Enzymic Generation of Volatile Aroma Compounds from Fresh Fish

0-15. 1. Josephson et al. (23-24)T. 2 Present in surface slime/water extracts. 3 Reported for .... desirable (51-53). In another .... fish is provided...
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17 Enzymic Generation of Volatile Aroma Compounds from Fresh Fish David B. Josephson and R. C. Lindsay Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706

Characterizing aromas for freshly-harvested f i s h are derived from polyunsaturated f a t t y acids through lipoxygenase-mediated reactions, and include both v o l a t i l e alcohols and carbonyls. Short-chain alcohols and aldehydes were shown to suppress the a c t i v i t y of lipoxygenase which was derived from trout gill tissue, and may serve as a means to regulate the formation of fresh f i s h aroma compounds. Exploratory experiments revealed that plant-derived lipoxygenases have potential for the biogenesis of fresh f i s h f l a v o r s . Flavors and aromas commonly associated with seafoods have been intensively investigated in the past forty years (1-7), but the chemical basis of these f l a v o r s has proven elusive and d i f f i c u l t to e s t a b l i s h . Oxidized f i s h o i l s can be described as painty, rancid or c o d - l i v e r - o i l l i k e (8), and c e r t a i n v o l a t i l e carbonyls a r i s i n g from the autoxidation of polyunsaturated f a t t y acids have emerged as the p r i n c i p a l contributors to this type of f i s h - l i k e aroma (3, 5, 9-10). Since oxidized butterfat (9, 11-12) and oxidized soybean and linseed o i l s (13) also can develop s i m i l a r painty, f i s h - l i k e aromas, confusion has arisen over the compounds and processes that lead to f i s h - l i k e aromas. Some have believed that the aromas of f i s h simply r e s u l t from the random autoxidation of the polyunsaturated f a t t y acids of f i s h l i p i d s (14-17). This view has often been retained because no single compound appears to exhibit an unmistakable f i s h aroma. Still, evidence has been developed which indicates that a r e l a t i v e l y complex mixture of autoxidatively-derived v o l a t i l e s , including the 2,4-heptadienals, the 2,4-decadienals, and the 2,4,7-decatrienals together elicit unmistakable, oxidized f i s h - o i l aromas (3, 9, 18). A d d i t i o n a l l y , reports also suggest that contributions from (Z)-4-heptenal may add c h a r a c t e r i s t i c notes to the cold-store f l a v o r of c e r t a i n f i s h , e s p e c i a l l y cod (4-5). 0097-6156/86/0317-0201$06.00/ 0 © 1986 American Chemical Society Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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The d e f i n i t i o n of oxidized f i s h o i l - l i k e aromas s t i l l leave fresh f i s h aromas undefined. Various freshly harvested f i s h have distinguishing aromas, but they also are characterized by a common p l a n t - l i k e , seaweed-like aroma. Thus, compounds and reaction pathways d i f f e r e n t from random autoxidation appear l i k e l y and reasonable. Even c o n f l i c t i n g descriptions of f i s h y odors, i . e . , including roles f o r v o l a t i l e amines (2^, 19) and sulfur compounds (20-22), can be accommodated by the hypothesis that previously unrecognized biochemical reactions y i e l d characterizing fresh f i s h aromas. These premises led to investigations (23-26) which have resulted in the i d e n t i f i c a t i o n of a group of enzymically-derived v o l a t i l e aroma compounds that contribute fresh, p l a n t - l i k e aromas to freshly harvested f i s h (Table I ) . Eight-carbon alcohols and ketones have been previously i d e n t i f i e d in mushrooms (27-28), and occur in a l l species of f i s h surveyed to t h i s point (23-25, 29), in crustaceans (30-31), and in s h e l l f i s h (26). Although the eight-carbon v o l a t i l e compounds i n d i v i d u a l l y possess mushroom, geranium-like aroma notes, they contribute d i s t i n c t heavy, p l a n t - l i k e aromas to freshly harvested f i s h . The eight-carbon v o l a t i l e alcohols appear to occur in greater abundance than the corresponding ketones, and this is consistent with a similar o r i g i n for these v o l a t i l e s in mushrooms (27). Table I. Enzymically derived carbonyls and alcohols associated with freshly harvested f i s h . 1

Cone. Range ALCOHOLS l-Penten-3-ol (Z)-3-Hexen-l-ol 1- 0cten-3-ol 1,5-0ctadien-3-ol 2- 0cten-l-ol 2.5- 0ctadien-l-ol 6-Nonen-l-ol 3.6- Nonadien-l-ol

Cone. Range CARBONYLS (ppb) Hexanal 10-100 (E)-2-Hexenal 1-10 1- 0cten-3-one 0.1-10 1.5- 0ctadien-3-one 0.1-5 2- 0ctenal 0.1-5 (E)-2-Nonenal 0-25 (Ε,Z)-2,6-Nonadienal 0-35 6-Nonenal^ trace 3.6- Nonadienal-j trace 2

2

(PPb) 3-30 1-10 10-100 10-100 1-20 1-20 0-15 0-15

1

Josephson et a l . (23-24)T 2 Present in surface slime/water extracts. 3 Reported for the f i r s t time.

Nine-carbon v o l a t i l e alcohols and aldehydes, responsible for much of the characterizing aromas of cucumber and melon f r u i t s (32-35), occur only in some species of f i s h , such as whitefish (Coregonus clupeaformis), ciscoe (Coregonus a r t e d i ) , smelt (Osmerus mordax) (23-24, 36), and spawning P a c i f i c salmon (Oncorhynchus sp.) (unpublished data). In these species the nine-carbon compounds provide characterizing cucumber-, melon-like aromas. P a c i f i c oysters also produce both the eightand nine-carbon alcohols and carbonyls i d e n t i f i e d in fresh f i s h , but A t l a n t i c oysters biosynthesize only the eight-carbon alcohols and ketones (26). In seafoods the nine-carbon v o l a t i l e aldehydes

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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have been found to occur in greater abundance than the nine-carbon alcohols, and t h i s is consistent with t h e i r enzymic formation in cucumbers (37). Six-carbon v o l a t i l e alcohols and aldehydes have been found in a l l freshwater f i s h surveyed (23-24). However, these compounds have not been found in either salmon residing in saltwater (unpublished data) or in oysters (26). Hexanal has been found in modestly fresh (5-6 days old) saltwater f i s h (24), but i t s formation may be the result of autoxidation rather than v i a enzyme-mediated reactions. Thus, data for the occurrence of hexanal in freshly harvested saltwater f i s h remains to be developed. Hexanal and (E)-2-hexenal contribute coarse, green-plant-like, aldehydic aroma notes to freshly harvested f i n f i s h where their aroma dominates the o v e r a l l odors within seconds after the death of the f i s h . (Z)-3-Hexen-l-ol contributes a clean, green-grass-like aroma note. Hexanal always occurs in substantially greater abundance than 1-hexanol in f i s h . In addition to the six-carbon v o l a t i l e compounds, l-penten-3-ol is also found in a l l freshwater f i s h . However, concentrations of l-penten-3-ol in f i s h remain below i t s recognition threshold (400 ppb; 38), and therefore J. is u n l i k e l y that this v o l a t i l e contributes strongly to the c h a r a c t e r i s t i c aroma of freshly harvested f i s h . Generally, the v o l a t i l e carbonyls found in f i s h exhibit coarse, heavy aromas whereas the v o l a t i l e alcohols contribute smoother q u a l i t i e s . Lower threshold values for the v o l a t i l e carbonyls, especially l-octen-3-one (0.005 ppb; 39); 1.5- octadien-3-one (0.001 ppb; 12); (E)-2-nonenal (0.08ppb; 39), and QS,Z)-2,6-nonadienal (O.Olppb; 39), result in greater contributions to o v e r a l l fresh f i s h - l i k e odors than do the corresponding alcohols (l-octen-3-ol, l,5-octadien-3-ol, and 3.6- nonadien-l-ol) whose threshold values are each 10 ppb (28, 31, 39). In addition to the enzymically-derived v o l a t i l e aroma compounds (Table I ) , low l e v e l s of autoxidatively-derived carbonyls can also be detected in harvested f i s h held a day on i c e , and these v o l a t i l e s are l i s t e d in Table I I . The oxidatively-derived carbonyls modify the fresh p l a n t - l i k e aromas of fresh f i s h by providing o x i d i z e d - o i l - l i k e , s t a l i n g fish-type odor notes 0~5, 9). The formation of hexanal in freshly harvested f i s h appears to be enzymic because the concentration of t h i s compound can be diminished by lipoxygenase i n h i b i t o r s (25). However, when fresh f i s h are stored on ice or are held under frozen storage, hexanal concentrations also increase because of autoxidative processes (40). Trimethylamine and other amines have often been variously associated with the aromas of f i s h (2, 19, 41). Much of the trimethylamine found in fresh f i s h arises from the microbial reduction of trimethylamine oxide (42-44) which is found abundantly in only marine f i s h (45-47). On the other hand, dimethylamine is an abundant product of an endogenous enzymic action on trimethylamine oxide in marine f i s h muscle, and J. is readily produced even under high-sub-freezing conditions in marine f i s h (48). Both trimethylamine and dimethylamine

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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contribute s i g n i f i c e n t l y to the stale, fishy aromas of aging fresh and frozen marine f i s h as well as to o v e r a l l boiling-crab, f i s h house-type aromas. However, freshly harvested f i s h contain l i t t l e of this compound. Table I I .

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CARBONYLS

Oxidatively-derived carbonyls i d e n t i f i e d in fresh f i s h stored on i c e . Cone. Range (ppb) 1

CARBONYLS

Cone. Range (ppb) 1

***2 0.1- •2 Hexanal (Ε,Ε)·-3,5--Octadiene-2-one 1-•5 (Ε,Z)-2,4-Heptadienal 1-10 (Ε,Ζ)--2,4- -Decadienal 1--5 (E,E)-2,4-Heptadienal 1-10 (Ε,Ε)·-2,4- -Decadienal (E,Z)-3,50.1-2 Octadien-2-one •Present in surface slime/water extracts. •Hexanal is formed both enzymically and autoxidatively, which makes contributions d i f f i c u l t to delineate. Sulfur-containing compounds have also been shown to contribute to deteriorative aromas associated with seafood spoilage, and other instances of o f f - f l a v o r occurrences. Dimethyl s u l f i d e has been i d e n t i f i e d as the causative agent in one case of non-spoilage o f f - f l a v o r in seafoods where J. was formed either from the enzymic or thermal degradation of dimethyl-B-propiothetin (49-50). S t i l l , dimethyl s u l f i d e provides a characterizing top-note aroma to cooking or stewing clams and oysters where i t s flavor contribution is expected and desirable (51-53). In another instance, Shiomi et a l . (22) have shown that methyl mercaptan and dimethyl d i s u l f i d e are responsible for the offensive odor of freshly-captured flat-head ( C a l l i u r i c h t h y s doryssus), and in t h i s case these two v o l a t i l e s appear to be formed by endogenous enzymes. The apparent enzymic formation of methyl mercaptan and dimethyl d i s u l f i d e in the flat-head d i f f e r s s u b s t a n t i a l l y from the usual microbial o r i g i n of these compounds in s p o i l i n g fresh f i s h (54-55). A g a r l i c - l i k e o f f - f l a v o r caused by bis-(methylthio)-methane has been reported for several species of prawns and the sand lobster (Ibacus p e r o n i i ) , and evidence also supports endogenous biosynthesis in these cases (21). Biogenesis of Fresh Fish Carbonyls and Alcohols Although for many years lipoxygenase a c t i v i t y in f i s h was discounted (14-17), plant lipoxygenases were e a s i l y demonstrated, and became well-accepted. Research into the biosynthesis of the six-, eight-, and nine-carbon v o l a t i l e aroma compounds of mushrooms (27, 56) and cucumber/melon-fruits (37, 57-58) showed the concerted a c t i v i t i e s of both s i t e - s p e c i f i c lipoxygenases and hydroperoxide lyases. Because animal lipoxygenases exhibit s e l f - i n a c t i v a t i o n properties (59-60), early experimentation f a i l e d to detect an active lipoxygenase from f i s h , and t h i s led to conclusions that autoxidation was responsible for the

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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17.

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LINDSAY

Volatile Aroma Compounds from Fresh Fish

production of a l l v o l a t i l e s derived from l i p i d s that were found in f i s h tissue. Tsukuda and Amano (61-62) and Tsukuda (63-64) believed they had observed lipoxygenase a c t i v i t y in f i s h by showing that fading or color deterioration associated with cartenoid pigment destruction in the skin of red f i s h could be delayed by introducing a variety of non-specific enzyme i n h i b i t o r s . Yet, previous skepticisms continued to p r e v a i l (17). D i f f i c u l t i e s in c l e a r l y demonstrating enzymic oxidative involvement in the biogenesis of fresh f i s h v o l a t i l e aroma compounds were also encountered in our work with freshly-harvested f i s h held on i c e . To circumvent obscure i n i t i a l enzyme a c t i v i t y as well as subsequent autoxidation in even very fresh f i s h , techniques were developed which employ s a c r i f i c i n g l i v e f i s h at the i n i t i a t i o n of experiments which are designed to demonstrate lipoxygenase a c t i v i t y . Thus, by i n h i b i t i n g a c t i v i t y at the time of death through the use of lipoxygenase-specific i n h i b i t o r s , such as esculetin (65) and Sn(II)Cl (66), J. was possible to more c l e a r l y separate enzymic and autoxidative l i p i d degradations. The integration of t h i s approach with highly-sensitive Tenax GC headspace analysis (67) has provided a powerful biochemical probe technique for investigating the biogenesis of fresh f i s h f l a v o r s . Recently, German and K i n s e l l a (68-69) have demonstrated a c t i v i t y of a 12-lipoxygenase in the g i l l and skin tissues of trout (Salmo sp.), and these findings i d e n t i f y an appropriate precursor to some of the fresh f i s h aroma v o l a t i l e s . This allows association of an appropriate precursor with the occurrence of c e r t a i n v o l a t i l e s , and provides very strong support for the view that the v o l a t i l e aroma compounds characterizing the fresh, p l a n t - l i k e aromas of freshly harvested f i s h result from lipoxygenase-mediated bioconversions of polyunsaturated f a t t y acids. A summary of the current hypothesis for the formation of v o l a t i l e aroma compounds in freshly harvested f i s h is shown in Figure 1. These proposed pathways d i f f e r in part from those previously proposed (25), but they are consistent with a l l data. Evidence in support of the mechanism shown in Figure 1 can be derived from p a r a l l e l lipoxygenase-mediated reaction pathways which have been proposed for the formation of the eight-carbon v o l a t i l e s in mushrooms (71) and the nine-carbon v o l a t i l e s in cucumber f r u i t s (37). Even more direct evidence, comes from the recent i d e n t i f i c a t i o n of a 12-lipoxygenase in the skin and g i l l tissue of trout by German and K i n s e l l a (68-69). However, an unknown remains at t h i s time regarding the existence of the 15-lipoxygenase. If the carbon number and functional group of fresh f i s h v o l a t i l e aroma compounds are viewed as i n d i c a t i v e of a s p e c i f i c , required hydroperoxy-fatty acid precursor, the biogenesis of the f i v e - and six-carbon v o l a t i l e aroma compounds would depend on the existence of 15-lipoxygenase. In such a case, the reaction pathways f o r the formation of the f i v e - and six-carbon v o l a t i l e compounds would p a r a l l e l those for the eightand nine-carbon v o l a t i l e s once the s i t e - s p e c i f i c hydroperoxidation has occurred.

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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BIOGENERATION OF AROMAS

206

COOH ACID (C20:5 n-3)

EICOSAPENTAENOIC

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15-LIPOXYGENASE OOH

A = A H O

J

(z)-1,5-OCTADIEN-3-OL / \ _ / \ / \ /

C

H

O

(z)-3-HEXENAL

M /

1

H

0

(E)-2-HEXENAL

(E,z)-2,6-NONADIENAL

A=A=A

Λ = % Ο Η CHfiH

(Z,Z)-3,6-NONADCN-1-OL

(z)-3-HEXEN-1-OL

(Z)-1,5-OCTADIEN-3-ONE

F i g u r e 1.

Proposed mechanism f o r t h e b i o g e n e r a t i o n o f some f r e s h s e a f o o d aroma compounds.

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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An important difference exists in terms of f a t t y acid precursors that are available as substrates for plant and animal lipoxygenases. Plant lipoxygenases primarily have available as substrates only l i n o l e i c (C18:2, n-6) and l i n o l e n i c (C18:3, n-3) acids (72), whereas f i s h lipoxygenases have access to the more unsaturated f a t t y acids, including arachidonic (C20:4, n-6), eicosapentaenoic (C20:5, n-3), and docosahexaenoic (C22:6, n-3) acids, which are quite abundant in most f i s h (73-74). Thus, in the proposed scheme eicosapentaenoic acid would be available for enzymic conversion to monounsaturated f i v e - , and six-carbon, and diunsaturated eight-, and nine-carbon v o l a t i l e aroma compounds (Figure 1, Table I ) . Docosahexaenoic acid could also serve as a precursor for these v o l a t i l e s . Alternatively, arachidonic acid (an n-6 f a t t y acid) could serve as a precursor for the saturated six, and monounsaturated eight-, and nine-carbon v o l a t i l e s (Table I ) . Biogenesis of the f i v e - and eight-carbon v o l a t i l e s appears to involve lipoxygenase-mediated hydroperoxidations which are l i k e l y followed by a reductive rearrangement and cleavage to result in the formation of the secondary alcohols. Enzymic oxidation of the secondary alcohols would result in the formation of the f i v e - and eight-carbon ketones. The s i x - and nine-carbon v o l a t i l e compounds would be formed v i a the concerted a c t i v i t i e s of lipoxygenase and a hydroperoxide lyase to form (55,Z)-3,6-nonadienal [or (Z)-3-hexenal], followed by either enzymic isomerization to form (E,Z)-2,6-nonadienal [or (E)-2-hexenal] or dehydrogenase a c t i v i t y to form (_Z,Z)-3,6-nonadien-l-ol [or (Z)-3-hexen-l-ol]. Proposed Physiological Role for the Enzymic Formation of Fresh F i s h V o l a t i l e Carbonyls and Alcohols The biogeneration of v o l a t i l e aroma compounds in f i s h can be hypothetically related to mechanisms for the regulation of recently-recognized physiologically-active compounds, e s p e c i a l l y leukotrienes and hydroxy-fatty acids (75-79). This view provides the basis for the diagram shown in Figure 2 where the skin-water interface has been chosen as an example of a s i t e for this type of enzymic a c t i v i t y . Stress-related l i p i d bioconversions (80) would be expected to occur at this interface to maintain physiological conditions in the f i s h , and lipoxygenase a c t i v i t y has been detected recently in skin and g i l l tissues of trout (69). Further, enzymically-derived v o l a t i l e aroma compounds appear to occur in higher concentrations in the outer skin and mucus layer of f i s h when compared to muscle tissue per se (23-24). If the biochemical processes are viewed as i n i t i a l enzymic formations of physiologically-active or regulatory compounds ( i . e . , leukotrienes) from polyunsaturated f a t t y acids that are followed by their i n a c t i v a t i o n through indicated enzymic mechanisms ( i . e . , lyases), the result is a release of the v o l a t i l e aroma compounds that are associated with the aromas of freshly-harvested f i s h . D i f f e r i n g physiological demands of i n d i v i d u a l genus/species of f i n f i s h would account f o r at l a s t some of the varying concentrations and occurrences of the fresh f i s h v o l a t i l e s that have been observed (23-26, 36).

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

BIOGENERATION OF AROMAS

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Hydroperoxides formed by animal lipoxygenases serve as intermediates of p h y s i o l o g i c a l l y - a c t i v e compounds in f i s h (79), but these hydroperoxides also appear to catalyze the i n i t i a t i o n of free r a d i c a l autoxidation. Such a c t i v i t y has been recently proposed by German and K i n s e l l a (69) who isolated hydroxy-fatty acids formed by reduction of hydroperoxides generated v i a 12-lipoxygenase in the skin of trout. Further evidence for the hydroperoxide-induced formation of c e r t a i n v o l a t i l e s in fresh f i s h is provided in Table III where occurrences of the products of both enzymic and non-enzymic oxidation are presented. Data were obtained for juvenile hybrid muskellunge (4-5 gm each; Esox masquinongy) that were s a c r i f i c e d by homogenization either in d i s t i l l e d water (control-blend) or in the presence of e s c u l e t i n (65). Also included with these data are r e s u l t s obtained when surface extracts (control-mucus) were taken from recently s a c r i f i c e d (suffocated) f i s h . Enzymically-derived v o l a t i l e s were produced along with low l e v e l s of autoxidatively-derived carbonyls in d i s t i l l e d water homogenates. In the presence of the lipoxygenase i n h i b i t o r , e s c u l e t i n , neither fresh f i s h v o l a t i l e s nor the autoxidatively-derived v o l a t i l e s were present. Therefore, generation of autoxidatively-derived v o l a t i l e s appears to occur at least i n i t i a l l y from the reactions of susceptible l i p i d fractions with enzymically-derived hydroperoxides. Table I I I .

E f f e c t of a lipoxygenase-specific i n h i b i t o r on the occurrence of enzymically- and oxidatively-derived v o l a t i l e s in f i s h .

Volatile

Control blend

Treatment Esculetin (10 mM)

Control mucus

ENZYMICALLY-DERIVED l-0cten-3-one 1,5-0ctadien-3-one l-0cten-3-ol 1,5-Octadien-3-ol 2-0cten-l-ol 2,5-Octadien-l-ol (E)-2-Nonenal (Ε,Ζ)-2,6-Nonadienal 6-Nonen-l-ol 3,6-Nonadien-l-ol

+ + + + + + + + +

+ + + + + + + + +

AUTOXIDATIVELY-DERIVED (Ε,Z)-2,4-Heptadienal (Ε,E)-2,4-Heptadienal (Ε,Z)-2,4-Decadienal

+ + +

-/+2

1

-/+ -/+

+ = P o s i t i v e l y I d e n t i f i e d ; - - Not I d e n t i f i e d . Not found i n i t i a l l y ; l a t e r found a f t e r 2-days on i c e .

Surface extracts (mucus) of the recently s a c r i f i c e d f i s h contained only enzymically-derived v o l a t i l e s . However,

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LINDSAY

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autoxidatively-derived carbonyls along with enzymically-derived carbonyls and alcohols were subsequently found in surface (mucus) extracts from the s a c r i f i c e d f i s h after storage on i c e for two days. This indicates slower r e a c t i v i t y v i a autoxidation compared to enzymically-induced oxidations. These results are consistent with the observations of Yamaguchi ejt a l . , (81-82) and Yamaguchi and Toyomizu (83) who suggested that accelerated rates of l i p i d oxidation in f i s h skin extracts were caused by lipoxygenase activity. Current Investigations on the Contributions of Various Oxidative Enzymes to the Generation of Fresh Fish V o l a t i l e Carbonyls and Alcohols While lipoxygenases (EC// 1.13.11.12) catalyze the s i t e - s p e c i f i c i n s e r t i o n of molecular oxygen into polyunsaturated f a t t y acids, cytochrome P-450 monooxygenases (EC# 1.14.14.1) and other oxidative enzymes share a common t r a i t of catalyzing non-specific oxidation of organic compounds, including polyunsaturated f a t t y acids. The l a t e r oxygenases result in the formation of variously positioned epoxy-, and hydroxy-fatty acids (84). Thus, enzymes catalyzing s i t e - s p e c i f i c hydroperoxidations of polyunsaturated f a t t y acids should be regarded as lipoxygenases, and measurements of v o l a t i l e aroma compounds which result from the cleavage of predictable hydroperoxy-fatty acids appear appropriate as probes into this type of biochemical reaction in f i s h . The high s e l e c t i v i t y of fused s i l i c a c a p i l l a r y gas chromatography allows use of this information to d i f f e r e n t i a t e between v o l a t i l e s generated v i a lipoxygenase-mediation from v o l a t i l e s generated through random autoxidation. E a r l i e r we reported that the biogenesis of fresh f i s h v o l a t i l e aroma compounds were suppressed by the addition of asprin (25), a potent i n h i b i t o r of cyclooxygenase (EC# 1.14.99.1; 85). Cyclooxygenase converts polyunsaturated f a t t y acids into p h y s i o l o g i c a l l y - a c t i v e prostaglandins. However, recent experiments have demonstrated that lower pH e f f e c t s from acetyl s a l i c y l i c acid rather than s p e c i f i c cyclooxygenase i n h i b i t i o n were primarily responsible for the suppression observed (70). Thus, at t h i s time unambiguous data for the involvement of prostaglandins in the biogenesis of fresh f i s h v o l a t i l e aroma compounds either through enzymic regulation mechanisms or d i r e c t l y from prostaglandins themselves as precursors to some fresh f i s h v o l a t i l e compounds are not available. However, hydroperoxy-fatty acids are now implicated in the production of fresh f i s h aroma v o l a t i l e s , and these compounds are also converted to leukotrienes or other p h y s i o l o g i c a l l y - a c t i v e hydroxy-fatty acid derivatives (75-79). Potential Technical D i f f i c u l t i e s in Applying Lipoxygenases the Controlled Biogeneration of Fresh F i s h Aromas

for

Although l i t t l e is known about many of the important enzymic parameters of the flavor generating systems that are endogenous in f i s h , the s e l f - i n a c t i v a t i o n or s u i c i d a l nature of animal

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lipoxygenases (59-60) is also c h a r a c t e r i s t i c of the lipoxygenases from f i s h (68-6"9J. L i p i d hydroperoxides formed by animal lipoxygenase are required to c a t a l y t i c a l l y activate the enzyme, but the hydroperoxides are also responsible f o r causing the s e l f - i n a c t i v a t i o n of the enzyme (60). The very rapid rate of s e l f - i n a c t i v a t i o n of animal lipoxygenases (60, 68), which delayed recognition of these enzymes in f i s h (14-16), can be suppressed by addition of reduced glutathione (1 mM; 68; Figure 3). In these systems, glutathione functions by chemically reducing hydroperoxy-fatty acid products (animal-lipoxygenase i n h i b i t o r s ) to hydroxy-fatty acids. Without such additions, J. has been d i f f i c u l t to maintain a c t i v i t y in order to demonstrate e f f e c t s of various treatments on actual enzyme a c t i v i t y . When crude trout g i l l lipoxygenase preparations were stored at 0°C, a c t i v i t i e s could be sustained over a s i x to eight hour period i f glutathione was present, but t h i s appeared to vary with d i f f e r e n t preparations. However, gradual losses of enzyme a c t i v i t y were s t i l l noted over this time span. Since a l l experiments on g i l l lipoxygenase to date have employed crude supernatant preparations (centrifuged at 15,000 χ g), J. remains to be determined whether further p u r i f i c a t i o n w i l l enhance or diminish the o v e r a l l a c t i v i t y of the enzyme. The involvement of lipoxygenases in the o v e r a l l flavor-generating scheme for f i s h encompasses only the hydroperoxidation of free f a t t y acids, and l i t t l e is known about the additional enzymes that are l i k e l y involved. At a minimum, these probably include lyases, isomerases, and dehydrogenases. Another major consideration regarding the lipoxygenase-mediated biogeneration of v o l a t i l e aroma compounds in f i s h is the hydroperoxidative nature of the enzyme products themselves. Since any l i p i d hydroperoxide is capable of p a r t i c i p a t i n g in subsequent random autoxidative deterioration of polyunsaturated f a t t y acids following dismutation to alkoxy (R0 ) and hydroxy ( Ό Η ) r a d i c a l s , the uncontrolled generation of l i p i d hydroperoxides by lipoxygenases w i l l lead to the production of undesirable amounts of c l a s s i c a l oxidized f i s h - l i k e aroma compounds (Table I I I ; 3-5, 9^, 11-12). However, microencapsulation (86-87) of enzyme systems which separates the flavor-generating reactions from the bulk of the food and allows passage of the v o l a t i l e compounds that are generated into the food may f i n d application f o r controlled seafood-flavor generations (88). In foods where uncontrolled e

hydroperoxide-initiated autoxidation might not l i m i t the q u a l i t y of the o v e r a l l flavor of the product, then perhaps direct incorporation of a lipoxygenase flavor-generating system might be exploited. The r e l a t i v e r a t i o s of alcohols and carbonyls f o r the s i x - , eight- and nine-carbon v o l a t i l e s in f i s h (23-24) and oysters (26) p a r a l l e l those encountered in cucumber f r u i t s (37) and mushrooms (27, 56) i f the two systems are combined. Therefore, the use of plant-based enzyme systems f o r the controlled generation of fresh seafood flavors and aromas has been under consideration in our laboratory as a means to overcome some of the s e l f - i n a c t i v a t i n g problems associated with f i s h lipoxygenases.

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Volatile Aroma Compounds from Fresh Fish

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J O S E P H S O N A N D LINDSAY

C20:4 ENZYME PREP. J jw/o GLUTATHIONE

24(H

(A)

ENZYME PREP. |w/ GLUTATHIONE

1ί (B)

OXYGEN CONC. (uM) OH TIME Figure 3.

5 min

The effect of reduced glutathione on f i s h lipoxygenase a c t i v i t y as measured by oxygen consumption employing a Johnson-type oxygen electrode.

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Exploratory Experiments on the Means to Assist in the Regulation of Lipoxygenases in Tissue Extracts' Mitsuda £t a l . (89) have demonstrated i n h i b i t i o n of soybean lipoxygenase a c t i v i t y by the addition of monohydric alcohols (n-butanol, n-pentanol, n-hexanol, and n-heptanol), and noted a marked increase in i n h i b i t i o n with increasing chain-length. Evidence obtained for other alcohols, including straight-chain, secondary and t e r t i a r y alcohols, strongly suggested the i n h i b i t i o n was caused by non-specific hydrophobic bonding between the alcohol and enzyme. These observations stimulated exploration of the p o s s i b i l i t y that naturally occurring fresh f i s h v o l a t i l e s might be involved in regulating enzymic a c t i v i t y . Further, J. was of interest to determine whether the presence of certain fresh f i s h v o l a t i l e s might exert regulating action on lipoxygenase in flavor generating systems. To test the effect of fresh f i s h v o l a t i l e compounds on trout g i l l lipoxygenase a c t i v i t y , a crude preparation of the enzyme was obtained from rainbow (Salmo gairdneri; 400 gm each) or brown (Salmo t r u t t a ; 300 gm each) trout (68). These preparations proved to be sensitive to c l a s s i c lipoxygenase i n h i b i t o r s , i . e . , esculetin and t i n (II) chloride, as reported by German and K i n s e l l a (69). For each enzyme preparation, the g i l l s were removed from f i v e l i v e trout and homogenized in 50 ml of t r i s buffer (0.05M; pH 7.8; 0°C) which contained 1 mM reduced glutathione. Homogenates were subsequently centrifuged at 15,000 χ g for 10 min., and f i l t e r e d through cheesecloth to remove f r e e - f l o a t i n g l i p i d s (69), and then used d i r e c t l y in enzyme assays. For assays of trout g i l l lipoxygenase, 1.5 ml of the preparation was equilibrated (5 min) with 50 u l of either a solution of ethyl acetate containing an i n h i b i t o r or neat ethyl acetate (control). One ml of the equilibrated extract was then added to a v i a l containing 2 ml of T r i s buffer (0.05M; pH 7.8), 100 nmoles of sodium arachidonate (in ethanol), and a Johnson-type l e a d - s i l v e r electrode (90-91) was used for the measurement of oxygen consumption. A l l extractions and measurements were performed in a walk-in cooler at 4°C. When the n-alkanols, n-butanol, n-hexanol, and n-heptanol (10-100 mM), were preincubated with trout g i l l lipoxygenase preparations, some degee of i n h i b i t i o n of oxygen consumption was observed as compared to control determinations. These data suggested that the alcohols inactivated f i s h lipoxygenase in a manner similar to that observed for soybean lipoxygenase (89). A d d i t i o n a l l y , a number of enzymically-derived v o l a t i l e s i d e n t i f i e d in fresh f i s h were tested f o r i n h i b i t o r y a c t i v i t y on trout g i l l lipoxygenase, including l-penten-3-ol, l-octen-3-ol, 6-nonen-l-ol, hexanal, and l-octen-3-one. In a l l cases the fresh f i s h v o l a t i l e compounds also exhibited some i n h i b i t o r y influence on trout g i l l lipoxygenase preparations. l-0cten-3-ol and l-octen-3-one at a concentration of 10 mM each provided about 50% i n h i b i t i o n of trout g i l l lipoxygenase a c t i v i t y . The similar i n h i b i t o r y properties of l-octen-3-ol and l-octen-3-one suggest a non-specific binding for these v o l a t i l e s to lipoxygenase.

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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J O S E P H S O N A N D LINDSAY

Volatile Aroma Compounds from Fresh Fish

213

Hexanal (to 150 mM) and 6-nonen-l-ol (to 100 mM) were less e f f e c t i v e in i n h i b i t i n g g i l l lipoxygenase, while l-penten-3-ol (to 300 mM) proved to be the least e f f e c t i v e . The range of concentrations of v o l a t i l e s which caused some degree of i n h i b i t i o n of trout g i l l lipoxygenase were in the mM range ( i . e . , 1000-3000 ppm). Therefore, r e l a t i v e l y high concentrations of these compounds are necessary to substantially reduce the a c t i v i t y of trout g i l l lipoxygenase in crude enzyme preparations. However, s o l u b i l i t y factors may influence the apparent effectiveness of the longer-chain compounds. Experiments also have shown that ethanol and ethyl acetate do not influence the a c t i v i t y of trout g i l l lipoxygenase up to about 1.2M (6.6%) and 0.75M (6.6%), respectively. Thus, ethanol and ethyl acetate provided a t t r a c t i v e solvents for d i l u t i n g both substrates and i n h i b i t o r y compounds in lipoxygenase i n h i b i t i o n studies. Although selected v o l a t i l e s might be exploited for providing contributions of b e n e f i c i a l flavor effects as well as lipoxygenase-control, the high concentrations required for lipoxygenase control w i l l require special manipulation in microencapsulated systems to allow p r a c t i c a l applications. Exploratory Model Employing Surimi to Evaluate Plant-Based Seafood Flavor Generation Since plant lipoxygenases lack the s e l f - i n a c t i v a t i o n properties of animal lipoxygenases, plant-based enzyme flavor generating systems have a greater potential for early application in the biogeneration of fresh seafood aromas and flavors than those of f i s h . At this time some of the most desirable, characterizing fresh seafood-like aroma/flavor compounds appear to be provided by combinations of the eight-carbon v o l a t i l e s , and these are also produced in mushrooms and geranium leaves. Thus, the models for i n i t i a l experiments included crude enzyme preparations from plants that were incorporated into pollack surimi which is a washed, minced f i s h f l e s h product widely used as a base for seafood analogs such as imitation crab. I t exhibits r e l a t i v e l y mild f i s h - l i k e aromas, and i t s aroma seems to respond well to added plant-based enzyme preparations. Flavor-generating systems have included those from mushrooms (mascreated; Agaricus bisporus) and cucumber f r u i t s (mascreated; Cucumis sativus) which were each added to surimi at 1%. A d d i t i o n a l l y , single geranium leaves (either crushed or uncrushed; Pelargonium sp.) were placed into 100 gm of surimi. The descriptions of aromas produced in these systems are shown in Table IV. The p r i n c i p a l compounds which contribute to the i n i t i a l f i s h - l i k e aromas of surimi appear to be the enzymically-derived eight-carbon carbonyls and alcohols in combination with some oxidized fishy aroma undertones that are caused by very low levels of autoxidatively-derived carbonyls, including the 2,4-heptadienals and the 2,4-decadienals (unpublished data). When geranium leaves were macerated before addition to surimi, the six-carbon v o l a t i l e compounds dominated the o v e r a l l aroma, and the desirable contributions associated

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Table IV.

Exploratory applications of plant-derived lipoxygenases for f l a v o r modifications of pollack surimi.

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Surimi Sample Treatment No treatment

Aroma Quality After Incubation Probable Dominant Contributing Compounds (12 h at 4°C) C 7 - , and C i Q - d i e n a l s , Mild, clean fishy C3 Alcohol/ketones

Uncrushed geranium leaves

I n i t i a l l y "marine green"; Then potent fresh trout

1,5-Octadien-3-one, Possibly unknowns

Mascerated mushroom

Masked f i s h i n e s s ; Oyster-like

C3

Mascerated cucumber

Lacks masking e f f e c t ; Green-vine-like

C9 Aldehydes

Alcohols/ketones

with the presence of l,5-octadien-3-one were overwhelmed. However, when uncrushed geranium leaves provided l,5-octadine-3-one to the aroma of fresh surimi, a marine-green aroma often found in fresh lake or ocean breezes was observed upon opening the container. As the sample warmed from 4°C, the marine-green-like aroma was replaced by an aroma that was c h a r a c t e r i s t i c of fresh trout, and t h i s persisted f o r several hours. When mushroom homogenates were incubated with surimi, enhanced p l a n t - l i k e aromas somewhat reminiscent of oysters were produced, and t h i s treatment also resulted in the masking of some of the f i s h - l i k e aromas of the surimi. Cucumber homogenates developed strong cucumber, cardboard-like aromas which appear to be contributed p r i n c i p a l l y by 2-nonenal and 2,6-nonadienal. As a r e s u l t , the cucumber homogenates caused undesirable and unbalanced aromas that did not suppress unpleasant f i s h i n e s s . Watermelon f r u i t extracts behaved s i m i l a r l y , and also provided unbalanced sweet aromas to surimi. Tests to date have been limited to short-term incubations of crude enzyme preparations with surimi. Further exploration of more p u r i f i e d and controlled plant-based flavor-generating enzyme systems for the production of fresh seafood-like aromas, and e s p e c i a l l y those for the eight-carbon v o l a t i l e aroma compounds, appear warrented. Other Future Directions In Seafood Aroma Research Although polyunsaturated f a t t y acids have h i s t o r i c a l l y received the most attention as precursors for the formation of v o l a t i l e s associated with the aromas of seafoods, another source of characterizing f l a v o r s for c e r t a i n seafoods appears to exist in the naturally-occurring carotenoid pigments found in some f i s h t i s s u e . Lipoxygenase can co-oxidize carotenoid pigments while catalyzing the hydroperoxidation of f a t t y acids (92-94), and t h i s reaction has been routinely exploited in bleaching wheat f l o u r

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17. JOSEPHSON AND LINDSAY

215Fish Volatile Aroma Compounds from Fresh

for breadmaking (95). In this respect, some of the unique flavor of cooked salmon now appears to result from selected oxidative reactions involving carotenoid pigments and polyunsaturated fatty acids in the flesh of these fish. Overall, the development of microencapsulation techniques which allow biogenerations of fresh seafood aroma compounds while separating the potentially damaging lipoxygenase-derived hydroperoxides from the bulk food consituents appears to be a promising area of future research. Combinations of such techniques with controllable plant-based enzyme systems seem within technological reach at this time. If fish lipoxygenases can be stabilized against self-inactivation, these enzymes potentially could be exploited also. However, regulating the rate of selective production of important volatile aroma compounds will remain one of the biggest hurdles in developing commercially feasible processes, and every means of controlling the formation of enzymic products should be carefully considered in the next stages of research and development. Acknowledgments This research was supported by the College of Agricultural and Life Sciences and the Sea Grant College Program, Univeristy of Wisconsin-Madison. Literature Cited 1. Obata, Y., Yamanishi, T. and Ishida, M. Bull. Jap. Soc. Sci. Fish. 1950. 15, 551-553. 2. Jones, N.R. In "Symposium on foods: The Chemistry and Physiology of Flavors"; Schultz, H.W., Day, E.A. and Libbey, L.M. Eds.; AVI; Westport, Conn., 1967; pp. 267-295. 3. Meijboom, P.W. and Stroink, T.B.A. J. Am. Oil Chem. Soc. 1972, 49, 555-558. 4. McGill, A.S., Hardy, R., Burt, J.R., and Gunstone, F.D. J. Sci. Food Agric. 1974, 25, 1477-1489. 5. McGill, A.S., Hardy, R., and Gunstone, F.D. J. Sci. Food Agric. 1977, 28, 200-215. 6. Aitken, A. and Connell J.J. In "Effects of Heating on Foodstuffs"; Priestly, J.R. Ed.; Applied Sciences: London, 1979; pp. 219-254. 7. Ikeda, S. In "Advances in Fish Science Technology"; Connell, J.J., Ed.; Fishing News Book; Farnham, Eng. 1980; pp. 111-123. 8. Yu, T.C., Day, E.A., and Sinnhuber, R.O. J. Food Sci. 1961, 26, 192-197. 9. Badings, H.T. J. Am. Oil Chem. Soc. 1973, 50, 334. 10. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. J. Food Sci. 1983, 48, 1064-1067. 11. Badings, H.T. Neth. Milk Dairy J. 1970, 24, 147-256. 12. Swoboda, P.A.T. and Peers, K.E. J. Sci. Food Agric. 1977, 28, 1010-1018.

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13. Seals, R.G. and Hammond, E.G. J. Am. Oil Chem. Soc. 1970, 47, 278-280. 14. Tappel, A.L. Food Res. 1952, 17, 550-559. 15. Tappel, A.L. Food Res. 1953, 18, 104-108. 16. Tappel, A.L. In "Autoxidation and Antioxidants, Vol. 1"; Lundberg, W.O. Ed.; Wiley: New York, 1961, p. 325. 17. Gardner, H.W. J. Agric. Food Chem. 1975, 23, 129-136. 18. Ke, P.J., Ackman, R.G. and Linke, B.A. J. Am. Oil Chem. Soc. 1975, 52, 349-353. 19. Moncrieff, R.W. In "The Chemical Senses"; John Wiley and Sons: New York, 1944; 424p. 20. Ackman, R.G., Hingley, J. and MacKay, K.T. J. Fish Res. Board Can. 1972, 25, 267-284. 21. Whitfield, F.B., Freeman, D.J., Last, J.H., and Bannister, P.A. Chem. Ind. (London) 1981, 5, 158-159. 22. Shiomi, K., Noguchi, Α., Yamanaka, H., Kikuchi, T. and Iida, H. Comp. Biochem. Physiol. 1982, 71B, 29-31. 23. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. J. Agric. Food Chem. 1983, 31, 326-330. 24. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. J. Agric. Food Chem. 1984, 32., 1344-1347. 25. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. J. Agric. Food Chem. 1984, 32., 1347-1352. 26. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. J. Food Sci. 1985, 50, 5-9. 27. Tressl, R., Bahri, D. and Engel, K. -H. J. Agric. Food Chem. 1982, 30, 89-93. 28. Pyysalo, H. and Suihko, M. Leb. Wiss. Tech. 1976, 9, 371-373. 29. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A. "Abstracts of Papers"; The 185th National Meeting of the American Chemical Society, Seattle, WA 1983; American Chemical Society: Washington, DC. AGFD # 28. 30. Whitfield, F.B. and Freeman, D.J. Wat. Sci. Tech. 1983, 15, 85-95. 31. Whitfield, F.B., Freeman, D.J., Last, J.H., Bannister, P.A. and Kennett, B.H. Aust. J. Chem. 1982, 35, 373-383. 32. Kemp, T.R., Knavel, D.E. and Stoltz, L.P. Phytochem. 1971, 10, 1925-1928. 33. Kemp, T.R., Knavel, D.E. and Stoltz, L.P. J. Agric. Food Chem. 1974, 22, 717-718. 34. Kemp, T.R., Knavel, D.E., Stoltz, L.P. and Lundin, R.E. Phytochem. 1974, 3, 1167-1170. 35. Kemp, T.R. Phytochem. 1975, 14, 2637-2638. 36. Suyama, Μ., Hirano, T. and Yamazaki, S. Bull. Jap. Soc. Sci. Fish. 1985, 51, 287-294. 37. Hatanaka, Α., Kajiwara, T. and Harada, T. Phytochem. 1975, 14, 2589-2592. 38. Frazzalari, F.A. (Ed.) In "Compilations of Odor and Taste Threshold Value Data"; American Society for Testing and Materials: Philadelphia, PA, 1978; p. 130. 39. Buttery, R.G. In "Flavor Research-Recent Advances"; Teranishi, R., Flath, R.A.; Sugisawa, H. Ed.; Marcel Dekker: New York, 1981; pp. 193-210.

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17. JOSEPHSON AND LINDSAY

Volatile Aroma Compounds from Fresh217 Fish

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