A comparison between conventional and fluorescence detection methods of cookinginduced damage to tuna fish lipids
Comparison of lipid damage measures with fluorescence shift during cooking of tuna fishes
Santiago Aubourg, Isabel Medina and Ricardo Pérez-Martín
Instituto de Investigaciones Marinas (CSIC) c/Eduardo Cabello, 6
36208-VIGO (Spain)
FAX: 34-86-292762
Running title: Lipid damages and fluorescence shift
Keywords: Tuna fishes, lipid damages, hydroperoxides, carbonyls, interaction compounds, fluorescence shift
Vergleich von Lipidschädenmaßnahme und Fluorescenzverschiebung während des Kochens von tuna Fische
ZUSAMMENFASSUNG

2
Lipidschäden während des Abkochens von zwei tuna Fische (Thunnus obesus und
Thunnus thynnus) wurden untersucht. Fluorescenzmeßung in verschiedenen Wellenlängen wurde mit anderen Methoden verglichen, die in der Bestimmung der Lipidschäden gebraucht werden. Als Ergebnis der Wärmebehandlung wurde eine Fluorescenzverschiebung zu höheren
Wellenlängenmaxima offenbar; gleichzeitig zeigten die Qualitätsanzeichen eine vergrößerte
Lipidbeschädigung (Karbonils- und Bräunungsentwicklung, sowie Zunahme von freien
Fettsäuren). Eine besondere Aufmerksamkeit wurde auf dem Fluorescenzsverhältnis zwischen
393/460 und 327/415 nm Maxima gewährt. Vermehrungen in diesem Verhältnis während des
Kochens zeigten weniger Abhängigkeit bei den verschiedenen Lipidmustern als bei den anderen Lipidschädensmeßungen.
ABSTRACT
Lipid damages during big eye and bluefin tunas cooking were investigated.
Measurement of fluorescence at different excitation/emission maxima was compared to other methods employed in determining lipid alterations. As a result of processing, a fluorescence shift to higher wavelengths was observed at the time that quality indexes indicated an increased lipid damage (carbonyl formation, browning development and increase in free fatty acid content). A special stress was given to the fluorescence ratio between 393/460 and
327/415 nm maxima; increases in this ratio as a result of cooking showed to be less dependent on samples than the other lipid damage measures.
INTRODUCTION

3
Marine foods possess a lipid composition constituted by a high content of polyunsaturated fatty acids (PUFA) (1). This unsaturated composition may lead to many undesirable changes caused by storage and/or processing with a special incidence over the final product quality (2-4).
Lipid degradation has been followed by a large quantity of methods focused mainly in order to assert the primary (hydroperoxides) and secondary (carbonyl compounds) oxidation products (5, 6). It has been proved that both kinds of damage products may react with aminated food constituents (proteins, peptides, free aminoacids, phospholipids) to produce fluorescent compounds, browning and flavour compounds (7-12). The study of such interaction structures by fluorescence determination has become a common complementary way of measuring lipid damages (13-15); however, experiences carried out account only for studies undergone at a single excitation/emission maximum.
Previous experiences of our research group have been aimed at the fluorescence formation in fish samples at different excitation/emission maxima. A model system study between acetaldehyde and albacore tuna muscle led to a fluorescence shift as a result of increasing acetaldehyde content and time and temperature of reaction (16). Further, a fluorescence analysis during albacore canning at different sterilization conditions revealed a fluorescence shift to higher wavelength maxima that was specially great in the case of the most energic sterilization treatment (17).
On the basis of both previous experiences the present work was aimed to compare this fluorescence shift with traditional lipid damage indexes. Changes occurred after cooking in two different tuna species (big eye and bluefin) were studied. The fluorescence shift to higher wavelength maxima was compared with methods of measuring lipid hydrolysis (free fatty acid content) and oxidation (conjugated diene and carbonyl content) and interactions between damaged lipids and other biological molecules (browning development).

4
MATERIALS AND METHODS
1. Raw material and processing
Two commercial tuna species were employed: big eye (Thunnus obesus) and bluefin
(Thunnus thynnus). The fish were purchased at a commercial centre. Upon arrival to our laboratory, the fish were kept frozen at -20°C prior to analysis.
The fish were beheaded and eviscerated. Five big eye (BE) fishes and four bluefin (BF) fishes were selected. Steam cooking was performed in our pilot plant (102-103 °C) to a final backbone temperature of 65°C; the fish were then cooled at room temperature (14°C) for about five hours.
Individual fishes were considered separately. An individual study of changes occurred during cooking was undergone in order to compare the evolution of changes measured by each of the methods employed along the present experience.
2. Lipid extraction
Lipids were extracted from raw and cooked samples by a mixture of chloroformmethanol-water following the Bligh and Dyer method (18) and were then stored at -20°C until analysis.
3. Fluorescence measurements
The excitation and emission spectra of the lipid extracts were determined using a
Perkin-Elmer LS 3B Fluorescence spectrophotometer. Measurements were made at the following excitation/emission maxima: 327/415, 356/416, 393/463, 446/490 and 479/516 nm.
Quinine sulphate solution (1
 g/mL in 0.05 M H2SO4) was used as standard of fluorescence intensity (S). The fluorescence of the sample relative to the standard was calculated using the following formula (19):
Fluorescence (mL/mg) = F x V / S x w

5 where V denotes the volume of the extract (5 mL) giving fluorescence F, and w is the weight
(mg) of the lipid sample employed.
4. Spectrophotometric measurements
All spectrophotometric determinations were carried out in a Beckman DV-64 spectrophotometer.
The content of free fatty acids (FFA) was determined by the Lowry and Tinsley (20) method, based on a complex formation with (AcO)2Cu-pyridine. Measurement of brown colour formation in lipid extracts was achieved at 420 nm (21, 22). Conjugated diene formation was followed at 233 nm (6). Carbonyl development was measured following the
Chiba et al. (23) method.
Results shown in Table 3 for conjugated diene and carbonyl determinations were calculated using the following formula (19):
Absorbance = B x V / w where B is the absorbance reading obtained, V denotes the volume of the sample (5 mL) and w is the weight (mg) of the lipid extract measured. In the case of brown colour formation, the same formula was employed, although Table 3 shows 1000 x Absorbance values.
RESULTS AND DISCUSSION
Fluorescence spectra of the different samples were studied. According to previous experiences, different excitation/emission maxima were determined. Values obtained for the

6 maximum 479/516 nm were very low, and were not considered in order to discuss results in the present paper. This maximum had showed to be interesting in the model system between acetaldehyde and tuna muscle (16). However, the present experience accounts for a real processing and progress in the interaction between oxidated lipids and biological aminated compounds has not been so large.
Table 1 shows the different values obtained in four excitation/emission maxima for raw and cooked samples. As a result of thermal processing a general increase in the four excitation/emission maxima has been found. As a general rule, increases showed to be bigger the higher the wavelength maxima being considered.
In the mentioned model system (16) between albacore muscle and acetaldehyde, a fluorescence shift to higher wavelength maxima was obtained as a result of acetaldehyde concentration, time and temperature of reaction. In another previous experience the fluorescence formation during albacore canning was followed (17); results indicated that the most drastic the sterilization step the highest increases in fluorescence were obtained, specially in the highest excitation/emission maxima.
Solid sample spectrofluorometry has been employed in order to assert oxidative deterioration in fish samples (24). For it, two kinds of fluorescence measures were undergone
(370/460 and 450/500 nm); according to our results, fluorescence intensity of the second maximum increased with progress of oxidation while the first one remained unchanged.
With the aim of observing the fluorescence shift to higher wavelength maxima ratios between fluorescence contents were studied (Table 2). It could be concluded that as a result of cooking fluorescence formation has been developed and mainly in the case of fluorescence wavelength of the highest values. Increases in the two highest excitation/emission maxima were particulary high. However, and according to the degree of damage of the actual cooking process (a real technological process), the F3/F1 ratio showed to be the most sensible, and was therefore chosen for further comparations. This fluorescence ratio had already been found useful by indicating significant differences between raw, cooked and sterilized lipid samples of albacore tuna (17).

7
The fluorescence shift to higher excitation/emission maxima, agrees with the general theory of lipid damages and the progressive formation of Schiff bases and other interaction products with increasing molecular weights and unsaturation bounds (7, 8, 25, 26). For a long time it had been assumed that malondialdehyde (MDA) played the main role in the formation of fluorescent compounds (8, 27) but many recent works have proved that other carbonyl compounds (unsaturated and with oxigenated functions, mainly) may participate in the fluorescence formation with a higher contribution than MDA itself (9, 10, 28, 29).
In order to compare with the fluorescence results obtained, traditional methods of measuring lipid hydrolysis (content on free fatty acids, FFA) and oxidation (diene and carbonyl levels), as well as further oxidated lipid interactions (browning development) were analyzed. All these results, as well as the lipid content are summarized in Table 3.
Conjugated diene content did not show a general pattern. In the case of big eye tuna a general decrease was observed as a result of cooking; however, a not defined result was obtained for bluefin tuna. In previous works (5, 30) the assessment of primary oxidation products (hydroperoxides) showed not to be sensible enough in order to measure lipid damages during thermal treatments. Peroxides would break down very quickly and interact with other biological compounds (31).
The carbonyl and browning evolution showed an increase in both kinds of compounds as a result of cooking. Shimasaki et al. (32) showed that during lipid oxidation, the amount of conjugated diene hydroperoxides decreased, while the TBA numbers and the formation of fluorescent products increased. Browning development has succesfully been employed for a long time as a way of measuring interactions between oxidated lipids and biological molecules (8, 22, 33).
The analysis of FFA content has also demonstrated to be very sensitive (34). In our results (Table 3) an increase from raw to cooked samples was obtained. Starting samples showed different FFA levels (low: BF3; medium: BE4, BF1, BF2 and BF4; and high: BE1,
BE2, BE3 and BE5) indicating different material quality.
It can be concluded that the fluorescence shift to higher wavelength maxima has been accompanied (according to Table 3 results) by an increase in FFA content (lipid hydrolysis),

8 carbonyl compounds development (lipid oxidation) and browning (interaction compounds formation).
The behaviour of each quality method along the different individual fishes was analyzed. Table 4 shows the ratios between the cooked and raw measurements for each of the methods employed, including the F3/F1 ratio. Mean values, standard deviations and variation coefficients have been calculated in order to assert the variation of the measure method along the nine individual fishes.
Variations in the case of dienes were very large, supporting the idea that it is not a sensitive enough method to follow lipid alterations during thermal treatments. Browning development showed a stated increase although large variations were obtained between the different samples. Best results were obtained by the FFA and carbonyls evaluations. Both showed large increases and variation coefficients smaller than the former analyses.
The fluorescence shift (F3/F1 ratio) was also evaluated. Its variation coefficient along the nine samples studied showed the lowest value, and proved that evolution of this measure along cooking was less dependent on the initial sample than the other lipid damage measures.
For many years, fluorescence development has been considered as a complementary way of measuring lipid damages. However, bibliography accounts for experiences where a fluorescence measure at a single excitation/emission maximum is undergone. Results obtained herein reinforce the role of fluorescence shift measurements as a fast and valid test of quality control in heat treated foods.
Further studies by comparing the fluorescence shift (F3/F1 ratio mainly) with nonlipidic quality measures (sensoric evaluation, total volatile bases, aliphatic and biogenic amines, and so on) along a complete processing (frozen storage, cooking and sterilization) are necessary in order to evaluate the significance of the fluorescence shift as a valid method for damage control in fish samples. A special stress will be given to the fluorescence shift comparison between frozen storage conditions and thermal treatments.
Another interesting aspect of studying the fluorescence formation is the close relation between these interaction compounds formed during storage and/or processing of foods and pigmented and fluorescent granules found in human and animal tissues (lipofuscin); these

9 compounds are supposed to be indicative of a degenerative process in cells such as ageing, environmental stress and vitamin E defficiency (35, 36).
ACKNOWLEDGMENT
We acknowledge financial support for Research Project ALI 90-0773 (1991-93) provided by the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Reserach
Project CEE UP.2.571 (of DG XIV) (1991-93).

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REFERENCES
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15. Miyagawa, K., Hirai, K. and Takezoe, R. (1991). J. Am. Oil Chem. Soc. 68: 163-166.
16. Aubourg, S., Pérez-Martín, R., Medina, I. and Gallardo, J. (1992). J. Agric. Food Chem.
40: 1805-1808.
17. Aubourg, S., Pérez-Martín, R., Medina, I. and Gallardo, J. (1992). Z. Lebensm. Unters.
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18. Bligh, E. and Dyer, W. (1959). Can. J. Biochem. Physiol. 4: 169-174.
19. Smith, G., Hole, M. and Hanson, S. (1990). J. Sci. Food Agric. 51: 193-205.
20. Lowry, R. and Tinsley, I. (1976). J. Am. Oil Chem. Soc. 53: 470-472.

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21. Kato, Y., Matsuda, T., Kato, N., Watanabe, K. and Nakamura, R. (1986). J. Agric. Food
Chem. 34: 351-355.
22. Labuza, T. and Massaro, S. (1990). J. Food Sci. 55: 821-826.
23. Chiba, T., Takazawa, M. and Fujimoto, K. (1989). J. Am. Oil Chem. Soc. 66: 1588-1592.
24. Hasegawa, K., Endo, Y. and Fujimoto, K. (1992). J. Food Sci. 57: 1123-1126.
25. Cho, R., Okitani, A. and Kato, H. (1986). In "Amino-carbonyl reaction in food and biological systems. Developments in Food Science 13. Editors: M. Fujiyaki, M.
Namiki and H. Kato. Kodansha Ltd. (Tokyo) and Elsevier.
26. Kikugawa, K. and Beppu, M. (1987). Chem. Phys. Lipids 44: 277-297.
27. Tappel, A. (1980). In "Free radicals in Biology" (ed. by W. Pryor). Academic Press, New
York.
28. Esterbauer, H., Koller, E., Slee, R. and Koster, J. (1986). Biochem. J. 239: 405-409.
29. Aubourg, S. (1993). Int. J. Food Sci. Technol. 28: 323-335.
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345.
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Technol. Vol. 1, pp.: 217-223.
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(ed. by W. Pryor). Academic Press, New York.
36. Sohal, R. (1987). Adv. Biosci. 64: 85-91.

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TABLE 1.- Fluorescence measurements* of raw (R) and cooked (C) samples at four different excitation/emission wavelength maxima
_______________________________________________________________
Sample
327/415
_________
356/416
_________
393/463
_________
446/490
__________
R C R C R C R C
_______________________________________________________________
BE1**
BE2
BE3
BE4
BE5
BF1**
BF2
BF3
BF4
0.60 1.43 0.63 1.78 0.33 1.23 0.12 0.41
0.59 1.48 0.60 1.73 0.31 1.21 0.11 0.49
0.61 1.14 0.76 1.86 0.52 1.28 0.20 0.49
0.30 0.97 0.40 2.16 0.26 1.57 0.10 0.39
0.63 1.68 0.65 2.45 0.41 1.78 0.15 0.64
0.48 0.53 0.62 0.73 0.33 0.57 0.10 0.19
0.46 1.49 0.58 2.25 0.34 1.84 0.11 0.76
0.18 0.76 0.17 1.01 0.10 0.69 0.03 0.26
0.33 1.01 0.46 1.55 0.29 1.13 0.10 0.42
_______________________________________________________________
* Obtained as expressed in the "Materials and Methods" section.
** BE 1-5 : five individual fish samples of big eye tuna; BF 1-4: four individual fish samples of bluefin tuna.

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TABLE 2.- Ratios between fluorescence measurements in raw (R) and cooked (C) samples
_______________________________________________________________
Sample
F2/F1*
_________
F3/F1*
_________
F4/F1*
__________
R C R C R C
_______________________________________________________________
BE1**
BE2
BE3
BE4
BE5
BF1**
BF2
BF3
BF4
1.05 1.24
1.02
1.33
1.29
0.94
1.17
1.25 1.63
2.23
1.03 1.46
1.38
1.26 1.51
1.33
1.39 1.53
0.55
0.53
0.85
0.88
0.65
0.68
0.74
0.57
0.86
0.82
1.12
1.12
1.06
1.08
1.24
0.91
0.89 1.12
0.20
0.19
0.34
0.33
0.24
0.21
0.23
0.18
0.29
0.33
0.43
0.40
0.38
0.36
0.51
0.35
0.31 0.42
_______________________________________________________________
* F1, F2, F3 and F4 are fluorescence measurements at 327/415, 356/416, 393/463 and
446/490 nm, respectively.
** Name of samples as specified in Table 1.

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TABLE 3.- Lipid measurements* in raw (R) and cooked (C) samples
______________________________________________________________________
LC
_________
FFA Dienes
__________ _________
Carbonyls
_________
Browning
___________
Sample R C R C R C R C R C
______________________________________________________________________
BE1**
BE2
BE3
BE4
BE5
BF1**
BF2
BF3
BF4
0.65 0.50 15.24 32.04 4.33 2.74 0.23 0.73 15.03 24.00
0.60 0.33 15.54 27.41 4.55 3.06 0.28 0.83 15.72 44.78
0.65 0.47 18.19 23.16 5.68 2.20 0.48 1.00 19.81 22.39
1.37 1.08 7.29 12.89 4.77 2.23 0.31 1.22 9.46 17.11
0.54 0.33 14.31 32.04 5.66 3.25 0.32 1.14 16.67 30.60
0.65 0.58 7.01 9.51 1.61 1.43 0.37 1.35 9.56 27.57
0.60 0.33 9.96 23.53 1.42 3.35 0.31 1.25 12.72 52.99
1.79 1.25 3.96 13.60 1.72 1.77 0.15 0.56 3.75 16.67
0.77 0.42 8.93 20.14 1.37 3.31 0.33 0.75 9.79 22.89
______________________________________________________________________
* LC: lipid content, expressed as g/100g wet muscle; FFA: free fatty acids, expressed as g/100g lipids. Dienes, carbonyls and browning were measured as expressed in the
"Materials and Methods" section.
** Name of samples as specified in Table 1.

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TABLE 4.- Ratios betweeen cooked and raw measurements in the different damage indexes
_________________________________________________________________
Sample FFA* Dienes Carbonyls Browning F3/F1*
_________________________________________________________________
BE1**
BE2
BE3
BE4
BE5
BF1**
BF2
BF3
BF4
2.10
1.76
1.27
1.77
2.24
1.36
2.36
3.44
2.26
0.63
0.67
0.39
0.47
0.57
0.89
2.36
1.03
2.42
3.17
2.96
2.08
3.94
3.56
3.65
4.03
3.73
2.27
1.60
2.85
1.13
1.81
1.83
2.88
4.17
4.45
2.34
1.56
1.55
1.32
1.27
1.63
1.59
1.68
1.60
1.26 average
SD***
2.06
0.61
1.05
0.74
3.27
0.67
2.56
1.08
1.50
0.15
VC*** 29.47 70.48 20.49 42.19 10.00
________________________________________________________________
* FFA: free fatty acids; F1 and F3 fluorescence measurements at 327/415 and 393/460 nm, respectively.
** Sample names as specified in Table 1.
*** SD: standard deviation; VC: variation coefficient.

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Relación entre las medidas en atún cocido y crudo de distintos índices de alteración
_________________________________________________________________
8
9
6
7
4
5
2
3
Muestra AGL* Dienos Carbonilos Pardeamiento F3/F1*
_________________________________________________________________
1** 2.10
1.76
0.63
0.67
3.17
2.96
1.60
2.85
1.56
1.55
1.27
1.77
0.39
0.47
2.08
3.94
1.13
1.81
1.32
1.27
2.24
1.36
0.57
0.89
3.56
3.65
1.83
2.88
1.63
1.59
2.36
3.44
2.36
1.03
4.03
3.73
4.17
4.45
1.68
1.60
2.26 2.42 2.27 2.34 1.26 media
DS***
2.06
0.61
1.05
0.74
3.27
0.67
2.56
1.08
1.50
0.15
CV*** 29.47 70.48 20.49 42.19 10.00
________________________________________________________________
* AGL: ácidos grasos libres; F1 and F3: medidas fluorescentes a 327/415 and 393/460 nm, respectivamente.
** Muestras correspondientes a nueve túnidos.
*** DS: desviación estándar; CV: coeficiente de variación.

17