## 3 Results and Discussion
### Effect of HP on the Bacterial Growth and the Shelf Life of Sea Bass Fillets  
Untreated and HP-treated sea bass fillets were stored in ice (0.0 \(\pm\) 0.2 \({}^{\circ}\)C) in the vacuum packaging used for HP processing. Fish samples that were treated at different pressures and at ambient temperature for 10 min were also stored in the same temperature but under aerobic conditions, after the laminated film packages were opened aseptically to simulate current commercial practices of distributing and selling fish in retail. Figure 2 shows the TVC growth of untreated and HP-treated sea bass fillets under different HP conditions, and Table 2 summarizes their growth parameters as they were estimated using the Baranyi and Roberts microbial growth model [39]. Regarding the effect of applied pressure on the growth of TVC under storage, it was observed that, in general, the higher the applied pressure, the lower was the estimated growth rate obtained (Table 2). An exception to this was the process with a pressure range above 200 MPa at 3 \({}^{\circ}\)C and 25 \({}^{\circ}\)C for 10 min. Under these processing conditions, a significantly longer lag phase was observed. These results are in accordance with previous studies which also confirmed that the application of pressure in the range of 200-400 MPa decreases the initial microbial counts by approximately 0.5-2.5 log CFU/g [42; 43; 44; 45] and affects the growth rate and lag phase of the dominant microflora, thus extending the shelf life of the final fish products. With respect to processing time, two different experiments in the pressure range of 100-400 MPa at a constant treatment temperature of 3 \({}^{\circ}\)C were performed, one for 5 min and one for 10 min (Figure 2b,c). An increase in the processing time by 5 min led to a significant (\(p<0.001\)) decrease in the growth rate or an increase in the duration of the lag phase (Table 2) and hence a significant (\(p<0.001\)) increase in the shelf life of the final product at 0 \({}^{\circ}\)C. Thus, in all other studied pressure-temperature combinations, HP treatment for 5 min was not investigated. Similar results have also been reported for mackerel fillets treated at 100-300 MPa for 2 and 5 min, showing that an increase of 3 min in the pressure holding time led to a slight yet significant decrease (i.e., 0.5 log CFU/g) in the total viable counts (TVCs) when a pressure of 100-300 MPa was applied, whereas when higher pressures were used, i.e., 500 MPa, the decrease in TVCs was higher than 1.0 log CFU/g, and for other spoilage microorganisms, e.g., H\({}_{2}\)S-producing bacteria, the corresponding decrease was equal to the non-measurable load [42]. observed only in the case in which 300 MPa was applied at the lowest temperature tested (3 \({}^{\circ}\)C), for which the highest growth rate was obtained. [PERSON] et al. [46] reported that the temperature of both the food and the pressure fluid play a significant role in the sensitivity of microorganisms under HP conditions and thus in their inactivation, with higher inactivation rates obtained either below or above the ambient temperature for mesophilic microorganisms. In fact, the pressure resistance of most microorganisms, spoilage and/or pathogens, is highest at their optimal growth temperature range, leading to higher inactivation at temperatures significantly lower or higher than their optimal growth temperature [47]. This is a result of the disruption of the cytoplasmic membrane, which becomes more rigid and changes from a liquid crystalline state to a semicrystalline gel state [28]. Similar results were also reported for the growth of psychotropic bacteria in fresh gifthead sea bream treated at 250 MPa at 3 or 15 \({}^{\circ}\)C for 5 min [29], in the inactivation of _Escherichia coli_ in beef mice pressurized at 300 MPa for 5 min and combined with temperatures of 0-20 \({}^{\circ}\)C [48], and for the total aerobic counts in minced chicken meat treated at 200-400 MPa and 0-40 \({}^{\circ}\)C for 5-15 min [49].