Z Lebensm Unters Forsch (1992) 195:332-335 Zeitschrift ffJr

�9 Springer-Verlag 1992

Original paper

Fluorescence formation during albacore (Thunnus alalunga) thermal processing Santiago Aubourg, Ricardo P6rez-Martin, Isabel Medina, and Jos6 M. Gallardo

Instituto de Investigaciones Marinas (CSIC), c/Eduardo Cabello, 6, E-36208 Vigo, Spain

Received March 10, 1992

Fluorescenz-Entwicklung wiihrend der W~irmebehandlung von Albacore ( Thunnus alalunga)

Zusammenfassung. Lipidsch~iden w/ihrend der Verarbei- tung von Albacore (Thunnus alalunga) wurden durch die Entwicklung yon Fluorescenz (Maximum vier verschie- dener Wellenl/ingen) und Br/iunung untersucht. Analy- sen wurden mit w~Brigen und organischen Extrakten so- wie 131mustern durchgefiihrt. Als Ergebnis des thermi- schen Prozesses wurde eine Fluorescenzverschiebung zu h6heren Wellenl/ingenmaxima offenbar. Gleichzeitig nahm die Br/iunung zu. Daher zeigte die h6chste Sterili- sationstemperatur die gr6Bten Sch/iden und Fluorescenz- verschiebungen zu h6heren Wellenl/ingenmaxima.

Summary. Lipid damage during albacore processing was investigated by following fluorescence formation at four different excitation/emission wavelength maxima and brown colour development. Analyses were made on aqueous and organic extracts, as well as in dipping oil samples. A fluorescence shift to higher wavelength maxima was observed as a result of processing. At the same time a marked increase in browning was obtained. The most energic sterilization treatment showed the highest damage and fluorescence shift to higher wave- length maxima.

Introduction

It has been demonstrated that primary and secondary products of lipid oxidation may react with aminated food constituents (proteins, amino acids, phospholipids) to produce fluorescent compounds [1, 2], browning [3-5] and flavour compounds [6, 7].

Besides the determination of primary (hydroperox- ides) and secondary (carbonyl compounds) oxidation

Correspondence to: S. Aubourg

products [8-9], fluorescent compounds have been also used as an analytical method for quantification ofperoxi- dation damage to foods during processing and storage [10]. Fluorescence determinations of treated samples have been shown on organic extracts [11, 12], aqueous ex- tracts [13, 14] and both types of extracts [15, 16].

Fluorescent compounds formed during processing have also attracted a great attention because of their close relationship with pigmented and fluorescent granules found in human and animal tissues [17, 18]; these com- pounds are supposed to be indicative of a degenerative process in ceils such as ageing, environmental stress and vitamin E deficiency [19, 20].

In the present work fluorescence formation during al- bacore (Thunnus alalunga) processing was studied; four different conditions of sterilization were investigated. Fluorescence measurements were achieved on aqueous and lipid extracts of fish muscle and in the dipping oils of cans. Analyses were carried out at four different excita- tion/emission wavelength maxima. Fluorescence ana- lyses were complemented by measuring non-enzymatic browning.

Materials and methods

1 Raw material and processing. The albacore tuna (Thunnus ala- lunga) used was caught by a tuna fishing vessel on the Atlantic Ocean (round 43~ and 27~ The fish were kept in boxes and transported on ice for 10 days. After arrival at our laboratory, the fish were frozen at -40 ~ C and stored at -20 ~ C for 1 month prior to analysis. The fish were beheaded, eviscerated and divided into four batches in order to achieve statistical analysis. Steam cooking was performed in our pilot plant (102-103 ~ C) to a final backbone temperature of 65 ~ C (90 min); the fish were then cooled at room temperature (14 ~ C) for about 5 h.

Portions of 80-90 g of cooked muscle were placed in RO-100 cans (6.52 cm diameter, 3 cm height) and soybean oil (20 ml) and salt (2 g) added. Cans containing only oil were also prepared (can- ned oil blanks). The cans were vacuum sealed and sterilized in a re- tort under four different conditions: E-1 (110 ~ C, 120 min); E-2 (130 ~ C, 120 min); E-3 (115 ~ C, 60 min); and E-4 (130 ~ C, 27 min). E-l, E-3 and E-4 correspond to industrial conditions (F 0 = 7 min)

333

differing in time and temperature. E-2 has a much higher Fo value and was employed in order to compare with the other treatments. After 4 months of storage at room temperature the cans were opened and the liquid part was drained off carefully.

Table 1. Fluorescence measurements of trichloro acetic acid (TCA) ex- tracts from flesh muscle a

Sample b Wavelength (nm)

327/415 356/416 393/463 446/490

R 1.83 a 1.24 a 0.44" 0.12 a (0.15) (0.12) (0.05) (0.02)

C 2.70 ab 2.42 u 1.06 a 0.14" (0.36) (0.27) (0.25) (0.03)

E- 1 3.09 "b 3.68 ~ 3.15 b~ 0.31 b (0.28) (0.26) (0.53) (0.06)

E-2 2.61 ab 4.65 d 4.53 c 0.49 ~ (1.48) (0.67) (1.24) (0.18)

E-3 3.44 b 3.88 c 2.84 b 0.33 b~ (0.35) (0.16) (0.76) (0.05)

E-4 3.17 "b 3.80 ~ 2.82 b 0.31 b (0.17) (0.36) (0.61) (0.03)

2 Preparation of samples. Portions (20 g) of albacore muscle (raw, cooked and canned) were extracted and made up to a 100 ml volume with 5% trichloroacetic acid (TCA). Lipid extraction was carried out by the method of Bligh and Dyer [21]; 2 g portions of albacore muscle (raw, cooked and canned) were extracted and made up to a 5 ml volume with chloroform. The dipping oils of the flesh cans were dried with anhydrous NaSO4 and made up to a 50 ml volume with chloroform. Portions of 20 ml of the initial oil and of the canned oil blanks were made up to a 50 ml volume with chloroform.

3 Fluorescence measurements. The excitation and emission spectra of the aqueous, lipid and oil samples were determined using a Per- kin-Elmer LS 3B Fluorescence spectrophotometer. Measurements were made at the following excitation/emission maxima: 327/415, 356/416, 393/463 and 446/490 nm. Quinine sulphate solution (1 gg/ ml in 0.05 mol sulphuric acid) was used as a standard of fluorescence intensity (S). The fluorescence of the sample relative to the standard was calculated using the following formula [16]:

F . V Fluorescence (ml/g) = S. w '

where V is the volume of extract (5 ml for lipid extracts of flesh muscle, 100 ml for TCA extracts and 50 ml for oils) giving fluores- cence F, and w is the weight of the initial sample (2 g for flesh lipids, 20 g for TCA extracts and 18.5 g for oils).

a Mean of four determinations; values in the same column followed by different letters are significantly different (P<0.05). Standard deviation is indicated in brackets b Samples: raw (R), cooked (C) and canned (E-l, sterilized at 110 ~ C, 120 min; E-2, 130 ~ C, 120 min; E-3, 115 ~ C, 60 rain; and E-4, 130 ~ C, 27 rain)

4 Spectrophotometric measurement of brown colourformation. Mea- surement of brown colour formation in lipid extracts and oils was achieved at 420 nm [22, 23] in a Beckman DV-64 spectrophotom- eter. The results shown in Tables 2 and 3 were calculated using the following formula [16]:

B . V Absorbance -

W

where B is the absorbance reading obtained, V is the volume of the sample (5 ml for lipid extracts of flesh muscle, 50 ml for oils), and w is the weight of the initial sample (2 g for flesh lipids and 18.5 g for oils).

5 Statistical analysis. Data resulting from the fluorescence and spectrophotometric measures were subjected to the ANOVA one- way method, according to Sokal and Rohlf [24].

Results

1 TCA extracts

Table 2. Measurement of fluorescence and spectrophotometric absor- bance of lipid extracts from flesh muscle ~

Sample b Wavelength (nm)

327/415 356/416 393/463 446/490 420

R 0.55 a 0.3P 0.19 a 0.07 a 56.3 a (0.13) (0.05) (0.04) (0.02) (4.65)

0.74 a 0.77 .b 0.50 . 0.14 ab 61.3 ab (0.21) (0.25) (0.09) (0.02) (3.70)

1.26 b 1.37 b~ 1.13 b 0.23 ~ 96.3 c (0.09) (0.13) (0.12) (0.03) (2.68)

1.56 b 1.82 c 1.70 c 0.32 d 131.3 a (0.17) (0.31) (0.37) (0.07) (9.47)

1.45 b 1.72 c 1.27 b 0.28 cd 95.0 c (0.21) (0.57) (0.21) (0.06) (6.06)

C

E-1

E-2

E-3

A t 356/416 n m a m a r k e d i nc r ea se in f l u o r e s c e n c e c o n t e n t was o b t a i n e d a l o n g the d i f f e r en t s teps o f p roces s ing , r aw, c o o k e d a n d c a n n e d ( T a b l e 1). S a m p l e s c o r r e s p o n d i n g to t he s t r o n g e s t s t e r i l i za t ion t r e a t m e n t (E-2) s h o w e d the h ighes t v a l u e s o f f l uo rescence . A t 393/463 a n d 446/ 490 n m r a w a n d c o o k e d f ish w e r e n o t s ign i f i can t ly d i f fer - ent , b u t s h o w e d a l o w e r leve l t h a n c a n n e d samples . A g a i n , t he s t e r i l i za t ion t r e a t m e n t E-2 p r e s e n t e d the h ighes t va lue .

2 Lipid extracts

F l u o r e s c e n c e ana lys i s a t 327/415 n m r e v e a l e d a l o w e r v a l u e fo r t he r a w a n d c o o k e d s amp le s c o m p a r e d to t he

E-4 1.26 b 1.33 bc 1.08 b 0.21 ~ 88.8 b~ (0.08) (0.05) (0.04) (0.01) (2.38)

a Mean of four determinations; values in the same column followed by different letters are significantly different (P<0.05). Standard deviations are indicated in brackets b Sample names as expressed in Table 1

c a n n e d samples ( T a b l e 2). A t 356/416 n m a p r o g r e s s i v e inc rease in f l u o r e s c e n c e w i t h the p r o c e s s i n g was ob - t a ined ; r a w samp le s s h o w e d l o w e r levels t h a n a n y c a n n e d samples ; E-2 a n d E-3 p r e s e n t e d the h i g h e s t va lues . A t 393/463 n m the f o l l o w i n g i n c r e a s i n g s e q u e n c e was ob -

t a ined : r a w a n d c o o k e d < E - l , E-3 a n d E-4 < E-2. F l u o -

334

Table 3. Measurement of fluorescence and spectrophotometric absor- bance of oil samples a

Sample b Wavelength (nm)

327/415 356/416 393/463 446/490 420

INO 10.35 ~ 12.54" 4.33" 0.54 ~ 79.0 a (0.10) (0.10) (0.12) (0.05) (8.04)

10.00 ~ 15.62 b 6.60 b 0.83 ab 158.0 b (0.09) (0.12) (0.15) (0.06) (10.80)

9.88 c 15.21 b 6.97 b 0.94 bc 166.5 be (0.07) (0.I0) (0.05) (0.10) (9.47)

10.39 ~ 16.87 b 6.40 b 0.79 .b 148.3 b (0.12) (0.11) (0.07) (0.07) (6.99)

10.06 c 15.28 b 6.55 b 0.85 ab 177.0 bc (0.10) (0.05) (0.06) (0.08) (5.72)

8.0P b 11.66 a 6.4P 1.06 bc 198.5 ~ (0.28) (1.60) (1.15) (0.16) (27.53)

7.37" 11.04 a 6.88 b 1.22 c 218.3 e (0.21) (1.50) (1.12) (0.16) (19.47)

8.27 b 11.25 a 6.00 ab 1.00 bc 172.5 bc (0.47) (1.80) (1.07) (0.14) (15.00)

7.87 "b 12.18" 6.870 1.11 ~ 211.5 d (0.96) (1.30) (1.51) (0.37) (26.39)

E-01

E-02

E-03

E-04

E-1

E-2

E-3

E-4

a Mean of four determinations; values in the same column followed by different letters are significantly different (P<0.05). Standard deviations are indicated in brackets b Samples: initial oil (INO), canned oil blanks (E-01, sterilized at 110 ~ C, 120 min; E-02, 130 ~ C, 120 min; E-03, 115 ~ C, 60 rain; and E-04, 130 ~ C, 27 rain) and dipping oils (names as expressed in Table 1)

rescence a t 446/490 nm a n d b r o w n co lou r f o r m a t i o n showed a p rogress ive increase a long the process ing steps; s ter i l iza t ion a t 130 ~ C, 120 ra in (E-2) aga in showed the h ighest values.

3 Oil samples

Fluorescence m e a s u r e m e n t s a t 327/415 n m showed lower levels o f d i p p i n g oils t han the canned oil b l anks (Table 3). In the case o f 356/416 nm, c a n n e d oil b l anks p resen ted h igher values t han the in i t ia l oil a n d the d ipp ing oils. F lu - orescence levels a t 393/463 n m showed an increase f rom ini t ia l oil to the o the r oil samples (d ipp ing oils a n d can- ned oil b lanks) . A t 446/490 n m a h igh increase in f luores- cence con ten t was o b t a i n e d by c o m p a r i n g the ini t ia l oil and d ipp ing oils. Browning values showed a s ignif icant difference be tween the ini t ia l oil a n d b o t h k inds o f can- ned oils (b l ank and d ipp ing oils); p rocess ing p r o d u c e d an increase, h ighes t in the case o f the d ipp ing oils.

Discussion

As a genera l b e h a v i o u r for each o f the exc i t a t ion /emis - s ion max ima , f luorescence analys is revealed an increase

a long the d i f ferent s teps o f processing. However , this a u g m e n t a t i o n was b igger the h igher the wave leng th m a x i m a be ing cons idered . W i t h the a im of observ ing the f luorescence shift to h igher wave length m a x i m a in all k inds o f samples , the re la t ionsh ip be tween f luorescence conten ts a t 393/463 n m and 327/415 n m was s tud ied (Table 4). A m a r k e d increase in this coeff ic ient was ob- ta ined for the T C A and l ip id ext rac ts a long the di f ferent p rocess ing steps. Oi l samples showed a s ignif icant aug- m e n t a t i o n f rom ini t ial oil to d ipp ing oils. Aga in , the ster- i l iza t ion t r ea tmen t E-2 showed the h ighest values, re la t - ing to the h ighest d a m a g e to samples processed in this way.

Prev ious ly [25] the in te rac t ion be tween a lbaco re muscle and ace t a ldehyde was s tud ied in a m o d e l system. A f luorescence shift to h igher wave leng th m a x i m a was also o b t a i n e d as a resul t o f ace t a ldehyde concen t ra t ion , t ime and t e m p e r a t u r e o f react ion .

A t the same t ime and as a resul t o f process ing , a g r a d u a l increase in b r o w n co lou r f o r m a t i o n was o b t a i n e d in l ipid ext rac ts (raw, c o o k e d and canned flesh) and in oils ( ini t ial oil, c anned oil b l anks a n d d ipp ing oils o f flesh muscle) . This increase, as well as the f luorescence shift to

Table 4. Relationship between the fluorescence measurements at 393/463 nm and 327/415 nm for TCA and lipid (LE) extracts and oils a

Sample b TCA LE Oils

R 0.24 a 0.35 a (0.01) (0.08)

C 0.39 ab 0.70 b (0.06) (0.12)

E-1 1.12 cd 0.89 ~ 0.80 boa (0.29) (0.04) (0.14)

E-2 1.44 d 1.09 d 0.93 d (0.38) (0.12) (0.15)

E-3 0.85 bc 0.88 uc 0.72 bcd

(0.30) (0.10) (0.09)

E-4 0.89 ~ 0,85 bc 0.90 Cd (0.19) (0.06) (0.33)

0.42 a (O.Ol)

o.668~c (0.01)

0.71 bed (0.01)

0.62 ab (0.01)

0.65 abe

(0.01)

INO

E-01

E-02

E-03

E-04

Mean of four determinations; values in the same column followed by different letters are significantly different (P<0.05). Standard deviations are indicated in brackets b Sample names as expressed in Tables 1 and 3

335

higher excitation/emission maxima, agrees with the gen- eral theory on the progressive formation of Schiff bases and other interaction products with increasing molecular weights and unsaturat ion bonds [3, 4].

As mentioned earlier, both organic and aqueous ex- tracts have been studied by measuring fluorescence for- mat ion [10-16]. Tables 1 and 2 show the fluorescence values to be higher in TCA extracts than in lipid extracts, al though the same conclusions could be obtained f rom previous results. Experimental evidence has been ob- tained demonstrat ing that fluorescence substances formed f rom oxidized membrane lipids with amino com- pounds remain attached to the amino constituent [26, 27]. In the case of amino substrates such as proteins and amino acids, fluorescent compounds could not be ex- tracted so easily by an organic solvent as when the amino substrate was a phospholipid. For this reason the extent of lipid oxidation might appear lower than the actual amount if organic extracts only are considered [3]. It can be concluded that the use of both kinds of extracts is nec- essary in order to ensure a correct and complete measure of food damage during processing. The results obtained reinforce the role of fluorescent measures as a test o f quality control in processed foods, and emphasize the value of studying fluorescence formation at different wavelength maxima.

Acknowledgement. We acknowledge financial support for the Re- search Project AL188-0145-C02-02 provided by the Comisi6n Inter- ministerial de Ciencia y Tecnologia (CICYT).

References

1. Leake L, Karel M (1985) J Food Biochem 9:117-136 2. Montfoort A, Bezstarosti K, Groh M, Koster J (1987) FEBS

Lett 226:101-104

3. Pokorny J (1977) Riv Ital Sost Grasse 54:389-393 4. Gardner H (1979) J Agric Food Chem 27:220-229 5. Pokorny J, Bulantov/t H, Davldek J (1981) Nahrung 25:531-

541 6. Kunert-KirchhoffJ, BaltesW(1990) Z LebensmUnters Forsch

190:14-16 7. Pokorny J (1990) Nahrung 34:887-897 8. Tsoukalas B, Grosch W (1977) J Am Oil Chem Soc 54:490-

493 9. Gray J (1978) J Am Oil Chem Soc 55:539-546

10. Melton S (1983) Food Technol 37:105-111,116 11. Bouzas J, Kamarei A, Karel M (1985) J Food Sci 50:1515-

1516 12. Maruf F, Ledward D, Neale R, Poulter R (1990) Int J Food Sci

Technol 25:66-77 13. Manwaring J, Csallany S (1981) J Nutr 111:2172 2179 14. Gonzfilez-Garza M, Montalvo I, Sotelo A (1990) J Agric Food

Chem 38:340-342 15. Pikul J, Leszczynski D, Niewiarowicz A, Kummerow J (1984)

J Food Technol 19:575-584 16. Smith G, Hole M, Hanson S (1990) J Sci Food Agric 51:193-

205 17. Tappel A (1980) In: Pryor W (ed) Free radicals in biology. Aca-

demic Press, New York 18. Kikugawa K, Beppu M (1987) Chem Phys Lipids 44:277-297 19. Miquel J, Or6 J, Bensch K, Johnson J Jr (1978) In: Pryor W (ed)

Free radicals in biology. Academic Press, New York 20. Sohal R (1987) Adv Biosci 64:85-91 21. Bligh E, Dyer W (1959) Can J Biochem Physiol 4:16%174 22. KatoY, MatsudaT, KatoN, Watanabe K, Nakamura R(1986)

J Agric Food Chem 34:351 355 23. Labuza T, Massaro S (1990) J Food Sci 55:821-826 24. Sokal R, Rohlf F (1981) Biometry. Freeman, San Francisco,

USA 25. Aubourg S, Medina I, P&ez-Martin R, Barreiro C, Gallardo J

(1991) Proc VI Euro Food Chem 2:585-590 26. Shimasaki H, Ueta N, Mowri H, Inove K (1984) Biochem Bio-

phis Acta 792:123-129 27. Iio T, Yoden K (1988) Lipids 23:65-67