GYA Grasas y Aceites Grasas y Aceites 1988-4214 0017-3495 Consejo Superior de Investigaciones Científicas gya.0215231 10.3989/gya.0215231 Research Effects of cold and hot smoking processes and the addition of natural Dunaliella salina polyphenol extract on the biochemical quality and shelf life of Sander lucioperca fillets after storage for 90 days Efecto de procesos de ahumado frío y caliente y la adición de extracto polifenólico natural de Dunaliella salina sobre la calidad bioquímica y la vida útil de filetes de Sander lucioperca almacenados durante 90 días https://orcid.org/0000-0001-6181-6896 Bouriga N. nawzet.bouriga@ispab.ucar.tn Ecology, Biology and Physiology Laboratory of Aquatic Organisms, Biology Department, Faculty of Sciences of Tunis, University of Tunis El Manar, 2092 Tunis, Tunisia. University of Carthage, Higher Institute of Fisheries and Aquaculture of Bizerte, Errimel, B.P.15, 7080 Bizerte, Tunisia. https://orcid.org/0000-0002-2625-8064 Mili S. Research Unit: Exploitation of Aquatic Environments, Higher Institute of Fisheries and Aquaculture of Bizerte, University of Carthage, Errimel B.P.15., 7080 Bizerte, Tunisia. https://orcid.org/0000-0002-7396-763X Troudi D. University of Carthage, Higher Institute of Fisheries and Aquaculture of Bizerte, Errimel, B.P.15, 7080 Bizerte, Tunisia. https://orcid.org/0000-0001-5051-2141 Ben Atitallah A. Enzymatic Engineering and Microbiology Laboratory, Algae Biotechnology Team, National School of Engineers of Sfax, University of Sfax, Sfax 3038, Tunisia. https://orcid.org/0000-0001-8416-8342 Bahri W.R. Laboratory of Biodiversity, Biotechnology and Climate change LR11ES09, Faculty of Sciences of Tunis, University Tunis El Manar, 2092 Tunis, Tunisia. https://orcid.org/0000-0002-7946-2763 Bejaoui S. University of Carthage, Higher Institute of Fisheries and Aquaculture of Bizerte, Errimel, B.P.15, 7080 Bizerte, Tunisia. https://orcid.org/0000-0002-0878-4220 Dridi M.A. Ecology, Biology and Physiology Laboratory of Aquatic Organisms, Biology Department, Faculty of Sciences of Tunis, University of Tunis El Manar, 2092 Tunis, Tunisia. https://orcid.org/0000-0001-9722-3566 Quignard J.-P. Ichthyology Laboratory, University of Montpellier II, Sciences et Techniques Languedoc, Place Eugène Bataillon, Case 102, 34095 Montpellier, Cedex 5, France. https://orcid.org/0000-0001-5291-6211 Trabelsi M. Ecology, Biology and Physiology Laboratory of Aquatic Organisms, Biology Department, Faculty of Sciences of Tunis, University of Tunis El Manar, 2092 Tunis, Tunisia. https://orcid.org/0000-0002-5368-4800 Ben-Attia M. Biomonitoring of the Environment Laboratory (LR01/ES14), Faculty of Sciences of Bizerte, University of Carthage, Tunisia. https://orcid.org/0000-0002-9325-0687 Shahin A.A.B. Department of Zoology, Faculty of Science, Minia University, El Minia, Egypt. 01 12 2023 12 2023 74 4 e530 16 02 2023 14 06 2023 19 12 2023 ©2023 CSIC 2023 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. Summary

The effects of cold and hot smoking and the addition of Dunaliella salina polyphenol extract on the biochemical quality and shelf-life of Sander lucioperca fillets after storage for 90 days at 0-4 °C were examined. The results showed a significant increase in protein, lipid, free fatty acid, and 1,1-diphenyl-2-picrylhydrazyl contents, and a decrease in peroxide and thiobarbituric acid reactive substances, and volatile base nitrogen levels in cold (CSF) and hot (HSF) smoked fillets covered with or without extract and stored for 1, 20, and 90 days compared to fresh fillets (FF). Saturated and monounsaturated fatty acids exhibited a significant increase in FF and CSF and HSF covered with or without extract. The total polyunsaturated fatty acids revealed a significant decrease in FF and CSF and HSF with or without extract. Therefore, cold and hot smoking and polyphenol extract improved the biochemical quality and storage shelf-life of fillets for 90 days at 0-4 °C.

 Resumen

Se examinaron los efectos del ahumado en frío y en caliente y la adición de extracto de polifenoles de Dunaliella salina sobre la calidad bioquímica y la vida útil de filetes de Sander lucioperca almacenados durante 90 días a 0-4 °C. Los resultados mostraron un aumento significativo en los contenidos de proteínas, lípidos, ácidos grasos libres y 1,1-difenil-2-picrilhidrazilo, y una disminución en las sustancias reactivas de peróxido y ácido tiobarbitúrico, y los niveles de nitrógeno básico volátil en frío (LCR) y caliente (HSF) de filetes ahumados cubiertos con o sin extracto y almacenados durante 1, 20 y 90 días en comparación con los filetes frescos (FF). Los ácidos grasos saturados y monoinsaturados exhibieron un aumento significativo en FF y LCR y HSF cubiertos con o sin extracto. Los ácidos grasos poliinsaturados totales revelaron una disminución significativa en FF y CSF y HSF con o sin extracto. Por lo tanto, el ahumado en frío y en caliente y el extracto de polifenoles mejoraron la calidad bioquímica y la vida útil durante el almacenamiento de los filetes durante 90 días a 0-4 °C.

 Antioxidants Cold and hot smoking Dunaliella salina microalgae Fatty acids Freshwater fish Polyphenols Ácidos grasos Ahumado en frío y en caliente Antioxidantes Microalga Dunaliella salina Pescado de agua dulce Polifenoles Introduction

Fish is a highly biodegradable food due to its susceptibility to oxidation, which assists in the growth of pathogenic microorganisms (Chaillou et al., 2015) and eventually leads to the formation of off-odor and flavor, and finally to rot (De Souza-Franco et al., 2010). Therefore, it must be precisely handled and preserved to retard its spoilage and to assure microbial safety and a marketable shelf-life (Amaral et al., 2021). Indeed, some chemical quality indices have so far been developed to assess the level and extension of fish spoilage, such as the total volatile basic-nitrogen (TVB-N) and trimethylamine-nitrogen (TMA-N), thiobarbituric acid (TBA) value, and the presence of biogenic amines (histamine, cadaverine, tyramine, and putrescine) produced by the decarboxylation of specific free amino acids by the action of microorganisms (Silbande et al., 2018). TVB-N implies the measurement of volatile basic nitrogenous compounds, such as trimethylamine (TMA), dimethylamine (DMA), and ammonia (NH3), which are produced by bacteria, from the action of enzymes or the deamination of amino acids (Kostaki et al., 2009). The proposed value of TVB-N for spoilage initiation is 30-35 mg N/100 g; however, some studies present lower levels depending on the fish species (Kostaki et al., 2009). TMA-N is the main constituent of non-protein nitrogen fraction, produced by the bacterial spoilage, enzymatic activity, and decomposition of TMA-N-oxide, and is responsible for the fishy odor. The upper limit of TMA-N values considered for spoilage acceptance is 10-15 mg TMA-N/100 g, but lower limits are also suggested by other authors (Kostaki et al., 2009). Regarding lipid oxidation, the TBA value is used to measure the malondialdehyde (MDA) content. The quality values range between 2-4 mg MDA/kg, but this value might not reflect the actual rate of lipid oxidation because MDA can interact with other components (Kostaki et al., 2009). In addition, several methods, including vacuum packaging, modified atmosphere packaging, active packaging, and chemical additives, such as organic acids and natural extracts, combined with freezing systems, have been applied to impede its decomposition (Amaral et al., 2021).

Extensive fish farming, as in the case of Sander lucioperca (pikeperch or zander), offers the opportunity for fishermen to cost-effectively harvest fish. Although the nutritional quality of S. lucioperca is high because it is rich in polyunsaturated fatty acids (PUFA), vitamins, and minerals (Bouriga et al., 2020), this species is not overly valued by consumers, mainly due to its undesirable taste and flavor compared to marine fish. The smoking process can offer such a marketing alternative to freshwater fish and result in high-quality and acceptable products (Bouriga et al., 2012). As far as known, smoking treatments (hot, cold, liquid, and electrostatic) have been documented as useful processes for preserving the quality of seafood and can be achieved through both traditional and innovative techniques (Karsli and Çağlak, 2021). Overall, some constituents of smoking substances are aldehydes, ketones, alcohols, acids, hydrocarbons, esters, phenols, and ethers. These substances are applied to the surface of the wires and then penetrate the muscle, giving the products their final color and taste. However, some reports have shown that smoking processes have a negative impact on the nutritional value of fish fillets (Bouriga et al., 2012). Thus, the addition of antioxidants can be a useful technique for preserving the quality of fillets.

Given that consumers are becoming more health conscious, there has been a strong demand for the use of functional foods and the addition of natural ingredients. For this reason, several authors have focused on microalgae as potential sources of compounds with functional, nutritional, antimicrobial, and antioxidant properties (Cakmak et al., 2014). Among the compounds that can be obtained from microalgae are antioxidants, which have been widely used as food conserves in the food industry (Madhavi et al., 1996 ). In addition, natural antioxidants, such as polyphenols, are now more extensively used due to their physiological benefits to human health. In this context, herbs have been the most valuable antioxidants used to protect smoked fillets, especially green algae of the genus Dunaliella. Of this genus, D. salina is a green, halophilic microalga commonly found in sea salt fields. This microalga is famous for its high commercial, economic, and industrial value due to its persistent capacity to produce large amounts of polyphenols and carotenoids, especially β-carotene, which has been widely used as an important natural antimicrobial and antioxidant for nutrient preservation in food, feed, and the pharmaceutical industry due to its high physiological properties, as well as biodiesel because of its high unsaturated fatty acid content (Cakmak et al., 2014). In addition, polyphenols and β-carotene function as scavenger compounds to protect the fillets from the generation of free radicals (Burton and Ingold, 1984).

Despite the immense number of works on smoking processes and their potential effects on fish fillets, the use of Dunaliella salina as a natural antioxidant has not been well explored. Hence, the current study was conducted to examine the effect of both cold and hot smoking processes and the addition of two graded concentrations (0.5 and 1% v/w) of natural Dunaliella salina polyphenol (pp) antioxidant extract on the biochemical quality and shelf-life and consumption of Sander lucioperca fillets during storage for 1, 20, and 90 days, respectively, in a refrigerator at 0-4 °C.

 Material and methods Polyphenol antioxidant extract

Dunaliella salina samples were collected in May 2019 from Chott El Djerid, an endorheic salt lake, situated in southern Tunisia (33°54′42.21′′N, 8°31′7.98′′E). The antioxidant extract was prepared following the method described by Messina et al. (2015), in which 10 g of dried and pulverized microalgae were extracted with 100 mL of distilled water and then incubated in a shaker for 24 h in the dark. Afterwards, the mixture was filtered and lyophilized. The final solution was prepared by dissolving 10 g of the freeze-dried extract in 1000 mL of distilled water (10 g·L−1 of distilled water), with a polyphenol content equal to 500 mg of gallic acid equivalents (GAE)/L (Messina et al., 2015).

 Sampling and smoking procedure of <italic>Sander lucioperca</italic>

S. lucioperca samples were collected in May 2019 during 10 fishing operations from Sidi El Barrak Reservoir (Beja Governorate, northwest Tunisia, 37°01’N, 09°39’E). A total of 45 fish samples, ranging from 12.5 to 28.6 cm (mean 24.3 ± 3.2 cm) in total length and 3400 to 1900 g (mean 700 ± 2.20 g) in total weight, were collected using a 30 mm mesh trammel net. The samples were preserved in ice and then transported to the laboratory where they were weighed, measured, de-capitated, and cleaned. These samples were longitudinally cut into fillets measuring 13.6 -18.2 cm (mean 15.8 ± 2.6 cm) in length, 3.2-4.4 cm (mean 3.1 ± 0.23 cm) in thickness, 7.4-9.8 cm (mean 6.78 ± 1.12 cm) in width, and 200.4-420 g (mean 264 ± 0.86 g) in weight. The fillets were divided into three groups (100 g each): the first consisted of fresh fillets without any additives, the second comprised fillets covered with a final concentration of 0.5%, i.e., 50 mL pp (v)/100 g fillets (w), of the polyphenol (pp) antioxidant D. salina extract using a micropipette, and the third included fillets that were treated with the polyphenol antioxidant extract as the second group, but with a final concentration of 1% pp, i.e., 100 mL pp (v)/100 g fillets (w), extract. Afterwards, the three groups were smoked in cold and hot conditions. The cold-smoked fillets were dried for 2 h in the smoking chamber at a temperature of 30-35 °C, while the hot-smoked fillets were introduced into the industrial smoking chamber using a peripheral smoke generator. The process of smoking the fillets was accomplished as follows: pre-drying the fish surface at 50-60 °C for 150 min, followed by hot smoking at 65-70 °C for 30 min, and finally steaming at a temperature of 68-72 °C. In both smoking processes, oak wood was used (Bouriga et al., 2020). Finally, the fillets were cooled in cold air at 10 °C. Subsequently, the smoked fillets were vacuum-sealed and stored in a refrigerator at 0-4 °C for 90 days. For the biochemical analyses, fresh and vacuum-sealed smoked fillet samples were gradually tested after 1, 6, 20, 50, and 90 days of storage at 0-4 °C. However, the results obtained from the storage periods 6, 20, 50, and 90 showed a similar significant gradual increase or decrease in each fillet type, as the case may be, so we reduced and summarized the data by presenting here only the results for three storage periods, namely 1, 20, and 90. All flesh samples were stored at -80 °C until analysis.

Biochemical analyses related to shelf-life Protein determination

The total protein content of S. lucioperca fillets was determined by estimating their total nitrogen content using the Kjeldahl method 981.10 of the AOAC. Approximately 1 g of raw material was hydrolyzed with 15 mL concentrated sulfuric acid (H2SO4) containing two copper catalyst tablets in a heat block (Kjeltec system 2020 digestor, Tecator Inc., Herndon, VA, USA) at 420 °C for 2 h. After cooling, H2O was added to the hydrolysates before neutralization and titration. The amount of total nitrogen obtained was multiplied with both the traditional conversion factor of 6.25 and species-specific conversion factors (Mariotti et al., 2008) in order to determine total protein content. The protein content was expressed as a mean percentage (%) of the wet weight (ww) of three replicates.

 Lipid determination

The total lipids were determined according to the protocol described by Bouriga et al. (2020). Briefly, 10 grams of fresh or smoked fillet samples were homogenized for 8-10 min at 4 °C in a mixture of chloroform: methanol (1:2) using a Polytron homogenizer (Malaysia). The homogenate was added to 5 mL NaCl saturated solution and 20 mL chloroform with Butylated hydroxytoluene (BHT; 50 ppm), and then homogenized for 7 and 5 min, respectively. Then, 20 mL of distilled water were added and the solution was homogenized again for 1 min. The obtained mixture was incubated in an ultrasound bath for 10 min, and a vacuum cleaner with Buchner funnel and chloroform. The organic fraction was extracted with a separating funnel, dried with sodium sulfate, and evaporated to dryness in the rotary evaporator (Stuart™, UK). The obtained oil was solubilized in a known volume of chloroform with BHT (50 ppm) and stored at -20 °C. The total lipids were expressed as a mean percentage (%) of the wet weight (ww) of six replicates.

 Peroxide value (PV)

The peroxide value of the fillet samples was determined according to the IDF standard method, 74A: 1991(9), with the ferric thiocyanate method based on the ability of lipid peroxides to oxidize ferrous ions at a low pH. The resulting ferric ions were reacted to thiocyanate and the concentration of the complex formed was determined by spectrophotometry (Jenway 6315, UK) at 500 nm. The standard sample was determined by the reaction of a series of aliquots of a 10-μg/ml iron (III) chloride standard solution, 10 mM ammonium thiocyanate, and a sufficient amount of chloroform/methanol mixture (7:3). The results were expressed as mequivalent of oxygen per kg of lipid (meq O2/kg) and the values were presented as a mean percentage (%) of three replicates.

 Thiobarbituric acid reactive substances (TBARS)

The production of thiobarbituric acid reactive substances (TBARS) was determined based on the AOAC (1998) method. Oil samples were dissolved in 1-butanol, mixed with 0.2% TBA in 1-butanol, incubated in a water bath for 2 h at 95 °C, then cooled under tap water. The absorbance was determined using a spectrophotometer at 532 nm, and the standard curve was established by the TBARS reaction of a series of aliquots of 0.2 mM TMP (1,1,3,3-tetramethoxypropane) prepared in 1-butanol. The results were expressed in mg MDA (malondialdehyde)/kg of oil and values were presented as a mean percentage (%) of three replicates.

 The 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant activity

To measure the antiradical activity, the synthetic radical DPPH (1,1-diphenyl-2-picrylhydrazyl) was used according to the method of Bersuder (Bersuder et al., 1998). Briefly, 10 mg of the fillet sample were suspended in 0.5 mL distilled water. Afterwards, 1.2 mL absolute ethanol and 0.2 mL DPPH solution (50 μM in ethanol) were mixed and incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm using a T70 UV-visible spectrophotometer. The results were expressed as a mean percentage (%) of three replicates of inhibition or trapping activity and were calculated by the following formula:

D P P H   t r a p p i n g   a c t i v i t y   =   ( ( c o n t r o l   O D   -   s a m p l e   O D ) / c o n t r o l   O D )   ×   100 . Total fatty acid (FA) determination

Fatty acid methyl esters (FAME) were determined following the AOAC 963.15 methodology (AOAC, 1990), with slight modification. In brief, the analysis was done in a Varian Agilent 6890 N gas chromatograph (Agilent Technologies, Santa Clara, USA), equipped with an auto-sampler and fitted with a split/splitless injector and a flame ionization detector (FID). Separation was performed in an Innowax 30 × 0.25 capillary column (25 m × 0.25 mm i.d., film thickness) (Agilent Technologies, Santa Clara, USA). The temperature was programmed from 180 to 200 °C at 4 °C/min, held for 10 min at 200 °C, heated to 210 °C at 4° C/min, and held at 210 °C for 14.5 min using an injector and FID at 250 °C. The fatty acid contents in the total lipids of the samples were estimated using nonadecanoic acid methyl ester C19:0 Me (Sigma Chemical Co. Ltd), as an internal standard (10 mg/mL) based on the ratio of the peak area. The fatty acid sequences ranged according to their chromatographic retention times, and the values are given as a mean percentage (%) of total fatty acid methyl esters of six replicates.

 Free fatty acids (FFA) determination

The samples (50 mg each) were homogenized with cyclohexane and copper acetate-pyridine reagent and stirred by vortex for 2 min, and then centrifuged at 9000 rpm for 20 min, and detected at 710 nm. The quantitative analysis of each fatty acid was performed for six replicates of each sample using nonadecanoic acid (C19:0, Sigma Chemical Co. Ltd), as an internal standard, and values were presented as a mean percentage (%) of three replicates.

 Total volatile base nitrogen (TVB-N)

The total volatile basic nitrogen (TVB-N) was measured by direct distillation of the homogenized samples according to the EU Commission Regulation (EC) No 2074/2005 (EEC, 2005). The sample was ground carefully by a meat grinder. Exactly 10 g ± 0.1 g of the ground sample were weighed in a suitable container, mixed with a 90-mL 6% perchloric acid solution, homogenized for two min with a blender, and then filtered. Steam distillation of 50 mL of the extract after sufficient alkalinization with 20% NaOH (6.5 mL) and the addition of several drops of phenolphthalein (1 g/100 mL 95% ethanol) and a few drops of silicone anti-foaming agent, began immediately. The steam distillation was regulated so that around 100 mL of distillate was produced within 10 min. The distillation outflow tube was submerged in a receiver with a 100-mL 3% boric acid solution, to which three to five drops of the indicator solution, Tashiro Mixed Indicator (2 g methyl-red and 1 g methylene-blue, dissolved in 1000 mL 95% ethanol), were added. After exactly 10 min the distillation was ended. The volatile bases contained in the receiver solution were determined by titration with a standard hydrochloric solution 0.01M till the pH reached 5.0 ± 0.1. The TVB-N (mg/100 g sample) = (V1 - V0) × 0.14 × 2 × 100/ M, where V1 = Volume of 0.01 M hydrochloric acid solution in mL for sample; V0 = Volume of 0.01 M HCL solution in mL for blank (50 mL 6% perchloric acid solution was used instead of the extract); M = sample wet weight (ww) in g. The values were expressed as a mean percentage (%) of the wet weight (ww) of three replicates.

 Statistical analysis

The results are presented as means ± standard deviation (SD). All biochemical indicators were statistically analyzed using SPSS software on triplicate samples, except total lipids and fatty acids, and free fatty acids, which were statistically analyzed on six triplicate samples. Normality and homogeneity of the variance of the results were confirmed by Kolmogorov-Smirnov and Levene’s tests, respectively. One-way ANOVA followed by the Tukey test was performed to determine the significant differences between fresh, cold-, and hot-smoked fillets with or without antioxidant extract and was set either at p < 0.05, p < 0.01, p < 0.001.

 Results and discussion Biochemical composition of fresh and smoked fillets

The biochemical composition of total proteins and lipids (%) in fresh and smoked S. lucioperca fillets are presented in Table 1. The percentage of total protein content remained nearly constant (range = 18.36-18.07%) in the fresh fillets (FF) during the 1, 20, and 90 days of storage, and showed a gradual significant increase (p < 0.001) in the levels (range = 41.27-51.31%) in both cold- (CSF) and hot-smoked (HSF) fillets covered with or without the two graded concentrations (0.5 and 1%) of D. salina polyphenol extract compared to FF during the three storage periods. However, this significant increase remained nearly constant in both CSF (41.27%) vs HSF (41.78%) without the covered extracts and CSF (42.57%) vs HSF (42.31%) covered with 0.5% of the polyphenol extract; while it increased in HSF (51.31%) covered with 1% of the polyphenol extract compared to CSF (42.31%) covered with 1% of the polyphenol extract during the three storage periods.

Variations in the biochemical composition of total proteins and lipids (in %) in fresh (FF), cold-smoked (CSF), hot-smoked (HSF) fillets of <italic>Sander lucioperca</italic>, and cold-smoked (CSF) and hot-smoked (HSF) fillets covered with <italic>Dunaliella salina</italic> polyphenol (pp) antioxidant extract at 0.5 and 1% concentrations during three storage periods.
Storage days FF CSF CSF+0.5% pp CSF+1% pp HSF HSF+0.5% pp HSF+1% pp
Proteins Lipids Proteins Lipids Proteins Lipids Proteins Lipids Proteins Lipids Proteins Lipids Proteins Lipids
1 18.36±1.13a 1.86±0.12a 41.27±2.57a 6.92±1.27a 42.57±1.13a 7.76±2.23a 42.31±2.57a 6.92±1.12a 41.78±3.27a 7.05±1.92a 42.57±1.13a 7.76±2.23a 51.31±3.22a 9.01±2.07a
20 18.20±1.54a 1.29±0.07a 39.94±2.68a 5.87±0.73a 40.20±1.54a 7.19±1.87a 41.98±1.27a 5.87±1.21a 40.36±2.38a 6.36±1.37a 40.20±1.54a 7.19±1.87a 46.07±2.39a 6.22±1.73a
90 18.07±1.17a 0.98±0.09a 39.07±1.31a 2.02±0.15a 39.01±0.22a 5.98±0.09b 41.04±1.72a 4.31±1.62a 38.27±1.43a 5.01±0.18b 39.01±0.22a 5.98±0.09b 44.69±1.86a 4.56±1.57a
ANOVA * *** *** *** * ** * ** ** ** * ** ** ***

Values are means ± standard deviation (n = 3 for proteins and n = 6 for lipids); Different superscript letters in the same column represent significant differences(p < 0.05, one-way ANOVA with Tukey test); Significant differences in total proteins and lipids percentages between FF and CSF and HSF with or without antioxidant extract during the three storage periods are presented (*p < 0.05, **p < 0.01, ***p < 0.001).

In addition, the total percentage of lipids revealed gradually decreased levels (1.86, 1.29, and 0.98%, respectively) in FF during the three storage periods and differentially significantly increased levels (p < 0.001) in both CSF and HSF with or without 0.5 or 1% of the polyphenol covering extracts during the three storage periods, with the highest similar levels in CSF and HSF covered with 0.5% of the extract and the highest level, especially after the day 1 of storage, in HSF covered with 1% of the extract (Table 1).

In the present study, the total proteins remained unaltered in FF over the 1, 20, and 90 days of storage. This stability in total protein content may be due to the retention of water in the fillets during these storage periods. However, its significant increase in both CSF and CSF, as well as in HSF and HSF, covered with 0.5 and 1% of the extracts, compared to FF during the three storage periods can be attributed not only to the decrease in moisture and water content, and the increase of ash associated with evaporation during the smoking process, but also to the effect of polyphenols, including phenolic acids and flavonoids, vitamin E, tocopherols, and tocotrienols, which are powerful antioxidants found in the D. salina extract and oil (Cakmak et al., 2014), which protect the cell membrane from oxidative damage and consequently prevent protein and lipid oxidation (Garavaglia et al., 2016). In addition, the significant decrease (p < 0.05) in protein levels in both HSF and HSF covered with 0.5% of the extract after the 20 and 90 days of storage may be due to signs of the enzymatic autolytic activity causing the spoilage (Ayeloja et al., 2020), which was discontinued in the HSF covered with 1% of the extract after e 90 days of storage.

On the other hand, the significant decrease (p < 0.001) in the lipid content in FF during the three storage periods can be attributed to the progression in reducing the antioxidant properties and total phenolic content, which reversibly increased in CSF and HSF with or without 0.5 and 1% of the extracts during the three storage periods. This significant increase can be explained in terms of the dehydration of the fillets during smoking and the incorporation of D. salina oil into the fillets covered with 0.5 and 1% of the extracts, which led to stopping the degradation of lipids by eliminating free radicals. Similar results were found with sardine samples canned in grape seed and olive oils (Bouriga et al., 2022). Nevertheless, the significant decrease in lipid levels in FF after the 20 and 90 days of storage suggests the hydrolysis of some lipid fractions associated with the progression of microbial spoilage (Burton and Ingold, 1984).

 Lipid oxidation indices in fresh and smoked fillets

The levels of the peroxide value (PV) and TBARS levels in the S. lucioperca FF and CSF and HSF covered with or without 0.5 and 1% of D. salina polyphenol antioxidant extracts are given in Table 2. The results showed that PV increased gradually during the storage periods from 1, 20 to 90 days, with the highest significant level (p < 0.001) found after 90 days of storage (Table 2). However, the PV in both CSF and CSF covered with 0.5 and 1% of the extract revealed a gradual decrease over the three storage periods compared to its level in FF. In HSF, although the PV indicated a significant increase (p < 0.05) after day 1 of storage and a significant decrease (p < 0.05) after the 20 days, it revealed a highly significant increase after 90 days compared to CSF. In HSF covered with 0.5 and 1% of the extract, compared with HSF, the PV displayed a slight decrease after 1 day of storage, a constant level at 1% concentration of the extract after the 20 days, and a significantly decreased level after 90 days; but this level was reduced significantly at 0.5% concentration of the extract. This significantly increased the PV level in FF after 90 days of storage and it is higher than that found in Sardinella gibbosa after 6 and 9 days of storage (Chaijan et al., 2006). In addition, the PV levels in both CSF and HSF with or without 0.5 and 1% of the polyphenol extracts during the three storage periods were higher than those reported for S. lucioperca by Bouriga et al. (2020), although these levels were significantly decreased by the addition of these polyphenols (Table 2). These higher levels of PV are likely due to the hot temperature of the smoking process and the higher content of PUFAs, which are highly sensitive to primary oxidative reactions induced by molecular oxygen, and fall within the acceptable limits (10-20 mEq/kg) declared by Connell (1995).

Variations in the peroxide (PV, in meq active O2/kg lipid), thiobarbi­turic acid reactive substances (TBARS, in mg MDA/kg lipid), and 1,1-diphenyl-2-picrylhydrazyl (DPPH, in g/100g lipids) values in fresh (FF), cold-smoked (CSF), hot-smoked (HSF) fillets of <italic>Sander lucioperca</italic>, and cold smoked (CSF), and hot smoked (HSF) fillets covered with <italic>Dunaliella salina</italic> polyphenol (pp) antioxidant extract at 0.5 and 1% concentrations during the three storage periods
Parameter Storage days FF CSF CSF+0.5% pp CSF+1% pp HSF HSF+0.5% pp HSF+1% pp ANOVA
PV 1 6.41±0.23a 4.13±0.27a 3.97±0.12a 3.01±0.57b 5.76±0.22b 4.52±0.42c 4.22±0.10c ***
20 10.06±1.04a 9.13±1.41a 6.08±1.06a 5.27±1.42b 7.27±0.47b 5.58±0.18c 6.73±0.76c ***
90 47.86±2.53a 26.21±1.43a 16.48±2.57b 15.78±1.27c 31.38±1.67b 17.98±1.79b 14.72±1.56c ***
TBARS 1 0.52±0.12a 0.96±0.17b 0.67±0.08a 0.55±0.03a 1.38±0.31b 1.17±0.38c 1.03±0.15c ***
20 4.06±0.13a 2.12±0.31c 2.07±0.08b 1.74±0.41b 3.27±0.38a 1.74±0.54b 2.36±0.42c ***
90 8.41±1.05a 3.68±0.35b 3.31±0.52c 2.11±0.72c 7.12±1.07a 5.03±0.67b 4.78±1.04c ***
DPPH 1 69.13±0.39a 72.0±0.58a 74.08±0.83a 77.48±0.23a 72.75±0.43b 81.94±0.95c 88.57±0.83c ***
20 33.72±0.57a 37.12±0.53c 44.12±0.43c 53.96±0.32c 39.52±0.26b 48.98±0.46a 57.14±0.35a ***
90 18.10±0.12a 21.89±0.45b 26.67±0.36c 28.56±0.13c 21.65±0.34a 27.98±0.27b 31.88±0.23b ***

Values are means ± standard deviation (n = 3); Different superscript letters in the same column represent significant differences (p < 0.05, one-way ANOVA with Tukey test); Significant differences between FF and CSF and HSF with or without antioxidant extract during the three storage periods are presented (***p < 0.001).

In S. lucioperca FF, Bouriga et al. (2020) reported that the TBARS original level was 0.49 mg MDA/kg oil. This level increased here to 0.52 mg MDA/kg after the 1 day of storage, indicating that lipid oxidation had occurred and progressively significantly increased (p < 0.001) after 20 and 90 days of storage (Table 2). This significant increase is consistent with that found in Trachurus trachurus during frozen storage (Aubourg et al., 2002) In CSF, the level was significantly higher than in FF, constant after 1 day of storage, and decreased significantly (p < 0.05) after 20 and 90 days in CSF covered with 0.5 and 1% of the extract. In HSF, the level revealed a gradual significant decline in SHF covered with 0.5 and 1% of the extract after the 20 and 90 days of storage. However, compared to its level in FF, it exhibited a significant elevation after 1 day of storage and a reduction in HSF and HSF covered with 0.5 and 1% of the extract after 20 and 90 days of storage. In comparison with both CFS and CSF covered with 0.5 and 1% of the extract, the TBARS level displayed a significant increase (p < 0.001), especially in HSF and HSF covered with 0.5% of the extract, and constant in HSF after 1 day of storage. However, its level was significantly higher in both HSF and HSF covered with 1% of the extract than in both CSF and CSF covered with 0.5 and 1% of the extract, constant in both CSF and HSF covered with 1% of the extract after 20 days of storage, and significantly higher in both HSF and HSF covered with 0.5 and 1% of the extract after 90 days of storage. This significant increase in the TBARS level beyond the acceptable limit (8 mg MDA/kg) in FF was significantly decreased in both CSF and HSF with or without the covering polyphenol antioxidant extracts during the three storage periods. Despite this decrease, the levels were significantly higher than those observed by Bouriga et al. (2020) and Bouriga et al. (2022) and lower than those found by Chaijan et al. (2006). This increase in TBARS reflects the increase in the formation of aldehydes, relatively polar secondary reaction products (Kolakowska, 2002) due to the increase in phospholipids. In addition, it is worth noting that the decrease in TBARS levels was concurrent with the gradual increase in PV during the three storage periods, a condition which is inconsistent with that described by Chaijan et al. (2006). This was probably due to an increase in hydroperoxides, especially aldehydes, in the later stages of secondary lipid oxidation as a result of the greater release of free iron and other prooxidants from the muscle which were excessively degraded when storage time was increased. However, this increase in hydroperoxides was reduced in both CSF and HSF by the addition of 0.5 and 1% of the covering polyphenol extracts.

 Free radical (DPPH) activity in fresh and smoked fillets

The DPPH activity in S. lucioperca FF, as well as in both CSF and HSF covered with or without 0.5 and 1% of D. salina polyphenol antioxidant extracts are shown in Table 2. Overall, the DPPH activity exhibited a significant decline in FF over the 1, 20, and 90 days of storage, respectively, while a progressive elevation in the activity was observed in both CSF and CSF covered with the two concentrations of the polyphenol antioxidant extracts during the three storage periods. Conversely, the activity showed a significant decrease (p < 0.05) in HSF compared to HSF covered with 0.5 and 1% of the extract during the three periods of storage. However, in comparison with its activity in FF, as well as in CSF and CSF covered with 0.5 and 1% of the extract, the activity level in HSF was significantly higher than in FF and lower than in CSF covered with 0.5 and 1% of the extract during the three periods of storage. On the other hand, the level in HSF covered with 0.5 and 1% of the extract was significantly higher than that in CSF covered with 0.5 and 1% of the extract, respectively, during the three periods of storage. Indeed, the DPPH free radical assay has been used to assess the antioxidant activity based on the electron transfer that produces a violet solution in ethanol (Huang et al., 2005). Overall, the DPPH activity showed a gradual significant decrease (p < 0.05) in both FF and CSF, as well as in HSF covered with or without 0.5 and 1% of the polyphenol extract, with an increase in the storage periods from 1 to 90 days. However, the levels of activity exhibited a significant increase in both CSF and HSF, compared to FF, with the addition of the polyphenol extract. Moreover, this increased activity was concurrent with the increase in lipid content. Therefore, we can assume that there was a close correlation between the polyphenol content, the DPPH activity, and the increase in lipid peroxidation.

 Total fatty acid (FA) composition in fresh, cold-, and hot-smoked fillets

The total FA composition in FF differed significantly during the three storage periods (1, 20, and 90), with a significant increase (p < 0.05) in both total saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), and a significant decrease (p < 0.05) in total polyunsaturated fatty acids (PUFAs) after the three storage periods (Table 3). This significant increase was confirmed by the considerable elevation of all SFAs (C14:0, C16:0, and C18:0) and MUFAs (C16:1n-7, C18:1n-7, and C18:1n-9), while the total decrease in PUFAs was only observed in C20:5n-3, C22:5n-3, C22:6n-3, and PUFA-n-3. Similarly, in both CSF and HSF, all SFAs and MUFAs exhibited a progressively significant increase (p < 0.05) in level during the three storage periods (1, 20, and 90). However, the total PUFAs, as well as PUFA-n-3 and -n-6, indicated a significant gradual decrease (p < 0.05) during these storage periods. Similarly, when CSF and HSF were covered with both concentrations of 0.5 and 1% of D. salina extract, all SFAs and MUFAs showed a remarkable increase (p < 0.05) over the 1, 20, and 90 days of storage, while the total PUFAs and PUFA-n-3 and -n-6 displayed a substantial significant decrease (p < 0.05) during these storage periods.

Total fatty acid (FA) composition (in %) in fresh (FF), cold-smoked (CSF), and hot-smoked (HSF) fillets of <italic>Sander lucioperca</italic>, and CSF and HSF fillets covered with <italic>Dunaliella salina</italic> polyphenol (pp) antioxidant extract at 0.5 and 1% concentrations during the three storage periods
FA FF CSF CSF+0.5% pp CSF+1% pp
1 day 20 days 90 days 1 day 20 days 90 days 1 day 20 days 90 days 1 day 20 days 90 days
C14:0 4.65±0.19a 5.09±1.12a 6.14±1.78b 5.72±0.25a 7.72±1.12b* 8.32±0.67c** 5.89±0.25a 6.41±1.52b 7.92±1.55c 6.72±1.05a* 7.12±1.78b** 8.01±1.23c***
C16:0 16.4±0.89a 20.46±1.52b 22.19±2.5c 17.65±2.14a 18.23±1.95b* 21.29±1.48c 16.5±0.87a 17.57±2.4b** 20.76±2.48c* 18.21±1.56a** 18.31±2.55b 19.7±2.37c
C18:0 2.76 ±0.66a 4.01±1.73b 7.15±3.45c 2.92 ±0.78a 4.57±0.73c 7.89±0.47c 3.19±0.78a 3.73±0.47b 6.37±1.51c* 4.22±0.96a*** 4.27±0.36a 6.03±0.58c**
C20:0 0.78 ± 0.03b 0.81 ± 0.05c 1.72 ± 0.18c 0.68 ± 0.04b 0.79 ± 0.07b 1.56 ± 0.11a 0.87 ± 0.02b 0.93 ± 0.01c 1.29 ± 0.06b 0.61 ± 0.03b 0.78 ± 0.02a 1.18 ± 0.21c
C22:0 0.52 ± 0.01b 1.12 ± 0.07b 1.41 ± 0.09b 0.34 ± 0.02c 0.57 ± 0.01b 1.47 ± 0.04b 1.22 ± 0.07b 1.07 ± 0.11b 1.63 ± 0.15b 1.22 ± 0.17c 1.41 ± 0.14b 1.57 ± 0.33b
C24:0 1.33 ± 0.08b 2.48 ± 0.15b 3.29 ± 0.21b 1.69 ± 0.13b 2.54 ± 0.27 3.66 ± 0.31c 1.53 ± 0.26b 1.27 ± 0.17c 2.7 ± 0.31c 1.86 ± 0.47b 2.68 ± 0.48b 2.29 ± 0.53b
Total SFA 26.44±2.03a 33.97±3.12c 40.49±2.30c 29.00±3.41a** 34.42±3.27b 44.19±3.06c*** 29.2±2.10a** 30.98±2.92a 40.67±2.94c 32.84±2.10a*** 34.57±3.41a 38.78±3.07b
C16:1n-7 6.75 ±1.51a 8.80±1.55a 9.984±2.47c 7.82 ±1.51a* 9.41±1.59b* 12.06±3.02c** 6.40±0.46a 7.37±1.42b 10.12±1.21c 5.92±0.47a* 6.68±1.25b** 9.42±1.36c
C18:1n-7 2.90±0.50a 4.25±0.58b 5.12±2.72c 3.51±0.78a* 4.12±0.68b 6.43 ±0.18c* 3.81±0.27a* 4.21±0.72b 6.87±1.36c* 3.36±0.27a 3.86±0.58a* 4.11±1.27b
C18:1n-9 12.23±2.40a 15.40±2.05a 17.95±3.22b 12.85±2.74a 13.78±2.02b* 16.08±0.87c* 13.53±1.13a 14.31±3.05b 17.31±2.87c 12.68±1.41a 13.57±2.42b* 19.52±2.31c**
C20:1 1.11±0.32a 1.96±0.65b 3.04±0.61c 1.27±0.12a 1.62±0.44a 2.08±0.41b 1.09±0.21a 1.51±0.66b 2.12±0.37a 1.31±0.20b 1.39±0.36a 0.97±0.02b
C22:1 1.02 ± 0.05a 1.68 ± 0.05b 2.74 ± 0.07b 1.08 ± 0.03a 1.31 ± 0.03a 1.55 ± 0.03a 1.01 ± 0.05a 1.32 ± 0.03a 1.83 ± 0.07a 0.98 ± 0.01a 1.09 ± 0.06b 0.65 ± 0.03a
C24:1 0.76 ± 0.01b 1.14 ± 0.06b 1.63 ± 0.04b 1.01 ± 0.02b 1.08 ± 0.06b 1.49 ± 0.02c 0.88 ± 0.03b 0.95 ± 0.04c 1.78 ± 0.06b 0.73 ± 0.01b 1.06 ± 0.02b 0.18 ± 0.01c
Total MUFA 24.77±2.05a 33.23±2.56b 40.47±3.67c 27.54±2.31a** 31.32±3.73b* 39.69±2.73c 26.72±1.58a* 29.70±3.41b* 40.03±3.21c 24.98±1.23a 27.65±2.73b** 34.85±3.14c***
C18:2n-6 (LA) 4.68±0.97a 5.40±1.96b 7.32±1.51b 5.69±0.82a* 3.78±1.25b*** 3.02±1.78b*** 6.32±2.48a** 4.92±1.63b 3.96±0.69c*** 7.01±1.57a*** 5.36±0.44b 4.00±0.73c***
C20:2n-6 0.96 ± 0.01a 0.42 ± 0.02b 0.01 ± 0.00a 1.48 ± 0.16a 1.09 ± 0.12b 0.36 ± 0.01c 0.09 ± 0.01b 1.34 ± 0.4c 0.46 ± 0.02b 1.39 ± 0.3c 1.19 ± 0.2b 0.75 ± 0.1c
C20:3n-3 1.06 ± 0.04b 0.51 ± 0.05c 0.03 ± 0.01b 1.29 ± 0.03c 1.12 ± 0.05b 0.49 ± 0.03b 0.03 ± 0.02b 1.2 ± 0.1b 0.57 ± 0.03b 0.99 ± 0.07b 1.2 ± 0.4b 0.46 ± 0.01c
C20:4n-6 (ARA) 11.24±2.35a 12.23±2.65b 13.05±2.29c 12.44±7.78a 11.22±2.42b* 11.23±1.65a* 9.98±1.75a* 7.78±1.57b** 9.52±1.38a*** 10.02±1.41a 9.08±1.36b*** 7.14±0.68c***
C20:5n-3 (EPA) 7.93 ±1.46a 3.49±0.51b 1.22±0.54c 8.57 ±1.33a* 4.98±1.94b* 1.74±2.31a 5.67±0.36a** 3.98±1.72b 3.21±0.42a*** 6.41±0.51a* 5.31±0.87b** 4.12±0.35c***
C22:4n-6 1.00 ± 0.03b 0.59 ± 0.06b 0.01 ± 0.00b 1.25 ± 0.07c 1.04 ± 0.05b 0.5 ± 0.02c 0.21 ± 0.01b 1.43 ± 0.6b 0.13 ± 0.01c 1.69 ± 0.5b 2.08 ± 0.8b 0.88 ± 0.02c
C22:5n-3 (DPA) 1.78±0.23a 1.27±0.72a 0.78±0.06b 2.42±0.27a** 1.63±0.12a 1.02±0,05a 1.31±0.68a 0.97±0.09b 0.98±0.08b 1.76±0.21a 1.36±0,01a 1.02±0.04b
C22:6n-3 (DHA) 13.68±2.78a 8.78±2.86b 1.67±0.57c 12.63±2.56a* 8.46±1.41b 6.98±2.97c*** 14.85±2.35a* 10.37±1.52b** 7.21±0.33c*** 13.78±2.27a 12.21±1.41b** 9.42±0.84c***
PUFA-n-3 23.39±1.15a 13.54±1.09c 3.67±0.08c 23.62±3.06a 15.07±0.76c** 9.74±0.63c*** 21.83±2.58a* 15.32±2.13c* 11.40±1.3c*** 21.95±1.61a* 18.88±1.2b*** 14.56±2.33c***
PUFA-n-6 15.92±1.22a 17.63±1.11b 20.37±1.98c 18.12±0.97a** 15.00±1.35a* 14.25±1.53b*** 16.30±1.00a 12.70±1.18b** 13.48±1.3b*** 17.03±1.22a* 14.44±1.37b** 11.14±0.51c***
Total PUFA 42.33±1.32a 32.69±2.78b 24.09±2.09c 45.77±3.02a* 33.32±2.78b 25.34±3.12c 37.8±3.1a*** 31.99±3.12b 26.04±3.07c 43.05±4.08a 37.79±2.44b 27.79±3.51b*
Continued
FA HSF HSF+0.5% pp HSF+1% pp
1 day 20 days 90 days 1 day 20 days 90 days 1 day 20 days 90 days
C14:0 5.28±0.63a* 6.92±1.51b* 9.01±1.44c*** 4.69±0.51a 5.08±1.03b 6.27±1.07c 4.73±0.27a 5.27±1.22b 6.55±1.43b**
C16:0 16.98±3.08a 17.86±2.44b** 22.18±1.55c 15.21±2.45a* 16.12±2.76b*** 20.36±1.28c** 15.39±2.64a* 15.68±2.31a*** 18.6±1.59b***
C18:0 3.26±0.92a 4.13±0.87b 7.41±0.51c 3.12±0.84a 4.01±0.56a 6.81±0.36c 3.58±0.72a 3.78±0.56a* 5.62±0.36b**
C20:0 0.61 ± 0.01a 0.73 ± 0.03b 1.59 ± 0.03b 0.24 ± 0.00a 0.84 ± 0.02b 1.42 ± 0.16a 0.57 ± 0.02b 0.69 ± 0.03b 1.09 ± 0.07b
C22:0 0.92 ± 0.04b 1.05 ± 0.13b 1.87 ± 0.05c 0.69 ± 0.01b 1.02 ± 0.07c 1.89 ± 0.12b 0.97 ± 0.06b 1.01 ± 0.01a 1.64 ± 0.08b
C24:0 1.61 ± 0.12c 2.21 ± 0.16b 2.55 ± 0.15b 2.1 ± 0.23c 1.9 ± 0.15b 1.91 ± 0.24b 1.7 ± 0.18b 1.86 ± 0.09a 2.08 ± 0.16b
Total SFA 28.66±3.24a** 32.90±3.15b 44.61±4.05c*** 26.05±2.54a 28.97±2.69b** 38.66±3.12c** 26.94±3.35a 28.29±2.56b** 35.58±2.86c***
C16:1n-7 8.43±1.26a** 8.78±1.21a 13.76±2.58c** 7.21±0.96a 7.92±1.26a 11.32±2.37c* 7.44±0.82a 7.66±1.41a 9.78±2.57b
C18:1n-9 11.46±2.52a 12.02±2.15b 17.45±2.41c 10.23±1.78a 11.12±2.27b 14.36±2.28c 10.51±1.45a 10.77±2.31b 11.90±2.45b
C18:1n-7 4.36±0.89a*** 4.99±0.38b 7.16±0.23c** 3.78±0.27a* 4.97±0.12b 6.46 ±0.47c 3.97±0.68a* 4.37±0.12b 5.86±0.47b
C20:1 1.21±0.57b 1.32±0.61a 1.38±0.72c 1.16±0.21c 1.48±0.22a 1.66±0.63a 1.2±0.18c 1.31±0.09b 1.43±0.12c
C22:1 1.07 ± 0.03a 1.46 ± 0.05a 1.16 ± 0.12b 1.01 ± 0.01b 1.10 ± 0.01b 1.31 ± 0.02b 1.16 ± 0.04b 1.68 ± 0.07b 1.63 ± 0.05b
C24:1 0.96 ± 0.04b 1.16 ± 0.07b 0.84 ± 0.01b 0.95 ± 0.00c 1 ± 0.12b 1.15 ± 0.03b 1.06 ± 0.01b 0.73 ± 0.02c 0.99 ± 0.03b
Total MUFA 27.49±2.72a** 29.73±4.21b 41.75±2.86c 24.34±2.15a 27.59±2.76b** 36.26±3.12c*** 25.37±2.68a 26.52±2.39b** 31.59±3.28b***
C18:2n-6 (LA) 6.21±0.74a*** 4.21±1.13a 2.27±0.14b*** 7.04±0.66a*** 6.54±0.76a* 4.31±0.21b*** 7.01±0.66a*** 6.81±0.15a** 6.01±0.18b**
C20:2n-6 1.22 ± 0.2b 1.51 ± 0.6c 0.89 ± 0.01b 1.96 ± 0.5b 1.79 ± 0.6c 1.44 ± 0.3b 1.65 ± 0.4c 2.48 ± 0.54b 1.22 ± 0.17c
C20:3n-3 1.94 ± 0.5a 1.27 ± 0.1b 0.87 ± 0.01b 1.59 ± 0.2c 1.55 ± 0.36b 0.87 ± 0.01a 1.61 ± 0.7c 1.73 ± 0.31c 1.12 ± 0.09a
C20:4n-6 (ARA) 13.25±2.31a** 12.08±1.31b 8.97±1.13a*** 11.78±2.58a 11.12±1.26a 7.33±1.09c*** 11.66±2.45a 11.36±1.13a 10.16±1.19c**
C20:5n-3 (EPA) 7.12±1.15a 5.96±1.26b** 1.21±0.27c 7.68±1.08a 6.96±1.37b*** 4.95±0.83c*** 7.51±1.11a 7.38±1.78b*** 6.06±0.27c***
C22:4n-6 2.05 ± 0.02b 1.89 ± 0.03c 0.92 ± 0.02b 2.47 ± 0.54c 2.58 ± 0.33b 2.39 ± 0.12b 3.01 ± 0.8a 3.44 ± 0.92b 1.16 ± 0.53b
C22:5n-3 (DPA) 2.51±0.14a** 2.04±0.17a* 1.02±0.01b 3.07±0.27a** 2.47±0.24a** 1.64±0.03b* 3.05±0.13a** 2.81±0.51a** 2.51±0.15b***
C22:6n-3 (DHA) 12.41±2.22a 11.09±1.28b*** 6.14±2.37c*** 13.61±2.52a 12.79±1.09b*** 9.26±1.99c*** 13.87±2.42a 13.16±1.28b*** 12.65±1.37c***
PUFA-n-3 22.06±1.14a 19.11±1.67b*** 8.37±0.78c*** 24.37±2.08a* 22.23±1.53b*** 15.86±1.47c*** 24.43±2.69a* 23.35±1.31a*** 21.23±1.70b
PUFA-n-6 19.46±1.42a** 16.31±1.27b* 11.25±0.99c*** 18.83±1.66a** 17.67±2.04a 11.64±1.38c*** 18.67±0.76a** 18.17±1.28a* 16.18±1.54b***
Total PUFA 46.71±4.31a** 40.05±3.51b** 22.29±2.05c 49.29±4.57a*** 45.80±2.48b*** 32.19±2.24c*** 49.37±3.34a*** 49.17±4.97b*** 40.89±2.51c***

Values are means ± standard deviation (n = 6); Different superscript letters in the same column represent significant differences (p < 0.05, one-way ANOVA with Tukey test); Significant differences between FF and CSF and HSF with or without antioxidant extract during the three storage periods are presented (*p < 0.05, **p < 0.01, ***p < 0.001); NS = non-significant, FAs = fatty acids, SFAs = saturated fatty acids, MUFAs = monounsaturated fatty acids, PUFAs = polyunsaturated fatty acids, LA = linoleic acid, ARA = arachidonic acid, EPA = eicosapentaenoic acid, DPA = docosapentaenoic acid, DHA = docosahexaenoic acid, PUFA-n-3 = Omega-3 fatty acid, PUFA-n-6 = Omega-6 fatty acid.

Regarding the variation in the FA composition in FF, it was shown that the significant increase in total SAFs was dominated by palmitic acid (C:16), which showed the highest increase (40.49%) after the 90 days of storage. This significant increase was also continued at different rates from day 1 to day 90 of storage in both CSF and HSF covered with or without 0.5 and 1% of the antioxidant extract. In addition, the significant increase in total MUFAs in FF was dominated by oleic acid (C18:1n-9), with the highest increase (17.95%) after 90 days of storage. This increase also persisted in differential proportions from day 1 to day 90 of storage in both CSF and HSF covered with or without 0.5 and 1% of the extract. However, the significant reduction in PUFAs was prevailed by docosahexaenoic acid (C22:6n-3) in FF, with the highest decrease (1.67%) found after the 90 days of storage. This decrease also proceeded to different percentages from day 1 to day 90 of storage in both CSF and HSF covered with or without 0.5 and 1% of the extract. Similar results of the significant reduction in PUFAs at the end of smoking (90 days) due to a decrease in PUFAs of the n-3 series (Tot n-3) and a simultaneous increase in MUFAs have been found in Argyrosomus regius fillets cold-smoked in combination with 1% Halocnemum strobilaceum antioxidant for 35 days of storage at 4 °C (Messina et al., 2021). In comparison with the previous work on S. lucioperca, the current increase in total SAFs is consistent with that also found in CSF and HSF by Bouriga et al. (2022). However, the increase in total MUFAs recorded here is not in line with that reported by these authors, as total MUFAs showed a significant decrease in CSF and HSF. Nevertheless, there is agreement about the decrease reported here and there in total PUFAs in both CSF and HSF. Therefore, we can assume that the D. salina polyphenol antioxidant extract had a significant effect not only on the increase in SAFs and MUFAs but also on their percentages in both CFS and HSF.

 Free fatty acid (FFA) content in fresh, cold-, and hot-smoked fillets

The profile of the FFA content in FF showed a progressive significant increase (p < 0.05) over the 1, 20, and 90 days of storage. This significant increase was gradually decreased in CSF covered with or without 0.5 and 1% of D. salina polyphenol antioxidant extract and then increased again in HSF during the three periods of storage but with the highest level in HSF (Table 4). Overall, the formation of FFA, as a marker of lipolysis in smoked oil fish fillets during storage, has been so far associated with fat content, lipolytic activity, and temperature (Bouriga et al., 2020). In S. lucioperca FF, FFA was represented by 1.24% of total lipids, a level which is within the range (1-7%) found in crude fish (Bimbo, 1998) and lower than that reported by Aidos et al. (2001) in herring oil. This level was significantly increased (7.57%) as the storage periods increased, with the highest increase (7.89%) shown in HSF after 90 days of storage. Similar results have previously been recorded by Bouriga et al. (2020). Such an increase, particularly in HSF, can be attributed to the lipolysis generated by lipases or phospholipases (Chaijan et al., 2006; Bouriga et al., 2020). In addition, the results obtained indicated that both smoking and the addition of 0.5 and 1% of the polyphenol extracts were below the acceptable limit (7 g/100g).

Variations in the free fatty acids (FFAs, in g/100g lipids) and total volatile basic nitrogen (<italic>TVB-N, in</italic> mg MDA/kg lipids) values in fresh (FF), cold-smoked (CSF), hot-smoked (HSF) fillets of <italic>Sander lucioperca</italic>, and cold-smoked (CSF), and hot-smoked (HSF) fillets covered with <italic>Dunaliella salina</italic> polyphenol (pp) antioxidant extract at 0.5 and 1% concentrations during the three storage periods
Parameter Storage days FF CSF CSF+0.5% pp CSF+1% pp HSF HSF+0.5% pp HSF+1% pp ANOVA
FFAs 1 1.24±0.07a 1.78±0.17a 1.52±0.04a 1.38±0.02a 3.42±0.10b 2.14±0.16c 2.04±0.37c ***
20 3.76±0.49a 2.39±0.71c 2.05±0.13c 1.69±0.39c 4.27±0.38b 3.47±0.44a 3.12±0.37a ***
90 7.57±1.21a 4.96±0.72b 4.23±0.47c 3.29±0.62c 7.89±1.53a 4.92±0.47b 4.47±1.36b ***
TVB-N 1 5.32±0.47a 3.73±0.06b 3.48±0.10b 2.78±0.10a 2.86±0.12a 2.48±0.09a 2.28±0.17b ***
20 10.06±0.09a 6.27±0.27b 5.2±0.02b 4.2±0.28a 6.05±1.23a 4,89±0.16a 3.21±1.69b ***
90 22.21±0.43a 19.42±1.22a 16.8±1.52b 15.7±1.02c 19.42±1.22a 13.62±1.38b 12.7±1.31b ***

Values are means ± standard deviation (n = 3); Diffrent superscript letters in the same column represent significant differences (p < 0.05, one-way ANOVA with Tukey test); Significant differences between FF and CSF and HSF with or without antioxidant extract during the three storage periods are presented (***p < 0.001).

 Total volatile base nitrogen (TVB-N) values in fresh, cold-, and hot-smoked fillets

The TVB-N indicated significantly increased levels (p < 0.001) in FF during the three storage periods, with the highest level found after 90 days of storage. In addition, differentially significant decreased levels (p < 0.05) were found in CSF and CSF covered with 0.5 and 1% of the extracts, as well as in HSF, and HSF covered with 0.5 and 1% of the extracts, during the three storage periods, with the highest similar level in CSF and HSF, especially after 90 days of storage, compared to CSF and HSF covered with 0.5 and 1% of the extracts (Table 4). This significant decrease (p < 0.001) in the level of TVB-N in CSF and HSF covered with or without 0.5 and 1% of the covering extracts, compared to FF during the three storage periods, is inconsistent with the findings of Karsli and Caglak (2021), who reported a gradual significant increase in TVB-N during the storage of the smoked fish. In this respect, Arous et al. (2014) reported that the increase in TVB-N levels resulted from the production of dimethylamine, trimethylamine, and ammonia associated with the destructive effect of microorganisms on proteins during storage. In addition, Karsli and Caglak (2021) attributed the increase in the TVB-N levels to the loss of water during smoking and the increase in proteolytic activity during salting and smoking. Therefore, in addition to the water loss during smoking, we can assume here that the significant decrease in the TVB-N levels in CSF and HSF with or without 0.5 and 1% of the covering extracts was due to increased deamination of adenosine monophosphate or amino acids, which led to an increase in ammonia release, resulting from the combined effect of smoking and the addition of D. salina extract (Bouriga et al., 2022).

 Conclusions

The results showed a significant increase in proteins, lipids, FFAs, and DDPH contents, and a decrease in PV, TBARS, and TVB-N levels in cold (CSF) and hot (HSF) smoked fillets of Sander lucioperca covered with or without 0.5 and 1% of Dunaliella salina polyphenol antioxidant extract and stored for 1, 20, and 90 days compared to fresh fillets (FF). Saturated (SFAs) and monounsaturated (MUFAs) fatty acids exhibited a significant increase in FF and CSF and HSF covered with or without polyphenol extract. Total polyunsaturated fatty acids (PUFAs) revealed a significant decrease in FF and CSF and HSF with or without the extract. Therefore, cold and hot smoking processes and the addition of 0.5 and 1% of natural D. salina polyphenol antioxidant extract was a valuable and promising method to improve the biochemical quality, shelf-life, and consumption of S. lucioperca fillets stored for up to 90 days in a refrigerator at 0-4 °C.

 Acknowledgments

The authors declare that they did not receive any specific grant from the funding agents for this work. The authors would like to express their gratitude and thanks to the fishermen who helped in catching the Sander lucioperca samples and preparing the fillets, as well as collecting the Dunaliella salina samples.

 References Aidos I Van-der-Padt A Boom RM Luten JB 2001 Upgrading of Maatjes herring byproducts: production of crude fish oil J. Agric. Food. Chem. 49 3697 3704 10.1021/jf001513s Amaral RA Pinto CA Lima V Tavares J Martins AP Fidalgo LG Silva AM Gil MM Teixeira P Barbosa J Barba FJ Saraiva JA 2021 Chemical-based methodologies to extend the shelf life of fresh fish-A Review Foods 10 2300 10.3390/foods10102300 AOAC 1990 AOAC official methods of analysis 15 Association of Official Analytical Chemists Arlington, VA AOAC 1998 Official Methods and Recommended Practices of the American Oil Chemists’ Society 5 Firestone D Official method Cd 19-90, 2-Thiobarbituric acid value Champaign, Ill Arous WH El-Bermawi NM Shaltout OE Essa MAE 2014 Effect of adding different carotenoid sources on growth performance, pigmentation, stress response and quality in red Tilapia (Oreochromis spp.) Middle East J. Appl. Sci. 4 4 988 999 Aubourg SP Lehmann I Gallardo JM 2002 Effect of previous chilled storage on rancidity development in frozen horse mackerel (Trachurus trachurus) J. Sci. Food. Agric. 82 1764 1771 10.1002/jsfa.1261 Ayeloja AA Jimoh WA Adetayo MB Abdullahi A 2020 Effect of storage time on the quality of smoked Oreochromis niloticus Heliyon 6 1 e03284 10.1016/j.heliyon.2020.e03284 Bersuder P Hole M Smith G 1998 Antioxidants from a heated histidine-glucose model system. I: Investigation of the antioxidant role of histidine and isolation of antioxidants by high-performance liquid chromatography J. Am. Oil. Chem. Soc. 75 181 187 10.1007/s11746-998-0030-y Bimbo AP 1998 Guidelines for characterizing food-grade fish oil Inter. News Fat. Oils R. Mat. 9 5 473 483 Bouriga N Ben Ismail H Gammoudi M Faure E Trabelsi M 2012 Effect of smoking-method on biochemical and microbiological quality of Nile Tilapia (Oreochromis niloticus) Am. J. Food Technol. 7 679 689 Bouriga N Bejaoui S Jemmali B Quignard JP Trabelsi M 2020 Effects of smoking processes on the nutritional value and fatty acid composition of Zander fish (Sander lucioperca) Grasas y Aceites 71 1 e340 10.3989/gya.1061182 Bouriga N Bahri WR Mili S Massoudi S Quignard JP Trabelsi M 2022 Variations in nutritional quality and fatty acids composition of sardine (Sardina pilchardus) during canning process in grape seed and olive oils J. Food Sci. Technol. 10.1007/s13197-022-05572-4 Burton GW Ingold KU 1984 Beta-carotene: an unusual type of lipid antioxidant Science 224 4649 569 573 Cakmak YS Kaya M Asan-Ozusaglam M 2014 Biochemical composition and bioactivity screening of various extracts from Dunaliella salina, a green microalga EXCLI J. 13 679 690 10.17877/DE290R-6669 Chaijan M Benjakul S Visessanguan W Faustman C 2006 Changes of lipids in sardine (Sardinella gibbosa) muscle during iced storage Food. Chem. 99 83 91 10.1016/j.foodchem.2005.07.022 Chaillou S Chaulot-Talmon A Caekebeke H Cardinal M Christieans S Denis C Desmonts MH Dousset X Feurer C Hamon E Joffraud J-J La Carbona S Leroi S Leroy S Lorre S Macé S Pilet M-F Prévost H Rivollier M Roux D Talon R Zagorec M Champomier-Vergès M-C 2015 Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage ISME J. 9 1105 1118 10.1038/ismej.2014.202 Commissione delle Comunità Europee 95/149/CE 1995 Decisione della Commissione, dell’8 marzo 1995, che fissa i Valori Limite di ABVT (Azoto Basico Volatile Totale) per Talune Categorie di Prodotti della Pesca e i Relativi Metodi D’analisi Commissione delle Comunità Europee Bruxelles, Belgium Connell JJ 1995 Control of fish quality Fishing News Books 4 Blackwell Science, Ltd Oxford DE Souza-Franco MS Viegas EMM Kronka SN Vidotti RM Assano M Gasparino E 2010 Effects of hot and cold smoking process on organoleptic properties, yield and composition of matrinxa fillet Rev. Bras. de Zootec. 39 4 695 700 10.1590/S1516-35982010000400001 EEC 2005 Total volatile basic nitrogen (TVB-N) limit values for certain categories of fishery products and analysis methods to be used. European Commission Regulation (EC) No 2074/2005 of 5 December 2005 Official J. Euro. Union 36 39 Garavaglia J Markoski MM Oliveira A Marcadenti A 2016 Grape seed oil compounds: biological and chemical actions for health Nutr. Metab. Insights 9 59 64 10.4137/NMI.S32910 Huang DJ Ou BX Prior RL 2005 The chemistry behind antioxidant capacity assays J. Agric. Food Chem. 53 6 1841 1856 10.1021/jf030723c Karsli B Çağlak E 2021 Combination effect of hot smoking and vacuum packaging on quality parameters of refrigerated thornback ray (Raja clavata Linnaeus, 1758) Int. J. Agric. Environ. Food. Sci. 5 1 42 50 10.31015/jaefs.2021.1.6 Kolakowska A 2002 Lipid oxidation in food systems Sikorski ZE Kolakowska A Chemical and functional properties of food lipids CRC Press FL, USA 133 160 Kostaki M Giatrakou V Savvaidis IN Kontominas MG 2009 Combined effect of MAP and thyme essential oil on the microbiological, chemical and sensory attributes of organically aquacultured sea bass (Dicentrarchus labrax) fillets Food Microbiol. 26 5 475 482 10.1016/j.fm.2009.02.008 Madhavi DL Singhal RS Kulkarni PR 1996 Technological aspects of food antioxidants Madhavi DL Deshpande SS Salunkhe DK Food Antioxidants: Technological, Toxicological, and Health Perspectives Marcel Dekker New York 159 Mariotti F Tome D Mirand PP 2008 Converting nitrogen into protein-Beyond 6.25 and Jones’ factors Crit. Rev. Food Sci. 48 177 184 10.1080/10408390701279749 Messina CM Bono G Renda G La Barbera L Santulli A 2015 Effect of natural antioxidants and modified atmosphere packaging in preventing lipid oxidation and increasing the shelf-life of common dolphinfish (Coryphaena hippurus) fillets LWT - Food Sci. Technol. 62 1 271 277 10.1016/j.lwt.2015.01.029 Messina CM Arena R Ficano G Randazzo M Morghese M La Barbera L Sadok S Santulli A 2021 Effect of cold smoking and natural antioxidants on quality traits, safety and shelf life of farmed meagre (Argyrosomus regius) fillets, as a strategy to diversify aquaculture products Foods 10 11 2522 10.3390/foods10112522 Silbande A Adenet S Chopin C Cornet J Smith-Ravin J Rochefort K Leroi F 2018 Effect of vacuum and modified atmosphere packaging on the microbiological, chemical and sensory properties of tropical red drum (Sciaenops ocellatus) fillets stored at 4°C Int. J. Food Microbiol 266 31 41 10.1016/j.ijfoodmicro.2017.10.015