## 3 Results and Discussion  
Results of _A. acidoterrestris_ spores' inactivation of strains A and B in orange juice pressurized at 500 and 600 MPa in combination with different temperature regimes (45, 60, and 70 \({}^{\circ}\)C) are illustrated in Figures 1 and 2, respectively. To prevent the effect of the initial load of spores (\(N_{0}\)), data are presented in terms of log (\(N\)/\(N_{0}\)), where \(N_{0}\) was ca. \(10^{6}\) CFU/mL in all experiments. Inactivation kinetics did not follow a first-order pattern but an upward concavity was noticeable at all temperatures assayed. A possible explanation for this inactivation pattern is that the microorganism population is composed by several subpopulations (spores and vegetative cells in different physiological state), each one presenting a distinct inactivation pattern, which causes the non-linear curves [37; 38]. In other words the presence of subpopulations have symmetric or asymmetric heat resistance distributions [35; 39]. Thus, upward concavity could be considered as evidence of quick inactivation of the sensitive cells of the population, whereas the remaining survivors were more resistant to the lethal agent. Similar upward inactivation curves were obtained for _A. acidoterrestris_ during temperature-assisted HHP processing of fruit juices with different soluble solids content (up to 20 \({}^{\circ}\)Brix) [5], as well as in orange juice where high pressure processing was combined with mild heat treatment (45-65 \({}^{\circ}\)C), although inactivation kinetics were simulated by the Bigelow model (first order) despite the fact that deviation from linearity was evident. The results obtained in this work demonstrated that temperature was a major parameter in the inactivation of _A. acidoterrestris_ spores. Thus for a given high pressure level, the higher the temperature the higher the spore inactivation. It needs to be noted that when high-pressure processing was undertaken at 500 MPa/25 \({}^{\circ}\)C and 600 MPa/25 \({}^{\circ}\)C the reduction in spore population was less than 1.0-1.5 log cycles throughout the process for both strains of the bacterium (Supplementary Table S1). These results are in agreement with previous researchers [19; 28; 32] who reported little or no inactivation of _A. acidoterrestris_ spores when HHP was applied at ambient temperature. For the wild-type strain A of _A. acidoterrestris_, results indicated that at 45 \({}^{\circ}\)C there was ca. 3 log-cycles reduction of spore counts after 30 min processing time at both 500 and 600 MPa, whereas at 60 \({}^{\circ}\)C the same reduction magnitude was achieved in 1-2 min in both high pressures employed. For complete inactivation of the spores the required time was 15 min at 600 MPa/70 \({}^{\circ}\)C and 30 min at 500 MPa/70 \({}^{\circ}\)C. It is worth noting that for strain A an initial decrease of ca. 2.5 log-cycles was observed within the first minute of processing at 500 MPa followed by an increase thereafter until 5 min (Figures 1 and 2). This could be attributed to the dormancy of spores that was interrupted by the implementation of HHP treatment, since HHP has proven to induce the germination of spores [40]. A two-step HHP treatment has been proposed to inactivate alicyclobacilli spores. In the first step, high pressure induces spore germination whereas in the second step high pressure causes the inactivation of germinated spores [27]. For the reference strain B of _A. acidoterrestris_, results revealed ca. 1.5 log-cycles reduction after treatment at 500 MPa/45 \({}^{\circ}\)C for 10 min and ca. 1.3 log-cycles reduction under 600 MPa/45 \({}^{\circ}\)C for the same time (Figures 1 and 2). This is in agreement with [PERSON] et al. [28] who inoculated _A. acidoterrestris_ spores in tomato sauce at various pH values and applied high-pressure treatment at 600 MPa/40 \({}^{\circ}\)C for 10 min and reported spores reduction of ca. 1.5 log-cycles for tomato sauce at pH 4.2.