d a d d i t i v e regulation o r by a r e g u l a t i o n a f f i r m i n g GRAS s t a t u s f o r p r o d u c t s d e r i v e d t h r o u g h rDNA t e c h n i q u e s ( 3 3 ) . A number o f t h e compounds u s e d f o r f l a v o r i n g materials are quite volatile, such as a c e t a l d e h y d e , and are r e a d i l y r e c o v e r a b l e i n pure form from existing m i c r o b i a l c u l t u r e s o r t h e i r d e r i v e d enzyme systems. T h u s t h e u s e o f non-GRAS m i c r o b i a l o r enzyme systems, w i t h i n reason, could presumably be u s e d t o p r o d u c e v o l a t i l e GRAS f l a v o r s u b s t a n c e s . A l t h o u g h i t s h o u l d be n o t e d t h a t t h e method o f m a n u f a c t u r e c o u l d be t a k e n into c o n s i d e r a t i o n i f a n y q u e s t i o n o f p u r i t y o f a GRAS substance produced by a n o n - G R A S b i o l o g i c a l s y s t e m i s u t i l i zed. I n t h e s y s t e m s d e v e l o p e d by u s d e s c r i b e d a b o v e , a t t e n t i o n has been d i r e c t e d a t c o m p l i a n c e with r e g u l a t o r y c o n s i d e r a t i o n s , p r i m a r i l y FDA. T h e p r o d u c t i o n o f e t h y l a c e t a t e a n d a c e t a l d e h y d e , b o t h GRAS f l a v o r s u b s t a n c e s , was a c c o m p l i s h e d u s i n g a GRAS y e a s t , C. u t i l i s . T h e medium c o n t a i n i n g b a s i c s a l t s and e i t h e r g l u c o s e o r f e r m e n t a t i o n e t h a n o l s h o u l d n o t r a i s e any questions regarding potential t o x i c i t i e s . The end p r o d u c t s b e i n g v e r y v o l a t i l e c a n be r e a d i l y isolated even i f t h e r e had been any c o n c e r n over t h e m i c r o b i a l system being employed. A d d i t i o n a l l y , the spent yeast produced i n t h i s p r o c e s s i s o n e o f o n l y t h r e e GRAS yeasts allowed f o r use i n food products d e s t i n e d f o r human c o n s u m p t i o n , and c a n t h e r e f o r e g i v e a b y - p r o d u c t c r e d i t to t h i s process as d i s c u s s e d e a r l i e r . I ti s t e m p t i n g t o g o i n t o n a t u r e t o i s o l a t e new m i c r o b i a l s y s t e m s b u t t h e p o t e n t i a l c o s t s c a n be s t a g g e r i n g f r o m a regulatory standpoint. A good example o f t h i s i st h e $60 m i l l i o n t h a t R a n k H o v i s M c D o u g a l l ' s company i n t h e U.K. s p e n t d e v e l o p i n g a n d s e e k i n g r e g u l a t o r y a p p r o v a l for a F u s a r i u m S C P p r o c e s s Λ3H ) . The n o n - a q u e o u s l i p a s e s y s t e m f o r f l a v o r esters d e v e l o p e d by o u r g r o u p u s e d c o m p o n e n t s a n d p r e p a r a t i v e t e c h n i q u e s f o r enzyme i m m o b i l i z a t i o n , t h a t would n o t o n l y be c o s t e f f e c t i v e a n d s i m p l e b u t a l s o m e e t regulatory requirements. The enzyme c o u l d have been i m m o b i l i z e d by a n u m b e r o f m e t h o d s h o w e v e r f o r t h e intended application only ( i ) adsorption ( i i ) ionic b o n d i n g o r ( i i i ) g l u t a r a l d e h y d e c r o s s - l i n k i n g w o u l d be


115


116


DEVELOPMENTS

appropriate. Adsorption of the l i p a s e to s i l i c a g e l was c h o s e n a s i t i s an e x t r e m e l y s i m p l e m e t h o d e l i m i n a t i n g the need f o r p o t e n t i a l l y d a n g e r o u s c h e m i c a l s . Strict regulations exist for food-related processes especially where s o l v e n t s are i n v o l v e d . H e x a n e was chosen for t h i s p r o c e s s as i t i s used a l m o s t u n i v e r s a l l y f o r p l a n t material extraction protocols (35). A typical allowable maximum s o l v e n t r e s i d u e i n n a t u r a l e x t r a c t i v e s i s 25 ppm (36). F i n a l l y , the l i p a s e from c y l i n d r a c a e had also had a p r e c e d e n t s e t f o r use i n food (cheese r i p e n i n g and butter fat modification). I n d u s t r i e s d e a l i n g w i t h f o o d or pharmaceutical p r o d u c t s are b e g i n n i n g to i n c o r p o r a t e biotechnology into t h e i r p r o d u c t development schemes a l t h o u g h the routes being taken are r a t h e r l i m i t e d . Large d e v i a t i o n from t h e u s e o f a s e l e c t s h o r t l i s t o f GRAS m i c r o o r g a n i s m s o r t h e i r d e r i v e d e n z y m e s i s n o t common p r a c t i c e (8). Where p o s s i b l e ' c l a s s i c * genetic and/or p h y s i o l o g i c a l m a n i p u l a t i o n of t h e s e o r g a n i s m s i s done i n o r d e r to f a c i l i t a t e r e g u l a t o r y c o m p l i a n c e and a p p r o v a l . As i n d u s t r y and g o v e r n m e n t r e g u l a t o r y a g e n c i e s work c l o s e r together t o c l e a r up t h e c o n c e r n s o v e r t h e u s e o f 'new biotechnology, including genetic engineering, more a p p l i c a t i o n s w i l l emerge. Apart from compliance with government r e g u l a t i o n s , i t i s c r i t i c a l t h a t the industry i t s e l f s e t i t s own h i g h s t a n d a r d s as t h e i r liabilities are not n e c e s s a r i l y p r o t e c t e d by g o v e r n m e n t guidelines (V2). 1

I N D U S T R I A L INVOLVEMENT DEVELOPMENT

IN

BIOTECHNOLOGICAL

FLAVORS

U n l i k e the p h a r m a c e u t i c a l i n d u s t r y , food processing i n d u s t r i e s t e n d t o be m a r k e t d r i v e n r a t h e r that t e c h n o l o g y d r i v e n (7J and i s much m o r e r e c e p t i v e to trends s u c h as t h e demand f o r n a t u r a l f l a v o r s . It is a n t i c i p a t e d t h a t t h e r e w i l l be m o r e i n v e s t m e n t i n b i o t e c h n o l o g i c a l f l a v o r d e v e l o p m e n t e s p e c i a l l y as the t e c h n o l o g y p r o v e s i t s e l f and that e x i s t i n g technology n e e d s c a n be s a t i s f i e d by t h e s e new approaches. The m a j o r i t y of l a r g e companies' involvement i n biotechnology i s i n t h e f o r m o f R&D c o n t r a c t s to small, e n t r e p r e n e u r i a l companies. This i s a s t r a t e g i c approach which allows f o r a minimal investment of c a p i t a l in order to t e s t i f the t e c h n o l o g y d e v e l o p e d can be a p p l i c a b l e t o t h e i r n e e d s (3^). I f the impact i s s i g n i f i c a n t , an i n - h o u s e p r o g r a m w o u l d t h e n e v o l v e . A number o f c o m p a n i e s h a v e t a k e n t h i s a p p r o a c h i n c l u d i n g : Firminech w i t h DNA Plant Technology f o r improved production o f f l a v o r s ; W.R. Grace with Synergen for development of m i c r o b i a l systems f o r f l a v o r s ; General F o o d s , a l t h o u g h t h e y do h a v e an i n - h o u s e p r o g r a m , h a v e e s t a b l i s h e d l i n k s w i t h ESCAGENETICS. F&C International (Canada) L t d . has e s t a b l i s h e d a r e s e a r c h collaboration


9.

ARMSTRONG ET A L


with the N a t i o n a l Research C o u n c i l , a Canadian Federal Government r e s e a r c h o r g a n i z a t i o n . With the p r o j e c t e d i n c r e a s e i n n a t u r a l f l a v o r s demand e x p e c t e d t o grow a t an a n n u a l r a t e o f a b o u t 20$ (12) and t h e c u r r e n t a v e r a g e p r i c e o f a b o u t $150-200/kg more c o m p a n i e s a r e e x p e c t e d to j o i n i n t h i s endeavor. At p r e s e n t o n l y about h a l f a dozen companies a r e involved i n developing natural flavor/aroma chemicals w i t h some r e t a i n i n g a n u m b e r o f t h e i r p r o d u c t s f o r c a p t i v e u s e i n t h e i r own f l a v o r b l e n d s . I t has been estimated ( 1 2 ) t h a t one o f t h e l a r g e r c o m p a n i e s h a s upwards o f 25-30 n a t u r a l compounds developed.


FUTURE

DIRECTIONS

A number research

of areas activity

w i l l see s i g n i f i c a n t i n c r e a s e s i n i n the f u t u r e and i n c l u d e :

• A greater understanding of the c a p a b i l i t i e s of b i o c a t a l y s t s w i l l a l l o w f o r an u p g r a d i n g and i n c r e a s e d c o m m e r c i a l e x p l o i t a t i o n o f many u n u t i l i z e d biological substances such as c e r t a i n components o f e s s e n t i a l oils. • The t r e n d t o w a r d more h e a l t h y l i f e s t y l e s h a s a l s o e n c o u r a g e d a demand f o r u n s a t u r a t e d a n d l o w f a t d i e t s which has i n c r e a s e d the use o f p l a n t r a t h e r than animal products. The p r e f e r e n c e o f 'meaty' f l a v o r s will n e c e s s i t a t e more u s e o f f e r m e n t a t i o n - d e r i v e d flavoring a g e n t s such as 5 ' - n u c l e o t i d e s i n c l u d i n g i n o s i n a t e and guanylate, along with hydrolyzed yeast. Overproduction o f e n z y m e s t o do t h e h y d r o l y s i s , by rDNA techniques, c o u l d a l l o w f o r use o f yeast e x t r a c t s or a u t o l y s a t e s d i r e c t l y without t h e n e e d f o r p u r i f i c a t i o n o f RNA by c h e m i c a l m e a n s (.20 ) . R e c e n t l y , a group i n S c o t l a n d has succeeded i n c l o n i n g genes f o r t h i s purpose i n yeast w h i c h c a n c o n v e r t RNA o f a n y o r i g i n t o t h e s e f l a v o r - e n h a n c i n g compounds ( 3 7 ) . • P o t e n t i a l development of ' i n d u s t r i a l l y robust' enzyme-mimetic systems to allow f o r s p e c i f i c biotransformations. T h e s e m i m i c s w o u l d be c a t a l y t i c a l l y a c t i v e b u t n o t be d e p e n d e n t upon t h e a m i n o a c i d backbone of e x i s t i n g enzymes. • An i n c r e a s e d u s e o f p l a n t c e l l c u l t u r e , f o r p r o d u c t i o n of plant-unique f l a v o r s , w i l l a p p e a r a s more i s understood on u n d e r l y i n g c o n t r o l and s y n t h e t i c mechanisms. E x p r e s s i o n of c e r t a i n p l a n t genes i n m i c r o o r g a n i s m s w i l l a l s o be d o n e . An e a r l y e x a m p l e o f t h i s i s the ' t a s t e a c t i v e ' p l a n t p r o t e i n thaumatin (2500-3000 times sweeter than s u c r o s e ) b e i n g expressed in m i c r o b i a l systems. For i n d u s t r i a l scale production many p r o b l e m s e x i s t w i t h l a r g e s c a l e p l a n t cell


117

118


DEVELOPMENTS


c u l t i v a t i o n as a r e s u l t of s e n s i t i v i t y to shear forces ( c e l l b r e a k a g e ) , and t h e i r t e n d a n c y t o clump thereby l i m i t i n g mass t r a n s f e r ( n u t r i e n t l i m i t a t i o n ) . Enabling technologies such as a p p r o p r i a t e b i o r e a c t o r d e s i g n and suitable production m e d i a must be d e v e l o p e d ( 1 0 ) . • N u m e r o u s f o o d p r o d u c t s w i l l be u p g r a d e d t o ' v a l u e - a d d e d * p r o d u c t s by t h e i n c o r p o r a t i o n o f f l a v o r i n g s with appropriate functional properties e x t e n d i n g beyond f l a v o r i n g . An i m p o r t a n t a r e a o f r e s e a r c h w i l l be i n t h o s e f l a v o r s i m p a r t i n g antioxidant and preservative f u n c t i o n (38 ) . Sage and rosemary e x t r a c t s h a v e b e e n shown t o f u n c t i o n a s a n t i o x i d a n t s w h i l e t h o s e o f n u t m e g , mace a n d b a y l e a f i m p a r t p r e s e r v a t i v e e f f e c t s i n h i b i t i n g growth of C l o s t r i d i u m botulinum. M i c r o b i a l o r enzyme p r o d u c t i o n of specific f l a v o r s with these p r o p e r t i e s should be p o s s i b l e . • C e r t a i n v o l a t i l e aroma compounds have b e e n shown t o b r i n g a b o u t p o s i t i v e c h a n g e s i n mood o r g e n e r a l well-being a n d i s e n c o m p a s s e d i n a new d i s c i p l i n e called a r o m a t h e r a p y . I t i s u n l i k e l y t h a t l a r g e d e m a n d s on c e r t a i n a r o m a - t h e r a p e u t i c c o m p o u n d s c o u l d be s a t i s f i e d by t r a d i t i o n a l t e c h n o l o g i e s . Biotechnological a p p r o a c h e s c o u l d s a t i s f y t h e s e r e q u i r e m e n t s f o r aroma therapeutics. • The f u t u r e f o r p r o d u c t i o n of natural f l a v o r s through t h e u s e o f b i o t e c h n o l o g i c a l m e a n s h o l d s much p r o m i s e . R e s e a r c h e r s and i n d u s t r i e s i n t e r e s t e d i n t h i s e x c i t i n g a r e a h o w e v e r , w i l l o n l y be s u c c e s s f u l i f t h e y m a i n t a i n an a w a r e n e s s o f m a r k e t t r e n d s , a n d o f e q u a l importance, knowledge of r e g u l a t o r y requirements.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Newell, Ν. Gen. Eng. News 1986, 6, 55. Hacking, A . J . Economic Aspects of Biotechnology; Cambridge Press, N.Y., 1986, p 29. Godfrey, T. In Industrial Enzymology; Godfrey, T.; Reichelt, J , Eds., Nature Press, N.Y., 1983; pp 305-314. Rijkens, F.; Boelens, H. Proc. Int. Symp. Aroma Res., 1975, p 203. Maarse, H. In Volatile Compounds in Food Quantitative Data; TNO, Netherlands, 1984, Vol. 1-3. Bedoukian, P.Z. Perfum. Flavor. 1985, 10, 1-27. Horton, H.W. Food Technol. 1987, 41, 80. Armstrong, D.W., Yamazaki, H. Trends Biotech. 1986, 4, 264-268. Newell, Ν.; Gordon, S. In Biotechnology in Food Processing; Harlander, S.K.; Labuza, T.P., Eds.; Noyes Publ., Park Ridge, ΝJ, 1986, pp 297-311.


9. ARMSTRONG ET A L

10. 11. 12. 13. 14.


15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25.

26.

27. 28. 29. 30. 31. 32. 33. 34. 35.


Whitaker, R . J . ; Evans, D.A. Food Technol. 1987, 41, 86. Tahara, S.; Fujinara, Κ.; Mizutani, J . Agr. Biol. Chem. 1973, 37, 2855. Anonymous. Chem. Marketing Reporter (5 Oct.), 1987, p 5. Tanabe Seiyaky Co. Ltd. 1979, Jap. examined patent 5, 407, 859. Armstrong, D.W. In Biogeneration of Aroma Compounds; Parliment, T., Croteau, R., Eds.; American Chemical Society, Washington, DC, 1986, pp 254-265. Armstrong, D.W.; Martin, S.M.; Yamazaki, H. Biotech. Lett. 1984, 6, 183-188. Byrne, B.; Sherman, G. Food Technol. 1984, 57. Dziezak, J.D. Food Technol. 1987, 104. Dhavlikar, R.S.; Albroscheit, G. Dragoco Rep. 1973, 12, 251-258. Anonymous. Chem. Marketing Reporter (14 March), 1988, 38. Trivedi, N.B. Gen. Eng. News 1986, 6(2), 1. Arbige, M.V.; Freund, P.R.; Silver, S.C.; Zelko, J.T. Food Technol. 1986, 40, 91. Godfrey, T. In Industrial Enzymology: The Application of Enzymes in Industry; Godfrey, T.; Riechelt, J., Eds.; Nature Press, N.Y., 1983, pp 424-427. Nelson, Ν.; Racker, E. Biochemistry 1973, 12, 563. Seo, C.W.; Yamada, Y.; Okada, H. Agric. Biol. Chem. 1982, 46, 405-409. Gillies, B.; Yamazaki, H.; Armstrong, D.W. In Biocatalysis in Organic Media; Laane, C.; Tramper, J.; L i l l y , M.D., Eds.; Elsevier, Amsterdam, 1987, pp 227-232. Laane, C.; Boeren, S.; Hilhorst, R.; Veeger, C. In Biocatalysis in Organic Media; Laane, C.; Tramper, J.; L i l l y , M.D., Eds.; Elsevier, Amsterdam, 1987, pp 65-95. Gillies, B.; Yamazaki, H.; Armstrong, D.W. Biotechnol. Lett. 1987, 9, 709-714. Anonymous. Bioprocess. Technol. 1987, 9(4), 1. Anonymous. Bioprocess. Technol. 1987, 9(9), 6. Title 49 Federal Reg. 50878 (31 Dec.), 1984. Korwek, E.L. Food Technol. 1986, 40, 67. Title 49 Federal Reg. 50879 (31 Dec.), 1984. McNamara, S. In Biotechnology in Food Processing; Harlander, S.K.; Labuza, T.P., Eds.; Noyes, Park Ridge, NJ, 1986, pp 15-27. Hacking, A . J . Economic Aspects of Biotechnology; Cambridge Press, U.K., 1986, p 29. Bosman, R.J. In Safe Uses of Solvents; Collins, A . J . ; Gluxon, Eds.; Academic Press, N.Y., 1982, pp 25-34.


119

120 36. 37. 38.

FLAVOR CHEMISTRY: TRENDS AND DEVELOPMENTS Newell, Ν. Gen. Eng. News 1986, 6(2), 5. Anonymous. Biobulletin 1987, 5(1), 2. Williams, S.K., Brown, W.L. Food Technol. 41(6), 76. November 8, 1988


RECEIVED

1987,


Natural Flavors Produced by Biotechnological Processing - American

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