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PARTIAL INHIBITION OF CHOLESTEROL OXIDES

FORMATION IN FROZEN FISH BY A PREVIOUS

PLANT EXTRACT TREATMENT Vera Lebovicsa, Andrea Lugasia, Judit Hóvaria and Santiago P. Aubourgb,*

a National Institute for Food Safety and Nutrition, H-1097 Budapest, Gyáli út 3/a b Department of Food Technology, Instituto de Investigaciones Marinas (CSIC), c/ E.

Cabello, 6, 36208-Vigo (Spain)

* Corresponding author: saubourg@iim.csic.es ; fax: +34986292762 34

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SUMMARY 1

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Formation of cholesterol oxide products (COPs) in frozen horse mackerel (Trachurus

trachurus) fillets was studied. The effect of a fish soaking treatment (45 minutes) in

either of two concentrations of an aqueous solution of an antioxidant plant extract prior

to the freezing step was determined. The fish fillets were sampled immediately after

freezing and at intervals throughout 12 months of frozen storage (-20ºC). Two control

groups consisting of untreated- and water-treated fish were stored and sampled under

the same conditions. Qualitative analysis showed the formation throughout the

experiment of the following COPs: 7-α-hydroxy-cholesterol, 7-β-hydroxy-cholesterol

and 7-keto-cholesterol. All treatment groups showed an increase (p<0.05) in the content

of all three COPs with storage time. As a result of pre-freezing antioxidant treatment, a

lower (p<0.05) COPs formation was observed when compared to both control samples;

this partial inhibition agreed with a longer shelf life time for the plant extract-treated

fish revealed by sensory analysis.

Running Title: Cholesterol oxide formation in frozen fish 19

Keywords: Horse mackerel, frozen storage, plant extract, antioxidant, rancidity,

cholesterol oxides, sensory analysis

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INTRODUCTION 1

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Cholesterol is an important member of neutral lipid class present in the human

body and the most prominent sterol found in food products of animal origin (Yeagle,

1985; Teshima, 1992). As an unsaturated alcohol, it may undergo oxidation to form a

wide range of cholesterol oxidation products (COPs) that can accumulate in the human

body by endogenous oxidation and may also be derived from food intake and further

absorption (Smith, 1987; Savage et al., 2002; Ohshima, 2002). It has been stated that

oxidised derivatives of cholesterol may exert a wide range of detrimental biological

activities in the human body such as disturbance of carcinogenesis, mutagenicity,

atherosclerosis and cytotoxicity (Guardiola et al., 1996; Linseisen & Wolfram, 1998).

Foods based on marine resources provide a high content on polyunsaturated fatty

acids (PUFA), among which, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic

acid) are the most abundant (Ackman, 1989). PUFA are known to be markedly

susceptible to peroxidation and be easily incorporated into the mechanism of lipid

peroxidation to yield free radicals and lipid peroxy radicals (Hsieh & Kinsella, 1989;

Porter et al., 1995). Such radical molecules have been reported to be implied in the

cholesterol peroxyl radical production that finally leads to the COPs formation. Thus,

previous research accounts for COPs formation in conjunction with PUFA oxidation in

different kinds of marine foods processed by heat treatment (grilling, boiling, roasting

and cooking), salting and drying (Ohshima et al., 1993; Li et al., 1996; Nan, 2002; Al-

Saghir et al., 2004).

Freezing and frozen storage have largely been employed to retain fish sensory

and nutritional properties, although enzymatic and nonenzymatic rancidity is known to

strongly develop (Harris & Tall, 1994; Erickson, 1997). To extend the shelf life of

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marine products, a great attention is being given to the employment of natural

antioxidants (Frankel, 1995; Kamal-Eldin & Appelqvist, 1996). In this sense, most

research on marine food has been focused on the PUFA oxidation inhibition, while little

research has been carried out concerning the effect of antioxidant treatment on the

COPs development (Shozen et al., 1997; Kim et al., 1997).

The present work concerns horse mackerel (Trachurus trachurus) trading as a

frozen product. Horse mackerel is a medium-fat content fish abundant in the Northeast

Atlantic that has recently captivated a great deal of commercial interest (FAO, 2006).

The study is aimed to investigate the effect of a plant extract pretreatment on the COPs

formation in horse mackerel fillets during frozen storage. As a plant extract, Rosmol-P

was chosen according to previous research where its inhibitory effect on lipid oxidation

development was already proved (Aubourg et al., 2004a; Lugasi et al., 2007).

MATERIALS AND METHODS 15

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Plant extract preparation

Plant extract was obtained as Rosmol-P (RP) (FitoChem Kft., Monor, Hungary).

For it, a 100 g mixture of dry hyssop (Hysoppus officinalis), brunella (Prunella

vulgaris), lemon balm (Melissa officinalis), and rosemary (Rosmarinus officinalis) was

percolated with 30% hydro-ethanolic solution to give 500 ml of brown percolate. The

solution was mixed with 2 g of activated coal, refrigerated and filtered. The hydro-

ethanolic solution was spray dried to get 30 g antioxidant powder. Maltodextrin was

used as carrier. Total polyphenol and rosmarinic acid contents were 66 g kg-1 and 21 g

kg-1, respectively (Lugasi et al., 2000). Concentrations of RP employed in the present

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experiment were chosen according to previous research (Aubourg et al., 2004a; Lugasi

et al., 2007).

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Raw fish, sampling and processing

Fresh horse mackerel (Trachurus trachurus) (n=87) were captured near the

Galician Atlantic coast (North-Western Spain) and kept on ice till arrival to the

laboratory (8 h). The fish were carefully dressed and filleted by hand and divided into

four groups. Each group consisted of 21 fishes. One fillet group was directly packaged

in individual polyethylene bags and immediately frozen at –80ºC (Blank Control; BC).

The other groups were immersed, respectively, in water (Water Control treatment; WC),

in a 0.332% aq. RP solution (RP-1 treatment) and in a 1.328% aq. RP solution (RP-2

treatment) in an isothermal room at 4ºC. After 45 min, the fillets were removed,

packaged in individual polyethylene bags and frozen at –80ºC. After 48 hours at –80ºC,

all fillets were placed at –20ºC. Sampling was undertaken on the initial material and at

months 0, 1, 3, 5, 7, 9 and 12 of frozen storage at –20ºC. For each fish pre-treatment

group, three different fillet batches were considered and studied separately to achieve

the statistical study (n = 3). Once fillets were subjected to sensory analysis, the white

muscle was separated and homogenised for carrying out the cholesterol oxide analyses.

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Preliminary analyses

Lipids were extracted by the Folch et al. (1957) method. Results were calculated

as g total lipids kg-1 wet muscle.

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Cholesterol oxide compounds analysis

Cholesterol oxide (5α-cholestan-3,5,6β-triol; 7α-hydroxy-cholesterol; 7β-

hydroxy-cholesterol; 7-keto-cholesterol; 5,6α-epoxy-cholesterol; 25-hydroxy-

cholesterol; 20α-hydroxy-cholesterol) and cholesterol standards were purchased from

Sigma (St. Louis, Mo, USA). The standard sterols were dissolved (1 mg ml-1) in

chloroform:methanol (2:1 v/v) and stored at –20ºC. Precoated Sil G HF254 thin-layer

chromatography (TLC) with concentrating zone (layer thickness 0.25 mm on 20x10 cm

glass plates) and Sil G 60 F254 (10x10 cm glass plates) and solvents were purchased

from E. Merck (Darmstadt, Germany).

Saponification of total lipids and isolation of cholesterol and COPs from the

non-saponifiable fraction were carried out according to Missler et al. (1985). The

separation of cholesterol and the individual cholesterol oxidation compounds was

performed by thin layer chromatography technique as described previously (Lebovics et

al., 1992). Detection and quantification (mg kg-1 wet muscle) was carried out by

enzymatic (cholesterol oxidase) assay combined to the thin layer chromatography

analysis (Lebovics et al., 1996).

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Sensory analyses

Sensory analyses were conducted on the thawed horse mackerel fillets by a taste

panel consisting of five experienced judges, according to the guidelines presented in

Table 1 (DOCE, 1989). Sensory assessment of fish included the following parameters:

general aspect (dryness, myotome breakdown, white spot presence), odour and colour.

Four categories were ranked: highest quality (E), good quality (A), fair quality (B) and

rejectable quality (C).

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Statistical analyses

Data from the different cholesterol oxide contents were subjected to the one-way

ANOVA method (p<0.05); comparison of means was performed using a least-squares

difference (LSD) method (Statsoft, 1994). Linear and non-linear (quadratic and

logarithmic) correlation analyses between frozen storage time and COPs content were

performed (Statsoft, 1994). Non-parametric analysis (Spearman test) was applied to

study the correlation between sensory scores and storage time.

RESULTS AND DISCUSSION 10

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Preliminary analyses

Formation of COPs throughout the present experiment was first studied by thin

layer chromatography of the different kinds of fish samples. Thus, Rf values and

characteristic colour spot development were analysed in comparison with different and

appropriate commercial cholesterol oxides.

Starting fresh fish and fish fillets taken after the freezing step (month 0 frozen

samples) did not provide detectable spots of oxysterols for any of the different kinds of

samples under study. However, when increasing the frozen storage time, different COPs

could be observed as a result of lipid oxidation development and accordingly, fillet

quality loss. Till the end of the experiment, three different COPs could be observed,

namely 7α-hydroxy-cholesterol (7α-OH-C), 7β-hydroxy-cholesterol (7β-OH-C) and 7-

keto-cholesterol (7-K-C). Figure 1 shows the presence of such COPs for fillet samples

corresponding to the different pre-treatments under study after a frozen storage time of

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7 months. Compound 7-K-C (marked as dotted line) was detected by UV-light (254

nm).

These three COPs have been described as being widely present in processed

marine products, such as salted, dried and thermally treated (Tai et al., 2000; Pickova &

Dutta, 2003; Echarte et al., 2004; Al-Saghir et al., 2004; Sampaio et al., 2006). The

cholesterol oxides most commonly reported have been those derived from the B-ring of

the main cholesterol chain, such as the three presently found in addition of both (α and

β) 5,6-epoxy-cholesterols; however, derivatives of the side chain, such as 20α-hydroxy-

cholesterol and 25-hydroxy-cholesterol have been detected in smaller concentrations.

The presence of COPs in food products has been related to the lipid content, in

the sense that a higher lipid content corresponds to a higher COPs formation. Thus, a

higher COPs formation during seafood grilling was produced when employing fatty fish

species as raw materials than in the case of starting from lean fish ones (Ohshima,

2002). On the other hand, the COPs formation has also been directly related to the

PUFA content according to an oxidation development supposed to be carried out in

conjunction (Ohshima et al., 1993; Li et al., 1996; Nan, 2002; Al-Saghir et al., 2004),

although some researchers have pointed out that lipid peroxidation would precede

cholesterol oxidation (Osada et al., 1993). Concerning the different kinds of processing,

heating has been reported to be the major responsible for cholesterol oxides generation,

mainly in extensively processed food (Tai et al., 2000).

In the present study, starting fresh fish was obtained in October, which

corresponds to the highest lipid content period of this species (Bandarra et al., 2001).

Accordingly, a lipid content range of 53-108 g kg-1 wet muscle was obtained that can be

considered a relatively high value for horse mackerel captured in the Galician Atlantic

coast. At the same time, cholesterol content showed a mean value of 505 mg kg-1 wet

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muscle, so that no significant (p>0.05) changes were obtained in its content as a result

of storage time or the preliminary treatment. The mentioned cholesterol content agrees

to common data on fish species (Piclet, 1987).

In a previous paper (Lugasi et al., 2007), quality loss of whole horse mackerel

during the frozen (-20ºC) storage was studied. In it, no COPs were detected up to month

12 of storage when applying the same analytical tools than in the present case. This

COPs formation lack can be explained by the fact that whole fish has shown to

deteriorate slower than fillet products, so that a lower rancidity development is

produced (Aubourg et al., 2004b; Aubourg et al., 2005).

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Quantitative analysis of COPs

According to the above mentioned qualitative analysis, these three COPs were

quantified and studied throughout the present experiment, with a special stress on the

effect of the previous antioxidant treatment.

The evolution of the 7α-OH-C (Figure 2) and the 7β-OH-C (Figure 3) contents

provided very similar patterns. Thus, no detection of both molecules was achieved in

starting samples and after the freezing step (month 0 frozen samples), according to

previous research that indicates no presence of COPs in fresh or high quality muscle

fish (Osada et al., 1993; Saldanha et al., 2006). However, from month 1 till the end of

the experiment, a progressive content increase with storage time could be observed for

both molecules in all kinds of samples. This increase was however higher (p<0.05) in

the case of both control samples (BC and WC) than in both antioxidant treated samples

(RP-1 and RP-2), so that a significant (p<0.05) inhibitory effect of the antioxidant

treatment could be assessed on the formation of 7α-OH-C and 7β-OH-C compounds.

Formation of both hydroxy compounds showed good correlation values (r2 = 0.92-0.94;

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Table 2) with frozen storage time under all treatments investigated, showing to be

accurate indices to follow the quality loss in the present experiment.

In the case of the 7-K-C compound (Figure 4), no formation could be assessed in

starting fish, nor in fish stored during a 0-3 month storage period. Then, a significant

(p<0.05) formation could be observed in all kinds of samples at month 5. From that time

up, its content increased till the end of the experiment. However, a different trend

formation could be outlined for the different kinds of samples; thus, control samples

(BC and WC) showed a great increase at month 9, while both antioxidant-treated

samples (RP-1 and RP-2) provided a progressive 7-K-C content increase throughout the

experiment. As a result, the 7-K-C content provided better correlation values with the

frozen storage time for the antioxidant-treated samples than for the control ones (Table

2).

7-K-C contents showed lower mean values for antioxidant-treated fish than in

both control samples for the 5-9 month period; significant differences (p<0.05) were

only attained at month 9 (for RP-1 and RP-2 treatments) and at month 7 (for RP-2

treatment). Comparison between both RP treatment samples, led to a higher 7-K-C

formation inhibition (p<0.05) for the RP-2 treatment at months 7 and 9.

The total COPs content for all kinds of samples was calculated and analysed.

Results are shown in Figure 5. According to above mentioned results on individual

oxidation compounds, no formation was found for starting fish samples and for those

resulting from the freezing step (month 0 frozen samples). Then, a progressive content

increase was observed till the end of the experiment in all kinds of samples. For control

samples (BC and WC), great increases (p<0.05) are found at month 5 and then at month

9, while antioxidant-treated fish provided a progressive content increase with the

storage time. Thus, the total COPs content led to good correlation values (r2 = 0.90-

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0.94; Table 2) with the storage time for the different kinds of samples. In this sense,

previous research (Scolari et al., 2000) has mentioned the employment of the total

COPs content as an accurate tool for assessing the rancidity status of fish products.

According to the total COPs values, an important inhibitory effect of the

antioxidant treatment was obtained, since lower (p<0.05) contents were reached in the

5-9 month period for RP-1 and RP-2 samples than in control ones (BC and WC);

antioxidant treatment showed to not exert (p>0.05) an inhibitory effect at month 12. On

the other hand, no differences (p>0.05) are found between fish samples corresponding

to both antioxidant concentrations, while the aqueous treatment (WC samples) did not

provide a significant (p>0.05) effect when compared to blank control.

In previous work, partial inhibition of COPs formation was obtained by

employing natural antioxidants (Li et al., 1996; Shozen et al., 1997; Kim et al., 1997;

Akhtar et al., 1998), although synthetic antioxidants showed to be more effective

(Shozen et al., 1997; Kim et al., 1997). In such cases, antioxidant treatment was applied

previously to salting, drying or thermal processing, or included as a previous dietary

supplementation in cultivated fish (Ohshima, 2002). However, no preliminary research

accounts for an antioxidant pre-treatment focused on the COPs formation reduction in a

frozen fish product. According to the major concern in public health related to the COPs

effect on human beings, the actual experiment presents a profitable soaking treatment to

be employed as a previous step to the commercialisation of safe frozen fish products.

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Sensory analysis

Progressive score decreases (Table 3) were observed throughout the experiment

for all kinds of samples, so that fair correlation values (r2 = 0.86-0.95; Table 2) were

obtained with the storage time in all cases. A different evolution pattern throughout the

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experiment could be outlined for sensory scores and COPs formation. Thus, logarithmic

fittings were obtained in most cases for sensory results, while cuadratic fittings were

obtained in many cases for COPs values.

The sensory analysis results (Table 3) showed a short shelf life time for the

blank control fillets, that were unacceptable at month 3. Both RP treatments enlarged

the shelf life time, so that fillets were still acceptable at month 5. At month 7, according

to an important COPs formation, fish fillets were found sensory unacceptable. Sensory

quality differences between both RP-treated fish were only observed at month 3 (B and

A scores for RP-1 and RP-2, respectively).

Water-treated fillets showed a longer shelf life than the untreated ones (Blank

Control), as were still acceptable at month 3. In the present experiment, lower mean

values in most COPs were obtained for the WC samples than for the BC ones, although

differences were not significant (p>0.05). In previous research, a lipid oxidation

inhibitory effect by a water treatment has been observed when traditional lipid oxidation

indices (peroxide value, thiobarbituric acid reactive substances, fluorescence

development) were checked (Undeland et al., 1998; Richards & Hultin, 2002; Aubourg

et al., 2004a). In such experiments, the authors explained the lower rancidity

development in water-treated fillets as a result of removal of hemeproteins and metal

ions included in the fish blood.

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Acknowledgements 1

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The authors thank Mr. Iván Jakóczi (FitoChem Kft, Monor, Hungary) for

providing the Rosmol-P product and Mr. Marcos Trigo and Mr. José M. Antonio for

technical assistance. The work was founded by the Academy of Sciences (Hungary)-

CSIC (Spain) Program (Project 2001 HU 0011), the Hungarian Ministry of Education

(NKFP 1/016/2001, "Széchenyi" Project) and the Comisión Interministerial de Ciencia

y Tecnología (CICyT; Spain) (Project ALI 99-0869).

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FIGURE LEGENDS 1

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Figure 1: Thin layer chromatography analysis of cholesterol oxidation products (COPs)

formation* after 7 months of frozen storage in horse mackerel that was pre-

treated under different conditions**

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* Cholesterol oxide standards: 5α-cholestan-3,5,6β-triol (1), 7α-hydroxy-cholesterol (2),

7β-hydroxy-cholesterol (3), 7-keto-cholesterol (4), 5,6α-epoxy-cholesterol (5),

25-hydroxy-cholesterol (6), 20α-hydroxy-cholesterol (7), cholesterol (8).

** Lanes identification: lane A (Blank Control), lane B (Water Control), Lane C (RP-1

treatment), lane D (RP-2 treatment) and lane E (cholesterol and cholesterol

oxide mixture).

Figure 2: Determination of 7–α–hydroxy–cholesterol in frozen horse mackerel fillets

that were pre-treated under different conditions*

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* Treatment names: Untreated (Blank Control, BC), water-treated (Water Control, WC),

and Rosmol P-treated (0.332% and 1.328%, RP-1 and RP-2, respectively).

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Figure 3: Determination of 7–β–hydroxy–cholesterol in frozen horse mackerel fillets

that were pre-treated under different conditions*

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* Treatment names as specified in Figure 2.

Figure 4: Determination of 7–keto–cholesterol in frozen horse mackerel fillets that

were pre-treated under different conditions*

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* Treatment names as specified in Figure 2.

Figure 5: Total content on cholesterol oxidation products (COPs) in frozen horse

mackerel fillets that were pre-treated under different conditions*

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* Treatment names as specified in Figure 2.

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REFERENCES 1

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15

16

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21

22

23

Ackman, R. (1989). Fatty acids. In: Marine Biogenic Lipids, Fats and Oils (edited by R.

Ackman). Pp. 103-137, vol. 1. Boca Raton, Florida, USA: CRC Press.

Akhtar, P., Gray, J., Booren, A. & Gomaa, E. (1998). The effects of dietary alpha-

tocopherol and surface application of oleoresin rosemary on lipid oxidation and

cholesterol oxide formation in cooked rainbow trout (Oncorhynchus kisutch)

muscle. Journal of Food Lipids, 5, 59-71.

Al-Saghir, S., Thurner, K., Wagner, K., Frisch, G., Luf, W., Raccaci-Fazeli, E. &

Elmadfa, I. (2004). Effects of different cooking procedures on lipid quality and

cholesterol oxidation of farmed salmon fish (Salmo salar). Journal of

Agricultural and Food Chemistry, 52, 5290-5296.

Aubourg, S., Lugasi, A., Hóvári, J., Piñeiro, C., Lebovics, V. & Jakóczi, I. (2004a).

Damage inhibition during frozen storage of horse mackerel (Trachurus

trachurus) fillets by a previous plant extract treatment. Journal of Food Science,

69, 136-141.

Aubourg, S., Piñeiro, C. & González, Mª J. (2004b). Quality loss related to rancidity

development during frozen storage of horse mackerel (Trachurus trachurus).

Journal of the American Oil Chemists’ Society, 81, 671-678.

Aubourg, S., Rodríguez, A. & Gallardo, J. (2005). Rancidity development during

mackerel (Scomber scombrus) frozen storage: Effect of catching season and

commercial presentation. European Journal of Lipid Science and Technology,

107, 316-323.

16

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Bandarra, N., Batista, I., Nunes, M. & Empis, J. (2001). Seasonal variation in the

chemical composition of horse mackerel (Trachurus trachurus). European Food

Research and Technology, 212, 535-539.

DOCE (1989). Baremo de Clasificación de Frescura. In: Diario Oficial de las

Comunidades Europeas (L5/21, 07.01.1989). Pp. 5-6. Brussels, Belgium:

European Commission.

Echarte, M., Zulet, M. & Astiasarán, I. (2004). Evaluation of the nutritional aspects and

cholesterol oxidation products of pork liver and fish patés. Food Chemistry, 86,

47-53.

Erickson, M. (1997). Lipid oxidation: Flavor and nutritional quality deterioration in

frozen foods. In: Quality in frozen food (edited by M. Erickson & Y-C. Hung).

Pp. 141-173. New York, USA: Chapman and Hall.

FAO (2006). FAO Yearbook 2004. Fishery statistics, Capture production, Vol. 98/1.

Pp. 273-274. Rome, Italy: Food and Agriculture Organization of the United

Nations.

Folch, I., Lees, M. & Stanley, G. (1957). A simple method for the isolation and

purification of total lipids from animal tissue. Journal of Biological Chemistry,

726, 497-509.

Frankel, E. (1995). Natural and biological antioxidants in foods and biological systems.

Their mechanism of action, applications and implications. Lipid Technology,

July, 77-80.

Guardiola, F., Codony, R., Addis, P., Rafecas, M. & Boatella, J. (1996). Biological

effects of oxysterols: Current status. Food Chemistry and Toxicity, 34, 193-211.

Harris, P. & Tall, J. (1994). Rancidity in fish. In: Rancidity in foods (edited by J. Allen

& R. Hamilton). Pp. 256-272. London, UK: Chapman and Hall.

17

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Hsieh, R. & Kinsella, J. (1989). Oxidation of polyunsaturated fatty acids: mechanisms,

products and inhibition with emphasis on fish. Advances in Food Research and

Nutrition Research, 33, 233-341.

Kamal-Eldin, A. & Appelqvist, L. (1996). The chemistry and antioxidant properties of

tocopherols and tocotrienols. Lipids, 31, 671-701.

Kim, Y., Lee, I., Lee, J. & Sung, N. (1997). Effect of ascorbic acid or BHA on the

formation of cholesterol oxidation products during storage of salted mackerel,

Scomber japonicus. Journal of the Korean Society of Food Science and

Nutrition, 26, 261-269.

Lebovics, V., Antal, M. & Gaál, Ö. (1996). Enzymatic determination of cholesterol

oxides. Journal of the Science of Food and Agriculture, 71, 22-26.

Lebovics, V., Gaál, Ö., Somogyi, L. & Farkas, J. (1992). Cholesterol oxides in γ-

irradiated spray-dried egg powder. Journal of the Science of Food and

Agriculture, 60, 251-254.

Li, S., Cherian, G., Ahn, D., Hardin, R. & Sim, J. (1996). Storage heating, and

tocopherols affect cholesterol oxide formation in food oils. Journal of

Agricultural and Food Chemistry, 44, 3830-3834.

Linseisen, J. & Wolfram, G. (1998). Origin, metabolism, and adverse health effects of

cholesterol oxidation products. Fett/Lipid, 100, 211-218.

Lugasi, A., Blázovics, A., Hagymási, K. & Jakóczi, I. (2000). Application of a natural

antioxidant as food ingredient. In: 10th Biennial Meeting of the International

Society for Free Radical Research (October 16-20). P. 223. Kyoto (Japan):

Abstract book.

Lugasi, A., Losada, V., Hóvári, J., Lebovics, V., Jakóczi, I. & Aubourg, S. (2007).

Effect of pre-soaking whole pelagic fish in a plant extract on sensory and

18

biochemical changes during subsequent frozen storage. Food Science and

Technology, 40, 930-936.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Missler, S., Wasilchuk, B. & Merrit, C. (1985). Separation and identification of

cholesterol oxidation products in dried egg preparation. Journal of Food

Science, 50, 595-598.

Nan, L. (2002). Distribution and origination of cholesterol oxides in marine products.

Food Fermentation Industry, 28, 27-31.

Ohshima, T. (2002). Formation and content of cholesterol oxidation products in seafood

and seafood products. In: Cholesterol and Phytosterol oxidation products (edited

by F. Guardiola, P. Dutta, R. Codony & G. Savage). Pp. 186-202. Champaign,

Illinois, USA: American Oil Chemists’ Society Press.

Ohshima, T., Li, N. & Koizumi, C. (1993). Oxidative decomposition of cholesterol in

fish products. Journal of the American Oil Chemists’ Society, 70, 595-599.

Osada, K., Kodama, T., Cui, L., Yamada, K. & Sugano, M. (1993). Levels and

formation of oxidized cholesterol in processed marine foods. Journal of the

Agricultural and Food Chemistry, 41, 1893-1898.

Pickova, J. & Dutta, P. (2003). Cholesterol oxidation in some processed fish products.

Journal of the American Oil Chemists’ Society, 80, 993-996.

Piclet, G. (1987). Le poisson aliment. Composition - intérêt nutritionnel. Cahiers de

Nutrition et Diététique, XXII, 317-335.

Porter, N., Caldwell, S. & Mills, K. (1995). Mechanisms of free radical oxidation of

unsaturated lipids. Journal of the American Oil Chemists’ Society, 30, 277-290.

Richards, M. & Hultin, H. (2002). Contributions of blood and blood components to

lipid oxidation in fish muscle. Journal of Agricultural and Food Chemistry, 50,

555-564.

19

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Saldanha, T., Frankland Sawaya, A., Nogueira Eberlin, M. & Bragagnolo, N. (2006).

HPLC separation and determination of 12 cholesterol oxidation products in fish:

Comparative study of RI, UV, and APCI-MS detectors. Journal of Agricultural

and Food Chemistry, 54, 4107-4113.

Sampaio, G., Bastos, D., Soares, R., Queiroz, Y. & Torres, E. (2006). Fatty acids and

cholesterol oxidation in salted and dried shrimp. Food Chemistry, 95, 344-351.

Savage, G., Dutta, P. & Rodríguez-Estrada, MªT. (2002). Cholesterol oxides: their

occurrence and methods to prevent their generation in foods. Asia Pacificic

Journal of Clinical Nutrition, 11, 72-78.

Scolari, M., Luzzana, U., Stefani, L., Mentasti, T., Nmoretti, V., López, C. & Hardy, R.

(2000). Quantification of cholesterol oxidation products in commercial fish

meals and their formation during storage. Aquaculture Research, 31, 785-791.

Shozen, K., Ohshima, T., Ushio, H. & Koizumi, C. (1997). Effects of antioxidants and

packing on cholesterol oxidation in processed anchovy during storage.

Lebensmittel Wissenschaft und Technologie, 30, 2-8.

Smith, L. (l987). Cholesterol autoxidation 1981-1986. Chemistry and Physics of Lipids,

44, 87-125.

Statsoft (1994). Statistica for Macintosh. Tulsa, Oklahoma (USA): Statsoft and its

licensors.

Tai, C., Chen, Y. & Chen, B. (2000). Analysis, formation and inhibition of cholesterol

oxidation products in foods: an overview (part 2). Journal of Food and Drug

Analysis, 8, 1-15.

Teshima, S. (1990). Sterols of Crustaceans, Molluscs and Fish. In: Physiology and

Biochemistry of Sterols (edited by G. Patterson & W. Nes). Pp. 229-256.

Champaign, Illinois, USA: American Oil Chemists’ Society Press.

20

1

2

3

4

5

6

7

Undeland, I., Ekstrand, B. & Lingnert, H. (1998). Lipid oxidation in minced herring

(Clupea harengus) during frozen storage. Effect of washing and precooking.

Journal of Agricultural and Food Chemistry, 46, 2319-2328.

Yeagle, P. (1985). Cholesterol in the cell membrane. Biochemical and Biophysical Acta,

822, 267-287.

21

TABLE 1 1 2 3 4 5 6 7

Scale employed for evaluating quality of frozen horse mackerel fillets

Attribute

E (Highest quality)

A (Good quality)

B (Fair quality)

C (Rejectable

quality) General Aspect

(dryness, myotomes

breakdown, white spots)

Strongly hydrated;

totally adhered myotomes; absence of white spots

Still hydrated; myotomes adhered;

absence of white spots

Slightly dry; myotomes adhered in

groups; small white spots

Dry; myotomes totally

separated; big white spots

Flesh Odour

Shellfish

Weakly Shellfish

Slightly sour or incipient rancidity

Sharply sour or rancid

Flesh Colour

Strongly pinky

Still pinky

Slightly pale

Yellowish

8 9

10

22

TABLE 2 1 2 3 4 5 6 7 8

Linear correlation values* between frozen storage time and COPs content and sensory acceptance for each of the treatments studied**

Parameter

BC

WC

RP-1

RP-2

7α-hydroxy-cholesterol

0.93

0.92

0.89 (0.94)

0.86 (0.93) a

7β-hydroxy-cholesterol

0.93

0.92

0.87 (0.94) a

0.88 (0.94) a

7-keto-cholesterol

0.86

0.83 (0.85) a

0.87 (0.94) a

0.84 (0.93) a

Total cholesterol

oxides

0.91

0.90

0.88 (0.94) a

0.86 (0.94) a

Sensory acceptance

0.72 (0.86) b

0.84 (0.94) b

0.90 (0.95) b

0.92

9 10 11 12 13

14

15

16

17 18 19

* Non-linear (quadratica and logarithmicb) correlation values are expressed in brackets

when superior to the linear ones. In all cases, significant (p<0.05) values were

obtained.

** Treatment name abbreviations as expressed in Figure 2.

23

TABLE 3 1 2 3 4 5 6 7 8

Sensory acceptance* of frozen horse mackerel fillets that were previously treated

under different conditions**

Frozen Storage Time

(months)

BC

WC

RP-1

RP-2

0 E E E E 1 B A A A 3 C B B A 5 C C B B 7 C C C C 9 C C C C 12 C C C C

9 10 11 12 13

14

15 16 17 18 19 20 21

* Category marks according to Table 1. Initial fish material was E category.

** Treatment name abbreviations as specified in Figure 2.

24

02468

101214161820

0 1 3 5 7 9 12

Frozen Storage Time (months)

7-al

pha-

OH

-cho

lest

erol

(mg

kg-1

mus

cle) BC

WC

RP-1

RP-2

Figura 2

0

2

4

6

8

10

12

14

16

18

0 1 3 5 7 9 12

Frozen Storage Time (months)

7-be

ta-O

H-c

hole

ster

ol (m

g kg

-1 m

uscl

e)

BCWC

RP-1RP-2

Figura 3

0

5

10

15

20

25

30

35

0 1 3 5 7 9 12

Frozen Storage Time (months)

7-ke

to-c

hole

ster

ol (m

g kg

-1 m

uscl

e)

BC

WC

RP-1

RP-2

Figura 4

0

10

20

30

40

50

60

70

0 1 3 5 7 9 12

Frozen Storage Time (months)

Tota

l cho

lest

erol

oxi

des

(mg

kg-1

mus

cle)

BC

WC

RP-1

RP-2

Figura 5