Characterization and Measurement of Flavor Compounds - American

2 Substances That Modify the Perception of Sweetness Michael A. Adams Downloaded by UNIV MASSACHUSETTS AMHERST on September 6, 2012 | http://pubs.acs.org Publication Date: November 8, 1985 | doi: 10.1021/bk-1985-0289.ch002

Monell Chemical Senses Center, University of Pennsylvania, Philadelphia, PA 19104

Human taste response is modified by several plant-derived substances. The detergent sodium dodecyl sulfate, as well as triterpene saponins from the leaves of several plant species (most notably Gymnema sylvestre and Ziziphus jujuba) will temporarily inhibit the sweet taste sensation in man; the duration of the effect being about one hour for G. sylvestre and about fifteen minutes for Z. jujuba. The mechanism of action seems to be related, in part, to the surfactant properties of the materials. Structures of the modifiers and possible mechanisms of action are discussed. The number of substances known to modify sweet taste i s quite small. M i r a c u l i n , a substance from the f r u i t of Synsepalum dulcificum, has the a b i l i t y to make sour s t i m u l i taste sweet. The f r u i t s of t h i s West A f r i c a n shrub ("miracle f r u i t " ) have been used by natives to improve the f l a v o r of maize bread, sour palm wine, and beer ( 1_). The taste modifying e f f e c t i s of quite long duration, sometimes l a s t i n g for more than three hours. M i r a c u l i n i s a glycoprotein with a molecular weight of about 4U,000 (2). Not much i s known about i t s structure or i t s mode of action, but i t has been proposed (3_) that t h i s substance binds to the taste c e l l surface, where i t s e f f e c t i s then manifested. I t was further suggested that exposure of the taste receptors to acid contained i n a food or beverage causes a conformational change i n the membrane enfolding the receptor, which allows the sugar moieties bound to miraculin to stimulate the sweetness receptor. The speed of response of miraculin i s on the order of milliseconds, and t h i s i s taken as evidence that the sweetness receptor l i e s on the outside of a taste c e l l , since the speed of response i s apparently too fast to allow for transport i n t o the c e l l i n t e r i o r . Additional evidence that the e x t e r i o r surface of the taste receptor c e l l plasma membrane i s the l o c a t i o n of the sweet receptor i s provided by the action of the chemostimulatory proteins, monellin and thaumatin. Monellin occurs i n the f r u i t of the African serendipity berry (Dioscoreophyllum cumminsii), and thaumatin i s found i n the f r u i t of Thaumatococcus d a n i e l l i i , also 0097-6156/ 85/ 0289-0011 $06.00/ 0 © 1985 American Chemical Society

In Characterization and Measurement of Flavor Compounds; Bills, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Downloaded by UNIV MASSACHUSETTS AMHERST on September 6, 2012 | http://pubs.acs.org Publication Date: November 8, 1985 | doi: 10.1021/bk-1985-0289.ch002

12

C H A R A C T E R I Z A T I O N A N D M E A S U R E M E N T OF FLAVOR C O M P O U N D S

from A f r i c a . Both of these proteins have an intensely sweet t a s t e . Studies have shown that they are e s s e n t i a l l y carbohydratefree, i n contrast with miraculin. Monellin i s a single peptide chain with a molecular weight of about 11,000, and thaumatin, believed also to be a s i n g l e peptide chain, has a weight of approximately 21,000. Evidence has been presented i n d i c a t i n g that the t e r t i a r y structures of these proteins are c r i t i c a l to the sweet taste , and Cagan (5) has suggested that the size of these molecules renders i t u n l i k e l y that they are being transported i n s i d e the taste receptor c e l l . I t i s reasonable to conclude that t h e i r taste-stimulating e f f e c t s are most l i k e l y being manifested at the c e l l surface. The e f f e c t s of miraculin, monellin and thaumatin, taken together, provide evidence that the sweetness receptor s i t e i s located at the surface of the taste receptor c e l l , i n or i n close proximity t o , the plasma membrane. Sweetness I n h i b i t o r s There are only a few sweetness i n h i b i t o r s known. This a r t i c l e w i l l focus on three: sodium dodecyl s u l f a t e (SDS), the gymnemic acids (GA) and the z i z i p h i n s (ZJ). Each of these substances has the a b i l i t y to diminish or eliminate the a b i l i t y to recognize sweetness. The i n t e n s i t y and duration of the effect varies with the i n h i b i t o r . The best-known sweetness i n h i b i t o r i s sodium dodecyl sulfate (SDS), also known as sodium l a u r y l s u l f a t e . This substance i s a twelve carbon surfactant that i s quite commonly used as a detergent i n toothpaste. The observation i s often made that a f t e r brushing one's teeth, the taste of orange juice i s unusually b i t t e r . This has been ascribed to the presence of SDS i n the d e n t i f r i c e (6). The duration of the e f f e c t , as measured i n psychophysical experiments, i s very b r i e f - on the order of minutes, and the suppression effect i s not complete. A substance i s o l a t e d from the Indian shrub Gymnema s y l v e s t r e , has a profound a b i l i t y to reduce perceived sweetness of sugar s o l u t i o n s . The e f f e c t was noticed over a century ago when two B r i t i s h inhabitants of an Indian v i l l a g e found that, a f t e r chewing the leaves of G_^ s y l v e s t r e , the sweetness of t h e i r tea disappeared (7). The sweetness suppressing a c t i v i t y i s due to a mixture of several triterpene saponins which have c o l l e c t i v e l y been termed the gymnemic acids. For most people exposed to the e f f e c t s of GA, sweetness suppression i s complete and the e f f e c t l a s t s for about an hour. Much work was done i n the I960's by S t o c k l i n , Sinsheimer and t h e i r coworkers to i s o l a t e , p u r i f y , and elucidate the structures of these taste i n h i b i t o r s (8-11). Many substances without antisweet a c t i v i t y were i s o l a t e d from the leaves of G^. s y l v e s t r e , including hydrocarbons, stigmasterol, 3-amyrin, lupeol and phytol, among others. A crude antisweet extract was, however, produced by mineral acid p r e c i p i t a t i o n of an aqueous l e a f extract. This extract, when dried, accounts f o r about 10% of the l e a f material. Chloroform extraction was then used to remove some remaining i n a c t i v e material. Pfaffmann (12) determined that the mixture of taste modifying substances was g l y c o s i d i c i n nature, as glucose,



2. A D A M S

Modifying the Perception of Sweetness

13

arabinose, and glucuronolactone were obtained upon hydrolysis. The hydrolysate was found to have no antisweet e f f e c t . Yackzan (7) postulated that the sweetness-inhibiting gymnemic acid must be a saponin, based on i t s foaming i n s o l u t i o n , a b i l i t y to produce red blood c e l l l y s i s , and i t s g l y c o s i d i c nature. Extraction of the crude l e a f f r a c t i o n with acetone removes a l l p h y s i o l o g i c a l l y active material. This acetone-soluble material was chromatographed on s i l i c a g e l by S t o c k l i n (9) to give the various c l o s e l y - r e l a t e d gymnemic acids. Further p u r i f i c a t i o n by Sinsheimer and Subba Rao led to the s t r u c t u r a l i d e n t i f i c a t i o n of gymnemagenin (I) and gymnestrogenin (II) as the aglycones of the gymnemic acids (Figure 1). The major taste active substance appears now to be gymnemagenin ( I ) , e s t e r i f i e d with glucuronic acid. This saponin i s c a l l e d gymnemic acid A, ( I I I , Figure 2 ) . The i d e n t i t y of the R groups i s presently unknown, although claims f o r formic, a c e t i c , i s o v a l e r i c and t i g l i c acids have been made UO, 13, 1M). No further s t r u c t u r a l work has been done with the gymnemic acids f o r the l a s t several years. Once a p u r i f i e d gymnemic acid became a v a i l a b l e , much psycho physical work was done to understand the nature of the sweetness i n h i b i t i o n e f f e c t . The work of Bartoshuk and co-workers i l l u s t r a t e s the course taken (15). The r e s u l t s of a t y p i c a l experiment are shown i n Figure 3. The sweetness of a sucrose s o l u t i o n was almost completely suppressed a f t e r holding a gymnemic acid solution i n the mouth f o r a few seconds. Further experiments were carried out to determine the e f f e c t of gymnemic acid on the other taste q u a l i t i e s (sour, b i t t e r and s a l t y ) . No e f f e c t of gymnemic acid on these tastes was observed. Early work with GA extracts had produced an apparent i n h i b i t i o n of b i t t e r taste, but t h i s effect was l a t e r a t t r i b u t e d to cross-adaptation to the taste of the crude l e a f extract, which was i t s e l f quite b i t t e r . Experiments with refined (and tasteless) extracts showed no bitterness suppression. A t h i r d group of sweetness i n h i b i t i n g substances has recently been i s o l a t e d from the leaves of the Middle Eastern t r e e , Ziziphus jujuba. This material, consisting of a mixture of 15-20 triterpene saponins, has a c t i v i t y s i m i l a r to that of the gymnemic acids, but the duration of the e f f e c t i s much shorter, on the order of 15 minutes f o r the average subject. The duration of suppression varies from subject to subject, and i s usually not complete. The i s o l a t i o n , p u r i f i c a t i o n , and s t r u c t u r a l characterization of these sweetness i n h i b i t o r s , which have been termed the z i z i p h i n s , i s under i n v e s t i g a t i o n i n our laboratory and some of our recent r e s u l t s are described below. Psychophysical and preliminary chemical studies were c a r r i e d out with 2^. jujuba (ZJ) extracts by Meiselman and co-workers (16). Their chromatographic investigations showed that the gymnemic acids were not present i n ΖJ l e a f extracts, but t h e i r observations of the physical properties of l e a f extract solutions d i d indicate that saponin-like substances were present. The e f f e c t of z i z i p h i n treatment of the tongue on sweet, sour, b i t t e r , and s a l t y tastes were examined. As with the gymnemic acids, ΖJ only reduced the i n t e n s i t y of sweet taste. A more detailed i n v e s t i g a t i o n of the z i z i p h i n s was carried out by Kennedy and Halpern (17). Included i n t h e i r work was a phylogenetic analysis of Z. jujuba and Gymnema sylvestre (Figure



14

C H A R A C T E R I Z A T I O N A N D M E A S U R E M E N T O F FLAVOR C O M P O U N D S

II Figure 1. Structure of gymnemagenin (I) and gymnestrogenin ( I I ) .

Figure 2. Structure of gymnemic acid A, ( I I I ) , the most abundant gymnemic a c i d .

before

χ after G s >
eop eophyto

Subdivision

Angiosperme»

Λ Dichotyledoneoe

Class

_ _ ____ : 1

Subclass

Archichlamydeae

Order

R h a m n o u :s

Family

Rhomnoceoe

A

Metochlomydeae

Gept

A

males

—

,

Asclepiodoceae piadai

Genus

Ziziphus

Gymnema ymnen

j\\ Species

sylvestre

jujubo /Ziziphus

jujuba]

/ Gymnema

sylvestre

Figure 4. Phylogenetic c l a s s i f i c a t i o n of Ziziphus jujuba and Gymnema s y l v e s t r e . Lineage. Since separation occurs e a r l y , a t the subclass l e v e l , these two species do not appear c l o s e l y r e l a t e d . S o l i d arrows indicate single pathways. Open arrows i n d i c a t e multiple pathways. Reproduced with permission from Ref. 17. Copyright 1980, ILR Press, Ltdo


J

ADAMS


Ground, dried leaves of


Ziziphus jujuba

MeOH-H 0 {2-\), 3x 2

residue

hexane ext.

aqueous Et 0 ext.

organic

2

X organic

organic 1. evap.MeOH 2. Et 0

aqueous BuOH ext.

aqueous

2

ppt.

supernatant

Crude antisweet fraction, ~ 4 % of leaf; C a . 1 5 - 2 0 components

Figure 5. Scheme f o r i s o l a t i o n of crude antisweet f r a c t i o n from the leaves of Ziziphus jujuba. Taste bioassays were performed at each step to a s c e r t a i n antisweet a c t i v i t y . An X i n d i c a t e s that no antisweet a c t i v i t y was detected.


18


Crude antisweet fraction


(4%

Flash chromatography (silica gel, solvent A )

of leaf; saponin containing)

ν

Y

DCC

Prep TLC

HPLC

(solvent A)

( reverse phase, C-18

(solvent A)

solvent Β or C )

Solvent A = GHCI : MeOhh PrOH= H 0 3

2

Solvent Β = MeOH=H 0 2

Solvent C = C H C N = H 0 3

2

Figure 6. Techniques used f o r i s o l a t i o n and p u r i f i c a t i o n of antisweet substances from the crude antisweet l e a f f r a c t i o n of Ziziphus jujuba*

IV

v

Figure 7o Structure of e b e l i n lactone (IV) and jujubogenin (V).



2.

ADAMS


acid-catalyzed rearrangement. Thus, i t presently appears that a l l of our saponins are jujubogenin-based, d i f f e r i n g only i n the type, number, and linkage of sugar moieties. From our hydrolysis studies we have i d e n t i f i e d glucose, xylose, fucose, arabinose and rhamnose as the sugars of the saponins. The smallest saponin we have i s o l a t e d thus f a r i s jujubogeninglucose-rhamnose and our general approach to structure i d e n t i f i c a t i o n i n t h i s system can be i l l u s t r a t e d using t h i s molecule as an example. This substance was i s o l a t e d from the crude antisweet f r a c t i o n by s i l i c a g e l f l a s h chromatography. I t was p u r i f i e d by HPLC on a reverse-phase s i l i c a g e l column, followed by prep-scale s i l i c a g e l TLC. Acid hydrolysis gave an aglycone (shown to be ebelin lactone by NMR and mass spectrometry) and the sugars glucose and rhamnose. The i d e n t i t i e s of the sugars were v e r i f i e d by t h i n layer chromatography, gas chromatography, and by comparison with sugar standards. We have used desorption-chemical i o n i z a t i o n mass spectrometry (DCI-MS) (21_) to obtain the order of sugar attachment to jujubogenin for t h i s molecule. DCI-MS i s an "in-beam" technique i n which vaporization-ionization of r e l a t i v e l y n o n - v o l a t i l e molecules i s f a c i l i t a t e d . The sample was deposited, as an aqueous s o l u t i o n , on a deactivated rhenium wire filament (Figure 8). A f t e r evaporation of the solvent, the filament was inserted into the mass spectrometer's electron beam. The wire was then rapidly heated to effect k i n e t i c desorption of the substance. An i o n i z i n g gas (ammonia i n t h i s case) was used to f a c i l i t a t e ion production. The sample desorbed very quickly and cleanly from the wire as shown i n the reconstructed ion chromatogram i n Figure 9 ( i n s e t ) . The mass spectrum i s shown i n Figure 9. A molecular ion was not seen under the run conditions. An ion corresponding to the ammonia adduct of jujubogenin-glucose i s seen at m/z 652, which indicates that rhamnose i s the ultimate sugar; glucose i s bonded d i r e c t l y to jujubogenin. The ion for the ammonia adduct of jujubogenin appears at m/z U90. This simple experiment permitted assignment of the carbohydrate linkage order, however, i t does not say anything about the nature of carbohydrate linkages themselves. This information may be obtained by methylation a n a l y s i s or c i r c u l a r dichroism techniques, and these determinations are currently being pursued i n our laboratory. Mechanism of Action Sodium dodecyl s u l f a t e , the gymnemic acids and the z i z i p h i n s have a l l been termed "surface a c t i v e " taste modifiers because they a l l possess detergent-like properties. These molecules a l l have a polar and a non-polar end and they are capable of penetrating the phospholipid membranes that are believed to be components of sweetness receptors. Any speculation about the mechanism of a c t i o n of these substances must take i n t o account the experimental observations concerning miraculin, monellin, and thaumatin, which were presented at the beginning of t h i s a r t i c l e . Those observations suggested that transport of the modifier to the c e l l ' s i n t e r i o r was not occurring and the i n h i b i t i o n e f f e c t i s manifested at the surface of the c e l l .


19


20


Figure 8 Preparing a sample f o r DCI mass spectrometry« Reproduced with permission from the Finnigan MAT Corporation. Copyright 1982, Finnigan MAT Corp. G

472

1ΘΘ.8-

jujubogenin

ebelin lactone 454

50.0 H

EL - H20 436

jujubogenin • NH4 490

634

*4* M/E

UjHillll^..fU.lfMiL 500

45Θ

550

652

600

650

Figure 9. DCI mass spectrum of jujubogenin-glucose-rhamnose. Conditions: ammonia i o n i z i n g gas at 0.25 t o r r ; filament heating rate 50 deg./sec; 0 3 sec/scan. Inset f i g u r e : reconstructed i o n chromatogram of jujubogenin-glucose-rhamnose. Reproduced w i t h o

permission

from R e f .

29.

C o p y r i g h t 1981,

Academic P r e s s ,

Inc.



2.

ADAMS


The size of the molecules and the speed with which the modifying effect appears argue against a c t i o n taking place i n the i n t e r i o r of a taste c e l l . DeSimone's work on the physicochemical e f f e c t s of tastants on phospholipid membranes provides i n s i g h t i n t o possible mechanisms of modifier action. He has shown that GA and SDS are about equivalent i n surfactant strength and that d i l u t e solutions of both substances are capable of penetrating phospholipid monolayers (6^). Surfactants are known to affect c e l l function by d i s r u p t i n g the structure or s p a t i a l arrangement of membrane l i p o p r o t e i n s (22). Surface-active taste modifiers are postulated to exert t h e i r e f f e c t s by a l t e r i n g a membrane-related process that r e l a t e s i n some way to the transduction of a taste s i g n a l . This d i s r u p t i o n of c e l l s t r u c t u r a l proteins, or related phospholipid membranes, might be a t t r i b u t e d to a l t e r a t i o n s i n the surface pressure (and surface free energy) of a membrane through i n s e r t i o n of the taste modifier into the membrane (23). Additional insight i n t o the mechanism of action of these i n h i b i t o r s i s provided by the k i n e t i c a n a l y s i s of Ray and B i r c h (2U). Two facts are known with c e r t a i n t y concerning molecules which produce taste e f f e c t s : they must be soluble i n s a l i v a and they must be able to occupy a receptor s i t e . To disrupt or i n h i b i t t a s t e , one or the other of these c h a r a c t e r i s t i c s can be i n t e r f e r e d with. Of the two, the l a t t e r i s the one most amenable to influence. From a k i n e t i c standpoint a stimulus molecule combines with a receptor to form a stimulus-receptor complex. This reaction occurs at a c e r t a i n rate. This complex may then break apart, g i v i n g back the receptor and the stimulus molecule; t h i s occurs at another rate. The formation of the stimulus-receptor complex eventually r e s u l t s i n the f i r i n g of a nerve and the production of a taste sensation. The rates of formation and breakdown of the complex are influenced by temperature. B i r c h has found that by increasing the temperature of tastant s o l u t i o n s , the perceived i n t e n s i t i e s of the solutions increase and the response-time p r o f i l e changes as w e l l (Figure 10). For a given temperature and sucrose concentration, taste sensation plateaus at a c e r t a i n time, suggesting that the sweetness receptors have become saturated ( i . e . that the rate of stimulus-receptor complex formation and breakdown are equal). A l i n e can be drawn on the graph from the beginning point of stimulus perception to the plateau point and the slope of a l i n e so drawn i s c a l l e d the magnitude estimation rate (MER). A plot of the r e c i p r o c a l s of MER values versus corresponding r e c i p r o c a l sucrose concentrations can be made; one obtains a Lineweaver-Burk type of p l o t (Figure 11). This plot indicates a low a f f i n i t y of sugar f o r receptor and a f f i n i t y values obtained by t h i s method agree generally with those of Cagan (25), which were determined by a r a d i o a c t i v e l y - l a b e l l e d sugar binding assay. In s i m i l a r fashion, a Lineweaver-Burk type plot for a gymnemic acid i n h i b i t e d tongue responding to sucrose solutions can be constructed. The appearance of t h i s p l o t i s quite d i f f e r e n t from that of Figure 12 and i s not c h a r a c t e r i s t i c f o r e i t h e r competetive or non-competitive i n h i b i t i o n . The r e s u l t s of the analysis seem to i n d i c a t e that i n h i b i t i o n of sweetness by gymnemic acid i s of a mixed k i n e t i c type which might r e s u l t from two i n h i b i t i o n mechanisms operating simultaneously. The i m p l i c a t i o n of t h i s i s that i n h i b i t i o n i s not immediately r e v e r s i b l e by increasing the


21

22

C H A R A C T E R I Z A T I O N A N D M E A S U R E M E N T OF FLAVOR C O M P O U N D S


20° C 30° C 40° C

Time of response

(sec)

Figure 10. Time-intensity p l o t f o r sugar sweetness. Magnitude estimation (ME) Reproduced with permission from Ref. 29. 0

Copyright

1981,

Academic P r e s s , I n c .

F i g u r e 11. R e c i p r o c a l p l o t of MER versus c o n c e n t r a t i o n of sweet s t i m u l u s . Reproduced w i t h p e r m i s s i o n from Ref. 29. Copyright 1981, Academic P r e s s , I n c .

Figure 12. Structure of methyl 4,6-dichloro-4,6-dideoxy-a-Dgalactopyranoside (VI), D i C l - g a l .



2.

ADAMS


concentration of sugar on the tongue. Thus, B i r c h postulates that sweetness suppression by gymnemic acid may be the r e s u l t of the i n h i b i t o r binding to the receptor for a period of time ( f o r up to an hour or so) during which time access of sugar molecules to the receptor i s blocked. Also, turnover of i n h i b i t o r i s much slower at the receptor than i s the turnover of sugar molecules. Therefore, a f t e r a receptor s i t e i s freed of bound gymnemic acid the sugar molecules must then compete with the proximate i n h i b i t o r for access and binding. A very powerful but s h o r t - l i v e d i n h i b i t i o n of sweet taste response i n the g e r b i l i s caused by methyl 4,6-dichloro-U,6dideoxy- α-D-galactopyranoside ( D i C l - g a l ) , VI, (Figure 12) a sugar d e r i v a t i v e synthesized and studied by Jakinovich (26). D i C l - g a l i s a potent sweetness i n h i b i t o r , but only when mixed with sucrose. I t has no long term i n h i b i t i n g e f f e c t on the sweet taste response. Also, pretreatment of the tongue with D i C l - g a l has no e f f e c t on subsequent response to sucrose s o l u t i o n s , i n contrast to the behaviors of SDS, GA and ZJ. K i n e t i c a n a l y s i s indicates that the substance i s a competetive i n h i b i t o r , but experiments with a r t i f i c i a l sweetners suggest that the mechanism of i n h i b i t i o n i s possibly more complex than t h i s . Prospects for Future Research There i s a growing body of knowledge r e l a t i n g structure to function for sweet t a s t e . In s p i t e of t h i s , many questions remain unanswered with regard to the physical nature of the sweetness receptor, including the question of whether i t i s indeed a s i n g l e receptor at a s i n g l e s i t e . Once the structures of several d i f f e r e n t i n h i b i t o r s are known i n d e t a i l i t should be possible to use them as probes of the receptors, to a s c e r t a i n the physical l i m i t s and s t e r i c - e l e c t r o s t a t i c requirements of the system. By systematically varying the structure of i n h i b i t o r s and measuring the psychophysical e f f e c t s , s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s can be constructed. These data, when taken together, may then allow construction of a s e l f - c o n s i s t e n t picture of the sweetness receptor. This, then, w i l l allow us to better understand the taste transduction process. F i n a l l y , i t i s i n t e r e s t i n g to speculate on what function sweetness i n h i b i t o r s such as the gymnemic acids and the z i z i p h i n s might perform for the plants i n which they occur. One thought i s that they might serve as feeding deterrents for predatory i n s e c t s . I t has been shown that gymnemic acids act as feeding deterrents to the Southern army worn (Prodenia eridana). The e f f e c t i s demonstrable with sugar-free d i e t s , thus the gymnemic acids do not appear to act on the sweet taste i n the i n s e c t , as they do i n humans (27,, 28). The sweetness i n h i b i t o r s may also function as defenses against mammalian herbivores: by d u l l i n g the sense of sweetness, the i n t r i n s i c b i t t e r n e s s of a plant might be accentuated, thus encouraging the browser to find t a s t i e r fare. Acknowledgments Much of the z i z i p h i n chemistry was carried out by Dr. Frank Koehn, and psychophysical studies were performed by Drs. C l a i r e Murphy and


23

24

CHARACTERIZATION AND MEASUREMENT OF FLAVOR COMPOUNDS

Carol Christensen. This work was p a r t i a l l y supported by NIH grant 5T32NS07176-05 (postdoctoral traineeship t o F.E.K.) and by the Ambrose Monell Foundation. Literature Cited 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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2. ADAMS

26. 27. 28. 29.


Jakinovich, W.; Science; 1983, 219, 408-410. Granich, M. S.; Halpern, B. P.; Eisner, T . ; J. Insect Physiol.; 1974, 20, 435-439. Harborne, J. B . ; "Introduction to Ecological Biochemistry"; Academic Press, New York, 1977; pp. 149-150. Birch, G. G . ; In "Biochemistry of Taste and Olfaction"; Cagan, R. H.; Kare, M. R . , E d s . ; Academic Press, New York, 1981; pp. 163-73.


RECEIVED August 14, 1985


Characterization and Measurement of Flavor Compounds - American

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