*3.1. Probiotics Viability Loss as a Function of Pressure and Temperature*

The viability loss of *Bifidobacterium bifidum* as a function of pressurization time in model systems of different pH values at various combinations of pressure (100−400 MPa) and temperature (20−40 ◦C) was described by first order kinetics (R<sup>2</sup> 0.84−0.99). The D-values were estimated at all studied pressure/temperature combinations (Table 1). The D-values decreased with increasing processing pressure and temperature at all levels tested, indicating the combined effect of temperature and pressure on the viability loss of these bacteria. In addition, *B. bifidum* exerted better survivability rates in high pH conditioned model systems, which corroborates previous studies reporting an optimal pH growth threshold in the range of 6.0−7.0 and a significant loss of survival for pH values below 4.5−5.0 [26,27].

At each temperature, the effect of pressure on the viability loss of *B. bifidum* was expressed through the pressure resistance constant (zP) (Table 1). When conducting the experiments in acid model system (4.80), the zP value ranged from 84.7 MPa for processing at 20 ◦C to 98 MPa for processing at 35 ◦C (R2 range: 0.88−0.97), while in model system with pH value close to the optimum pH of growth (6.50), the zP values ranged from 119 MPa when processing at 25 ◦C to 152 MPa when processing at 40 ◦C (R<sup>2</sup> range: 0.89−0.99). Taking into account that in both laboratory and industrial scale HP equipment the minimum pressure setting step is 50 MPa, the zP value for each model system was considered to be constant for the temperature range.


**Table 1.** Viability loss kinetic parameters of *Bifidobacterium bifidum.*

Values are means ± standard error of regression.

At each pressure, the effect of temperature on the viability loss of *B. bifidum* on the D-values was estimated using the Arrhenius equation and expressed in bacteriological terms by the thermal resistance constant (zT) (Table 1). All zT values were estimated for all pressures tested (R<sup>2</sup> range: 0.81–0.99), and it was observed that there is no specific trend when increasing the applied pressure, which is in accordance with findings in the literature for a variety of microorganisms [24,28].

The viability loss of *Lactobacillus casei* was studied in growth media of both pH values. Results indicate that in growth medium with pH value of 6.50 (Figure 1a) *L. casei* exhibited similar viability loss behavior to that of *B. bifidum*, regardless the pressure applied. However, when *L. casei* was studied in the acid growth medium (Figure 1b), significant difference (*p* < 0.05) was observed in its viability as compared to that of *B. bifidum*, especially in the pressure range of 100−200 MPa. When high pressures were applied (300−400 MPa) both probiotic microorganisms exhibited similar viability loss. Similar results were obtained for all the pressure/temperatures combinations tested.

The zP value of *L. casei* was found similar of that of *B. bifidum* when tested in acid environment (91.7 <sup>±</sup> 1.74 MPa in the temperature range of 20−35 ◦C, R2 range: 0.92−0.94), while when the experiments conducted in model system of high pH value (6.50), *L. casei* found to be slightly, yet significantly (*p* < 0.01), more baroresistant than the *B. bifidum* is (zP value equal to 208 ± 6.53 MPa in the temperature range of 25−<sup>40</sup> ◦C (R<sup>2</sup> range: 0.95−0.96). There are no available data in the literature that could explain the observed baroresistance of Lactobacilli; however, several shelf-life studies in fermented dairy products have showed that both microorganisms lose their viability in pH values below 4.5−5.0, and that in higher pH values Lactobacilli are significantly less sensitive as compared to Bifidobacteria [29–31]. All zT values of Lactobacilli were estimated for all pressures tested and, as in the case of *B. bifidum*, no specific trend was observed while increasing the applied pressure, with zT values ranging from 15.8 to 45.1 ◦C and from 23.4 to 40.7 ◦C when the bacteria were studied in growth medium of pH 4.80 and 6.50, respectively.

**Figure 1.** Comparison of *B. bifidum* (closed symbols-solid lines) and *L. casei* (open symbols-dashed lines) viability loss in broth model systems of pH value of (**a**) 6.5 and (**b**) 4.8, when pressurized at 30 ◦C (mean values ± standard deviation; linear lines represent the first order kinetics that describes the viability loss as a function of process time).
