Enzyme Methods to Assess Marine Food Quality

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Enzyme Methods to Assess Marine Food Quality FREDERICK D. JAHNS and ARTHUR G. RAND, JR. Department of Food Science and Technology, University of Rhode Island, Kingston, R.I. 02881

How fresh is a fresh fish? If we look at Figure 1, perhaps we can i l l u s t r a t e this point! In examining these f i s h , espec i a l l y the one at the bottom, one might conclude "this fish is putrid." However, this evaluation would be incorrect. This fish may not be fresh, but i t has been stored in ice at 0°C for only a few days and certainly is not spoiled. The belly is a l most completely digested in the lower fish but this fish is 6 days old postmortem. It is also possible to have fish only several hours postmortem in the same condition, because they weren't properly iced. While the tissue in the belly region on the lower fish was digested, a cooked fillet of this fish could be quite acceptable to many people. Note that the fish at the top of the picture was caught at the same time and was stored under the exact same conditions as the one at the bottom, yet somehow their appearance is different. It is this type of example that contributes to the elusiveness of evaluating the quality of seafood, particularly in trying to distinguish the fresh and edible stages. The inedible phase is usually quite clear to anyone. There have been many o r g a n o l e p t i c g u i d e l i n e s s e t f o r judgment o f fish q u a l i t y such as t e x t u r e , appearance, c o l o r , eye transparency, and odor. In many cases t h i s approach may give a general i n d i c a t i o n of q u a l i t y , but they are very s u b j e c t i v e (1)

(2). R e a l i z i n g the drawbacks i n using these criteria, and understanding the economic impact o f properly assessing seafood q u a l i t y , many i n v e s t i g a t o r s have set out to f i n d a more r e l i a b l e means. The most l o g i c a l approach was to view the issue through chemical t e s t s . In general most of the chemical t e s t s appear to have v a r i a b l e success, with l i m i t a t i o n s being placed on them by innate b i o l o g i c a l d i f f e r e n c e s from species to s p e c i e s . Some compounds such as trimethylamine, v o l a t i l e nitrogen bases, ammonia, and volatile reducing substances appear mostly i n the l a t t e r stages o f r e f r i g e r a t e d storage, which l i m i t s t h e i r value in that considerable period p r i o r to t h i s time, as i n d i c a t e d by 266 In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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Dugal (3). There are a number o f other techniques, such as an automated determination o f volatile bases (4), the Torry Fish Freshness Meter and the I n t e l e c t r o n Fish Tester (5)(6)(7) which have attempted t o s i m p l i f y q u a l i t y assessment and have met with some success. However, they are not without problems. For example, the Torry meter seems t o be l i m i t e d t o lean f r e s h fish. Frozen or f a t t y f i s h produce somewhat v a r i a b l e r e s u l t s (8). The automated determination o f volatile bases requires a l a b o r a t o r y situation. The c r i t e r i a o u t l i n e d by Shewan & Jones (9) continues t o be a sound b a s i s f o r a marine food q u a l i t y index. The method s e l e c t e d should detect a compound which: 1. W i l l be absent o r present i n constant amount i n f r e s h l y caught food; 2. Should accumulate o r disappear q u i c k l y at a steady rate during post-harvest aging and s p o i l a g e ; 3. Has the c a p a b i l i t y o f being simply and s p e e d i l y estimated. Enzyme methods which have the advantages o f s p e c i f i c i t y and r a p i d i t y meet t h i s basis q u i t e w e l l . The f i r s t enzyme methods to assess marine food q u a l i t y were reported i n 1964 (10)(11); t h i s was an a n a l y s i s f o r the compound hypoxanthine, a metabolic byproduct o f ATP breakdown, employing an enzyme method developed almost two decades before by Kalckar (12). The i n i t i a l work u t i l i z e d ion exchange chromatography to separate the n u c l e o t i d e bases i n a f i s h t i s s u e e x t r a c t . This enzyme technique then employed xanthine oxidase (EC 1.2.3.2) to convert the hypoxanthine f r a c t i o n t o u r i c a c i d , followed by d i f f e r e n t i a l spectrophotome t r y i n the UV f o r a n a l y s i s . There has been ample demonstration of the usefulness o f hypoxanthine as a measure o f f i s h q u a l i t y

in several papers (3)(10)(13)(14)(15).

Burt e t a l . (16) proposed a method o f automating the xant h i n e oxidase r e a c t i o n f o r hypoxanthine a n a l y s i s using the redox dye 2,6 dichlorophenolindophenol (DIP). This placed the method on a c o l o r i m e t r i c b a s i s , s i m p l i f i e d the equipment requirements, and increased sample c a p a c i t y . Figure 2 i l l u s t r a t e s the i n t e r a c t i o n between hypoxanthine, DIP, and xanthine oxidase. The hypoxanthine concentration can be r e l a t e d to the extent o f dec o l o r i z a t i o n o f the dye. The c o r r e l a t i o n between the automated c o l o r i m e t r i c methods, measuring at 600 nm using DIP, and the manual enzyme method measuring u r i c a c i d appearance a t 290 nm was e x c e l l e n t (16). Beuchat Q5) u t i l i z e d DIP i n a manual enzyme assay t o measure hypoxanthine d i r e c t l y i n a deproteinated e x t r a c t , and c o r r e l a t e d t h i s method with an o r g a n o l e p t i c e v a l u a t i o n o f c a t f i s h (Ictalurus punctatus). Jahns e t a]_. (17) a l s o used a s l i g h t l y modified xanthine oxidase-DIP method t o monitor hypoxanthine concentration i n iced Winter Flounder (Pseudopleuronectes americanus). A steady increase i n hypoxanthine concentration occurred with storage time u n t i l the values l e v e l e d o f f a t about 5 yM/g a f t e r 10 days where o r g a n o l e p t i c e v a l u a t i o n i n d i c a t e d the

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure 1. The appearance of red hake after six days of storage in ice (0°C)

DIP reduced

DIP oxidized BLUE Figure 2.

The reduction of DIP by xanthine oxidase in the presence of hypoxanthine


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f i s h were no longer f r e s h .


Semi-automated hypoxanthine

analysis

The manual xanthine oxidase c o l o r i m e t r i c method has been f u r t h e r modified f o r quick routine a n a l y s i s in the laboratory by employing an automatic p i p e t and a Spectronic 20 c o l o r i m e t e r , o u t f i t t e d with a f l o w - t h r u c u v e t t e . B r i e f l y , the method i s as follows: 1. A 10 g sample of f i s h i s blended f o r one minute with 90 ml of c o l d 0.6N p e r c h l o r i c a c i d . 2. The homogenate i s then f i l t e r e d through Whatman #2 paper to remove p r o t e i n and t i s s u e d e b r i s . 3. A 10 ml a l i q u o t of the f i l t r a t e i s n e u t r a l i z e d to pH 7.6 with 20% Κ0Η and buffered with 0.1M Phosphate b u f f e r , pH 7 . 6 , making the volume up to 100 ml. 4. Approximately 12 ml i s added to a c e n t r i f u g e tube. This i s c h i l l e d in a f r e e z e r u n t i l a layer of i c e begins to form at the top of the tube. 5. The sample i s then c e n t r i f u g e d i n a c l i n i c a l c e n t r i f u g e f o r approximately 3 minutes to remove KC10-. 6. The c e n t r i f u g a t e i s poured o f f and allowed to temper to 25°C. One ml i s used in the a n a l y s i s f o r hypo xanthine, and contains 10 mg of f i s h . 7. A 4 ml p o r t i o n of buffered xanthine oxidase-DIP s o l u t i o n at 25 C i s i n j e c t e d i n t o the 1 ml sample of f i s h e x t r a c t with an automatic p i p e t . The enzyme reagent i s comprised of 60 ml of 0.15M phosphate b u f f e r (pH 7.6) containing DIP @ 23 yg/ml, and 20 ml of xan thine oxidase s o l u t i o n @ 0.02-0.025 units per ml. 8. The absorbance at 600 nm i s read in the f l o w - t h r u cuvette o f the Spectronic 20 at e x a c t l y 2.5 min. The absorbance change i s compared against a standard p l o t obtained by analyzing known hypoxanthine concentra tions. Employing t h i s method we were able to assess the hypoxan thine values found in Whiting (Merluccium b i l i n e a r i s ) during i c e d storage, as shown in Figure 3. Hypoxanthine was monitored over a 32 day p e r i o d , in order t o evaluate concentration well past the f r e s h and acceptable p e r i o d s . Levels of hypoxanthine increased to a maximum of 6 yM/g between 17 and 21 days. It then grad u a l l y declined by 32 days to s l i g h t l y above 2 yM/g. Figure 4 depicts the r e s u l t s obtained from two separate t r i a l s using Tautog (Tautoga o n i t e s ) . Both t r i a l s showed very s i m i l a r r e s u l t s e x h i b i t i n g maximum hypoxanthine values near 3 yM/g at from 12-14 days in i c e d s t o r a g e . T r i a l one was caught in November and T r a i l two was taken during December. The values again diminish a f t e r peak hypoxanthine formation.



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TIME (DAYS) Figure 3. Hypoxanthine concentration in whiting during iced (0°C) storage. Each point represents the mean value for two fish.

TIME(DAYS)

Figure 4. Hypoxanthine concentrations in tautog during two iced (0°C) storage trials. Each point in trial one represents onefish,while trial two shows the mean value for two fish.


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Immobilized xanthine oxidase as an a n a l y t i c a l


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tool

Another laboratory method f o r hypoxanthine a n a l y s i s , which i s s t i l l i n the p r e l i m i n a r y stages o f development, i s the use of an immobilized xanthine oxidase column. Very few reports are a v a i l a b l e on the immobilization o f xanthine oxidase. Coughlan and Johnson (18) f i r s t reported on methods of preparation d e s c r i b ing attachment to c e l l u l o s e , Sepharose and g l a s s . However, they u t i l i z e d these preparations only to study the f l a v i n chromophore of the enzyme. P e t r i H i (19) and Singer and Warchut (20), working i n our l a b o r a t o r y , studied xanthine oxidase immobilization i n acrylamide gel and on glass and D u o l i t e * beads as a method f o r measuring hypoxanthine c o n c e n t r a t i o n . The acrylamide gel entrapment demonstrated the p o t e n t i a l of immobilized xanthine oxidase f o r hypoxanthine a n a l y s i s , but the enzyme was unstable during continuous a n a l y t i c a l use. In a comparison of xanthine oxidase immobilization on Corning z i r c o n i a - c l a d s i l i c a glass and D u o l i t e beads, maximum enzyme attachment occurred i n a s h o r t e r time with the glass than the D u o l i t e . However, the D u o l i t e appeared to r e t a i n higher enzyme a c t i v i t y of the two supports, and thus was chosen to prepare an experimental plug flow r e a c t o r f o r hypoxanthine a n a l y s i s . The immobilized enzyme column was connected to a pump and a continuous flow UV analyzer equipped with a recorder as presented i n Figure 5. The buffered s u b s t r a t e , which could be e i t h e r a sample o f f i s h e x t r a c t , o r a known hypoxanthine s o l u t i o n f o r c a l i b r a t i o n , was pumped i n t o the column. The e q u i l i b r i u m absorbance value f o r the product of u r i c a c i d can be d i r e c t l y r e l a t e d to hypoxanthine c o n c e n t r a t i o n , as i l l u s t r a t e d , over a range o f 0-30 yg/ml. This enzyme d e r i v a t i v e e x h i b i t e d remarkable s t a b i l i t y when u t i l i z e d repeatedly over a period of weeks, and r e t a i n e d high a c t i v i t y during months of r e f r i g e r a t e d storage. This a n a l y t i c a l t o o l has great p o t e n t i a l i n r o u t i n e monit o r i n g o f f i s h and marine food q u a l i t y . The equipment and r e agent requirements are minimal i n comparison to s i m i l a r approaches ( 4 ) ( 1 6 ) . The automatic processing and recording of r e s u l t s allows the i n v e s t i g a t o r time t o prepare samples and e l i m i n a t e s constant manual operation which i s g e n e r a l l y involved i n the standard c o l o r i m e t r i c procedure. Diamine-oxidase as an i n d i c a t o r of marine food s p o i l a g e An enzymatic spoilage t e s t that would help to c l a r i f y the hypoxanthine freshness a n a l y s i s f o r a given sample of f i s h was investigated. S p i n e l l i e t a l . (10) compared the pattern of hypoxanthine formation i n f i s h with some other t e s t s , i n c l u d i n g Total V o l a t i l e Bases (TVB). T h e i r r e s u l t s i n d i c a t e d the TVB *Duolite i s a phenolic-formaldehyde r e s i n product of Diamond Shamrock Co.


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HYPOXANTHINE (/ig/ml) Figure 5.

Immobilized xanthine oxidase for the analysis of hypoxanthine

ο HYPOXANTHINE

^

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 32 TIME (DAYS)

Figure 6. Hypoxanthine concentration and diamine presence in red hake during iced (0°C) storage. Each point represents the mean value for two fish.



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values d i d not begin to measurably increase u n t i l the hypoxan t h i n e development had peaked. The composition of the TVB material undoubtedly includes diamine compounds, which are produced by m i c r o b i a l s p o i l a g e (2J.). A recent report by Mietz and Karmas (22) s u c c e s s f u l l y f r a c t i o n a t e d and quantitated the diamines from tuna by high-pressure l i q u i d chromatography. T h e i r r e s u l t s confirmed that p u t r e s c i n e , cadaverine, and h i s t a mine a l l increased in concentration as the f i s h reached unaccep table levels. Thus, a n o n - s p e c i f i c enzyme t e s t f o r diamines could i n d i c a t e i f these compounds were present and c l a r i f y whether the hypoxanthine a n a l y s i s was i n d i c a t i n g f r e s h or s t a l e fish. A method f o r determining diamines with the enzyme diamine oxidase (EC 1 . 4 . 3 . 6 ) coupled to the o - t o l i d i n e - p e r o x i d a s e c o l o r i m e t r i c system was developed. The procedure e s t a b l i s h e d i s as follows: 1. A 25 g sample of f i s h i s blended f o r one minute with 75 ml of c o l d 0.6N P e r c h l o r i c A c i d . 2. This i s f i l t e r e d through a Whatman #2 paper. 3. A 25 ml p o r t i o n of the f i l t r a t e i s adjusted to pH 8.5 with 20% KOH and made up to 50 ml with 0.1M borate b u f f e r , pH 8 . 5 . 4. About 12 ml i s placed in a c e n t r i f u g e tube and allowed to c h i l l in a f r e e z e r u n t i l a layer of i c e j u s t begins to form on t o p . 5. The c h i l l e d sample i s c e n t r i f u g e d f o r approximately 3 min in a c l i n i c a l c e n t r i f u g e . 6. The c e n t r i f u g a t e was poured o f f and allowed to temper to ambient temperature. Each 1 ml contains 0.125 g of fish. 7. To 1.0 ml of the c e n t r i f u g a t e , 2.0 ml of diamine o x i dase reagent i s added with an automatic p i p e t , and the mixture i s incubated at 37 C f o r 30 min. The diamine oxidase reagent c o n s i s t s o f - - 1 0 ml of diamine oxidase s o l u t i o n @ 4 units/ml of 0.1M borate b u f f e r (pH 8 . 5 ) , 5 ml of o - t o l i d i n e d i h y d r o c h l o r i d e s o l u t i o n @ 500 yg/ml o f water, and 5 ml o f peroxidase s o l u t i o n @ 226 units/ml of pH 8.5 borate b u f f e r (0.1M). 8. The c o l o r development i s determined on a Spectronic 20 Colorimeter equipped with a f l o w - t h r u c u v e t t e . The r e s u l t s are evaluated with a standard curve prepared from the a n a l y s i s of known putrescine c o n c e n t r a t i o n s . Since the purpose of the diamine t e s t was only to support hypoxanthine a n a l y s i s , the r e s u l t s have been presented only in terms of whether diamines were present or not. Figure 6 presents a graph comparing the pattern of hypoxanthine concentration and diamine values in iced Red Hake (Urophycis chuss). In Red Hake the hypoxanthine values increased to a peak of approximately 4 μΜ/g at 17-18 days followed by the usual downward t r e n d . Posi t i v e diamine values were found only in samples more than 18


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days o l d , or a f t e r the hypoxanthine peak. The hypoxanthine and diamine a n a l y s i s procedures were a l s o compared on iced Winter Flounder, as shown in Figure 7. The c h a r a c t e r i s t i c r i s e and drop in hypoxanthine concentration with storage time i s again evident. The peak value of 7.5 yM/g occurred at approximately 11 days postmortem. In a pattern almost i d e n t i c a l to Red Hake, diamines in winter flounder were detected a f t e r the storage time f o r peak hypoxanthine development. Thus, the diamine a n a l y s i s does show value as a s p o i l a g e i n d i c a t o r with the f i s h t e s t e d . Combining the two enzyme procedures, xanthine oxidase f o r hypoxanthine c o n c e n t r a t i o n , and diamine oxidase f o r diamine detection, can c l a r i f y the i n t e r p r e t a t i o n of f i s h q u a l i t y . Figure 8 i l l u s t r a t e s how these two t e s t s g e n e r a l l y i n t e r a c t .


m

Rapid v i s u a l enzyme method The methods that have j u s t been discussed were designed f o r quick q u a n t i t a t i v e a n a l y s i s in the l a b o r a t o r y . However, the greatest need e x i s t s f o r a t e s t of marine food q u a l i t y that w i l l be simple, inexpensive, and lend i t s e l f to usage outside of the l a b o r a t o r y , e i t h e r on board s h i p , dockside, or in the food processing plant. Jahns et al_. (17) proposed a method f o r estimat i n g f i s h freshness which makes use of a s t r i p of absorbent paper impregnated with r e s a z u r i n , xanthine oxidase, and b u f f e r combination. When the enzyme t e s t s t r i p was moistened with flounder e x t r a c t s and allowed to react f o r 5 min at room temperature, the c o l o r change from blue to pink c o r r e l a t e d with the actual l a b o r a t o r y c o l o r i m e t r i c hypoxanthine a n a l y s i s of the same e x t r a c t s . This i s i n d i c a t e d in Figure 8 by the b l u e - p i n k - b l u e progression of c o l o r . As already i n d i c a t e d the hypoxanthine method i s b a s i c a l l y a freshness t e s t . I f the t e s t s t r i p was set to turn pink at a hypoxanthine concentration of 5 yM/g or 70 mg%, which would be an average value f o r the f i v e f i s h analyzed in t h i s study, then a simple c o l o r change from blue to a shade of pink could i n d i cate when f i s h can no longer be considered f r e s h . However, t h i s t e s t does not seem to be useful in estimating s p o i l a g e in s e a food. In the r e s u l t s obtained, the hypoxanthine concentration in a l l f i s h studied always begins to diminish a f t e r the f r e s h ness storage p e r i o d . This makes i t d i f f i c u l t , i f not impossible, to equate hypoxanthine concentration with storage time beyond 10-15 days in iced c o n d i t i o n s . In f a c t , the c o l o r changes from pink back toward b l u e , as the f i s h ages, could be m i s i n t e r p r e t e d . A second v i s u a l t e s t to i n d i c a t e spoilage would compliment the hypoxanthine enzyme t e s t s t r i p . Since diamines appear to develop a f t e r the hypoxanthine peak in a s i g n i f i c a n t q u a n t i t y , another v i s u a l t e s t could be employed on the same s t r i p as i n d i c a t e d in Figure 8. Essent i a l l y the hypoxanthine-diamine i n t e r a c t i o n can be d i v i d e d i n t o three c a t e g o r i e s , as i l l u s t r a t e d in Figure 9. Marine foods could


JAHNS

AND


RAND

Δ HYPOXANTHINE + DIAMINES DETECTED - DIAMINES NOT DETECTED

_


-

\ +

-

Δ

,/ Δ

Δ

+ ^ ^ - ^ ^

Δ

-

Δ 2 •

2

4

6

8

ι

ι

ι

ι

ι

ι

ι

ι

ΙΟ 12 14 16 18 20 22 24 26 28 30 32 TIME (DAYS)

Figure 7. Hypoxanthine concentration and diamine presence in winter flounder during iced (0°C) storage. Each point repre sents the results for at least two fish.



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f

/I/J

fill

DIAMINE

PINK

BLUE

HYPOXANTHINE

11IIIJ/11

lli/llllli

7

'

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BLUE

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Figure 9. The three phases of rapid visual enzyme analysis of marine foods for freshness (hypoxanthine) and spoilage (diamines)



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be simply and q u i c k l y e v a l u a t e d , based on the r e a c t i o n c o l o r s . Class 1 would be the f r e s h food of highest q u a l i t y , where hypoxanthine and diamine l e v e l s are low, i n d i c a t e d as blue i n the t e s t zones. Class 2 would be intermediate q u a l i t y i n which food i s acceptable but not f r e s h . Hypoxanthine l e v e l s are h i g h , i n d i cated by the pink t e s t zone and diamines are s t i l l low, with a blue t e s t zone. Class 3 occurs when hypoxanthine l e v e l s are now lower and a blue t e s t zone, while diamine l e v e l s have i n c r e a s e d , as i n d i c a t e d by the pink t e s t zone. Marine foods i n the 3rd c l a s s would be c l a s s i f i e d as marginal or not acceptable. It may even be p o s s i b l e to s u b - d i v i d e the 3 t e s t categories f u r t h e r by shades o f blue or pink to increase the s e n s i t i v i t y o f grading. Perhaps even a t h i r d i n d i c a t o r to produce a multi-phase s t r i p might be employed to v i s u a l l y i n d i c a t e the presence of t r i methylamine. This might be accomplished by employing the enzyme t r i m e t h y l ami ne dehydrogenase, which Large and McDougall (23) suggested could be used f o r the microestimation of t r i m e t h y l ami ne. When these enzyme t e s t s t r i p s are f i n a l l y employed i t should be p o s s i b l e to use these d i r e c t l y on the f o o d , employing the t i s s u e f l u i d to wet the t e s t zones. This would e l i m i n a t e the need to sample and prepare a p r o t e i n - f r e e e x t r a c t . The a p p l i c a t i o n of enzyme methods to marine food a n a l y s i s can provide o b j e c t i v e , inexpensive and r a p i d approaches to the assessment of seafood q u a l i t y . Adaptation of these methods to a v i s u a l basis permits extension of enzyme a n a l y s i s t o non-laborat o r y s i t u a t i o n s , such as dockside or on board s h i p s . Thus, enzymatic a n a l y s i s o f marine foods can be u t i l i z e d f o r q u a l i t y assessment at c r i t i c a l p o i n t s , where t h i s type of determination can have maximum impact f o r grading and purchase. Acknowledgements The authors would l i k e to acknowledge the c o n t r i b u t i o n s of James A. Hourigan, Richard J . C o d u r i , J r . and J e f f r e y L. Howe who provided encouragement and t e c h n i c a l a d v i s e . This research was supported i n part by the College of Resource Development, U n i v e r s i t y of Rhode I s l a n d ; by the Cooperative State Research S e r v i c e , U.S. Department of A g r i c u l t u r e ; and by the NOAA O f f i c e of Sea Grant, U.S. Department of Commerce under grants 04-6-158-44002 and 04-6-158-44085. Contribution No. 1709 of the Rhode Island A g r i c u l t u r a l Experiment S t a t i o n .

Literature cited 1.

Farber, L. G. in "Fish as Food" Vol. 4, pg. 65, G. Borgstrom, ed. Academic Press, NY, 1965.

2.

H i l l i g , F . , Shelton, L. R., J.A.O.A.C. (1962) 45, 724.

Jr.,

and Loughrey, J . H . ,



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