Rapid Multiparameters Approach to Differentiate Fresh Skinless Sea Bass (Dicentrarchus labrax) Fillets from Frozen-Thawed Ones

ABSTRACT Food authenticity is one of the major issues in the mind of today’s consumers. The sale of frozen-thawed fish fillets under the “fresh fillet” label is considered as a commercial fraud. However, their close sensory properties complicate the differentiation. This study focused on analyzing the composition of exudate (pressured flesh juice) in order to rapidly differentiate between fresh and frozen-thawed skinless fillets of sea bass (Dicentrarchus labrax). Protein concentration, α-D-glucosidase specific activity, nucleotides and related compounds (NRCs) concentration, and free calcium concentration were measured in exudates corresponding to fresh or frozen-thawed sea bass fillets. Significant increases of these four parameters were observed in exudates from frozen-thawed fillets, especially with a twofold increase in NRCs and free calcium concentrations. These results suggest that NRCs and free calcium concentrations can be promising indicators to rapidly detect mislabeling of fresh fillets.


Introduction
The consumption of fish has increased recently; in France for example, there was an increase of 24.9 kg per habitant in 2015, an increase of 0.9 kg in 5 years (FranceAgrimer, 2016). Consumers prefer to eat freshly caught fish, but frozen storage is widely used to protect these products against microorganism growth during the long transportation to consumers (Pavlov, 2007). Some businessmen are tempted to cheat and label thawed fillets as "fresh fillets" in the market. Indeed, such exercise could be very lucrative when thawed fish are sold out of the fishing season. Nevertheless, the final consumer should be appropriately informed when fishery products have been defrosted, according to article 28 of Regulation EU 1169. Several methods, such as the observation of eye lens opacity (Love, 1956;Yoshioka and Kitamikado, 1983) and sensory analysis (Parisi et al., 2002) can be used to differentiate frozen-thawed whole fish from fresh. A large amount of fish is sold in the form of fillet, and several instrumental methods have been tested to differentiate a fresh fillet from a frozen-thawed one. For example, the use of spectroscopy in visible and nearinfrared has shown conclusive results on grass carp (Ctenopharyngodon idella) (Cheng et al., 2015). Utilizing the front face fluorescence spectroscopy to specifically measure the Nicotinamide-Adenine Dinucleotide (NADH) fluorescence spectra might also be considered as a promising tool for testing whiting (Merlangius merlangus) (Karoui et al., 2006). The use of impedance spectroscopy to identify European sea bass (Dicentrarchus labrax), Atlantic salmon, and sea bream (Sparus aurata) frozen samples (Fernández-Segovia et al., 2012;Fuentes et al., 2013;Vidaček et al., 2008) has also been investigated. Others techniques such as the analysis of salmon DNA degradation by a Comet Assay have been used to distinguish frozen-thawed fillets (Le Grandois et al., 2013). Certain volatile compounds were also identified as potential markers to differentiate between fresh and frozen-thawed counterpart sea bream, cod (Gadus morhua), and European sea bass (Leduc et al., 2012). Elevated enzymatic activities of α-D-glucosidase (AG) (Duflos et al., 2002) and lactate dehydrogenase (Diop et al., 2016) in species like whiting (Merlangus merlangus), plaice (Pleuronectes platessa), mackerel (Scomber scombrus), and sea bream (Sparus aurata) also have been reported to highlight the thawing of fish. Among these methods, however, few tests have been carried out with the fish fillet exudate. The freezing of fish results in the leakage of enzymes into the exudate, and an increase in AG activity could then be measured (Duflos et al., 2002). For example, the protein composition of sea bass fillet exudate has been tested, and changes led to a differentiation of frozen-thawed fillets (Ethuin et al., 2015).
The aim of this work was to study the composition of the exudate in order to develop reliable, complementary, and fast tools to identify frozen-thawed sea bass fillets. Protein concentration and AG specific activity, already tested in other fish species exudates (Table 1), were measured in sea bass exudates. Furthermore, two additional parameters, nucleotides and related compounds (NRCs) concentration and free calcium concentration, were measured in this study. The use of these concentrations in order to differentiate fresh and frozen-thawed fillets was, to our knowledge, investigated for the first time ( Table 1).The use of these two parameters would allow fish industries to easily implement quick evaluation methods. This set of four indicators was also investigated in order to support industry for the rapid identification of potential fraudulent labelling of frozen-thawed fish fillet.

Fish material
The sea bass (Dicentrarchus labrax) sample was acquired from Aquanord sea farm (Gravelines, France). This local supplier of farmed sea bass allowed for a model with controlled environmental conditions and freshness. Strictly controlled growing and breeding conditions were: temperature 18 ± 5°C, pH 8.2, total ammonia <30 mol/L, and dissolved oxygen level over 99% (v/v) saturation (7°ppm). Sea bass (average body weight 500 ± 150 g) were slaughtered via asphyxia/hypothermia and were kept on ice (0-2°C). Fish samples were rapidly skinned (less than 90 min after slaughtering) and filleted by the Centre de Formation des Produits de la Mer et de la Terre (CFPMT: Training Center for sea and land products) (Boulogne-sur-Mer, France). Cling-film protected fillets were stored in polystyrene boxes with crushed ice and kept at 0-2°C. Ten fish produced 20 fillets that were divided in two groups: 10 fillets corresponding to the fresh condition were subjected to immediate analysis; 10 fillets corresponding to the frozen-thawed condition were frozen at −30°C, stored at −20°C for 40 days, then removed from the freezer, and stored between 0°C and +4°C for 24 h to thaw completely. Each analysis was performed on a fresh and a frozen-thawed fillet provided from the same fish.

Fish exudates preparation and protein extraction
Fish exudates were obtained from flesh juice (Morel, 1979) after centrifugation (12,500°rpm at 5°C) (Ayala et al., 2005;Duflos et al., 2002;Tironi et al., 2010), according to the method of Ethuin et al. (2015). Each exudate was prepared from a single fillet, and totally 10 exudates from fresh fillets and 10 exudates from frozen-thawed fillets were obtained. Protein concentration in exudate was determined with the Bradford method (Bradford, 1976) using the Bio-Rad reagent (Bio-Rad, Marnes-la-Coquette, France) and bovine serum albumin as standards. All analyses were performed in triplicate. Total analyzing time for fish exudates preparation and protein extraction was less than 1 h. Press juice Rehbein et al. (1978) α-glucosidase, β-glucuronidase, β-galactosidase, β-N-acetyl-glucosaminidase

Cod
Press juice Rehbein (1979)   Determination of AG specific activity AG (EC 3.2.1.20) specific activity was assayed in accordance with the method of Duflos et al. (2002). The reaction mixture was incubated at 37°C for 2 h and followed by the addition of 1 mL potassium hydroxide (0.2 M) to terminate the reaction. Control reactions were performed in the same way, but the exudate was added after the stopping reagent. The absorbance value was measured at 405 nm, and the specific enzyme activity was quantified in mg/protein/h. The analysis time was less than 3 h.

Detection of free calcium concentration
Free calcium concentration in the exudate was estimated with the Calcium Detection Kit (Abcam Company, Cambridge, UK) according to the manufacturer's protocol and previous report (Ansari et al., 2017). The analysis time was less than 30 min.

Determination of NRCs
The concentration of NRCs in exudates was assessed with UV spectrophotometry at 260 nm according to the method of Barcelo et al. (1986). Calibration curve was generated with denatured herring sperm DNA (ThermoFisher, Waltham, MA, USA). The analysis time was less than 30 min.

Statistical analysis
All experiments were performed in triplicate, and all values are given as means ± standard deviation. Statistical analyses were performed using "XLSTAT-Pro" 2014 (Addinsoft, Paris, France). Differences of means between fresh and frozen-thawed fillets, concerning the four studied parameters, were compared using the independent t-test (significance was defined at p < 0.05).

Results and discussion
In this study, four parameters were measured in exudates of fresh or frozen-thawed sea bass fillets in order to assess their effectiveness to differentiate these two storage conditions.

Protein concentration
Protein assays represented a necessary step for studying AG specific activity. Protein concentration (Figure 1) was about 1.7-fold higher in frozen-thawed fillets than in fresh fillets. Protein denaturation during frozen storage has been highlighted for many years (Dyer, 1951). The formation of large ice crystals during freezing led to cell membrane deterioration (Mazur, 2010). Release of intracellular proteins into the exudate could therefore have occurred. The increase of protein concentration observed in the frozen-thawed fillet exudate could be linked to such release (Ethuin et al., 2015). However, this conventional parameter in biochemistry cannot be used as the sole marker to certify the correctness of fish fillet labeling. Indeed, the protein content may vary according to fish species (Güner et al., 1998;Soriguer et al., 1997) and to the seasons (Albrecht-Ruiz and Salas-Maldonado, 2015; Jan et al., 2012), which could influence the labeling certification.

AG specific activity
The result showed an increase of AG specific activity (Figure 2) in frozen-thawed fillets. This increase of AG activity was already observed in thawed fillets of several fish species, including whiting and plaice (Duflos et al., 2002), sardine (Sardina pilchardus), horse mackerel, and anchovy (Engraulis engrasicolus) (Alberio et al., 2014). These observations indicated the disruption of membranes during the freeze-thawing. During thawing, the diluted external medium increases hydrostatic pressure in cells and induces the rupture of plasma membrane (Mazur, 2010;Takamatsu and Zawlodzka, 2006). This process leads to an exudate particularly enriched in intracellular enzymes. However, the observed statistically significant ratio of 1.47 between fresh and frozen-thawed fillets of sea bass was marginally highlighted due to important standard deviations.  Indeed, this parameter exhibited greater variations in fresh fillets (Supplementary data), which could potentially minimize the differences between fresh and frozen-thawed fillets. These greater standard deviations for AG activity were already observed in other species such as mackerel (Duflos et al., 2002) and cod (Benjakul et al., 2003). To our knowledge, the increase of AG specific activity in sea bass frozen-thawed fillets was reported for the first time. Nevertheless, the great standard deviation represented a limitation for using AG activity as a reliable indicator. Therefore, the use of other complementary markers will be essential to define a method that allows identification of mislabeled sea bass fillets.

Nucleotides and related compounds
NRCs are a group of compounds, including adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine 5ʹ-monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). After the death of fish, ATP is quickly transformed into IMP, via ADP and AMP degradation (Kassemsarn et al., 1963;Lakshmanam and Gopakumar, 1999). Postmortem nucleotide metabolism has been previously investigated in several fish species using chromatographic methods (Hong et al., 2015). In this study, the results showed NRC concentration ( Figure 3) twice as high in frozen-thawed fillets compared to fresh fillets. Özogul et al. (2005, 2010) studied contents of different nucleotide degradation compounds and observed an IMP concentration decrease parallel to an HxR and Hx increase. These measurements were realized in fresh sea bass stored on ice in a cold room maintained at 4 ± 1°C, but not in frozen sea bass. Moreover, the link between NRCs concentration and the freeze-thawing of fish fillets has not been greatly studied. For example, samples of milkfish (Chanos chanos) subjected to frozen storage at −20°C for 18 weeks showed a gradual decrease of ATP, ADP, AMP, and IMP levels, while HxR and Hx increased (Jiang et al., 1987). The ATP level also decreased in frozen-thawed gilthead sea bream (Mendes et al., 2001). NRCs concentration obtained in frozen-thawed sea bass fillets in this study could be correlated with results from studies mentioned above. According to current commercial practice, fresh fish can be stored 3-4 days on ice Bars indicate the standard deviation. * reported significant difference (p < 0.05) before sale. In the case of sea bass, such qualification of fish as "fresh" would remain to be investigated with the NRCs measurement described in this study. A twofold higher NRCs concentration in a frozen-thawed fillet could still be observed after storage on ice, even though the complete ATP degradation cycle in ice-stored sea bass proceeds at a slower pace than in most species (Kyrana and Lougovois, 2002). However, the objective of this NRCs measurement was to rapidly detect a freeze-thawing process but not to define the freshness loss of sea bass fillet during storage time. This increase of NRCs concentration in frozen-thawed sea bass fillets was probably due to the liberation of sarcoplasmic nucleotides linked to the DNA damage, which has been reported in thawed salmon (Le Grandois et al., 2013).

Free calcium concentration
Calcium (Ca 2+ ) is one of the main regulatory and signaling ions in all muscles. Therefore, it is interesting to investigate the free calcium concentration in sea bass fillets. An increase of free calcium concentration (Figure 4) was observed in frozen-thawed fillets with a ratio of 2 as compared with fresh fillets. To our knowledge, this parameter was never tested in exudates or in fish fillets. Calcium is stored in the endoplasmic reticulum, which is mainly responsible for the Ca 2+ signaling in muscle cells (Berridge, 2002). The membrane integrity of endoplasmic reticulum might have been disrupted during defrosting, and this could lead to a rise of free calcium. Moreover, myofibrillar proteins like tropomyosin are linked to Ca 2+ , and the frozen storage induced the denaturation of tropomyosin (Benjakul et al., 2003). A release of free calcium could result from a conformational change of the tropomyosin during this denaturation. This rapid assay of free calcium detection could be a promising indicator for industries to differentiate visually identical fresh fillets from frozenthawed ones.

Conclusion
The present study focused on measuring four parameters in the exudate in order to differentiate between fresh and frozen-thawed skinless fillets of sea bass. NRCs concentration and free calcium concentration were tested in sea bass exudates for the first time. Observation of higher value (twofold) in frozen-thawed fillets for both parameters highlights the potential of applying these two rapid and not expensive techniques as tools to differentiate fresh from frozen-thawed sea bass fillets. Moreover, NRCs and free calcium concentrations should be further investigated in other fish species in order to develop a rapid test and to improve the reliability of labeling for the consumer.