3.3.1. Viability of Starter Culture and Probiotic Bacteria in Yoghurt Samples

The total viable counts of the starter culture (*Str. thermophilus* and *L. bulgaricus*) in the plain probiotic yoghurt samples after HP treatment and during storage are given in Table 3.


**Table 3.** Viability of starter culture (as total numbers of *Str. thermophilus* and *L. bulgaricus*) in different treated plain and cherry-flavored yoghurt during storage.

\* Control samples were not HP-treated but only homogenized at 10 bar after the break of the coagulum. Letter D indicates the day of production. N.D. indicates microbial load below the acceptable limit of 7.0 log10 CFU/g of the total microbial counts for the starter culture at the end of shelf life. Different letters indicate significant difference (*p* < 0.05) between tested samples according to Duncan's mean values post hoc comparison test.

HP processing of the probiotic yoghurts induced a reduction of the starter culture total load accounting for ca. 0.2−0.5 log10 CFU/g for HP-treated products and ca. 0.2−0.3 log10CFU/g for Homo-HP-treated products. Throughout storage, starter culture cells underwent a pressure dependent decrease in their viability of about 0.2−0.5 log10CFU/g; yet their total counts were well above the recommended level of 7.0 log10 CFU/g [32]. When probiotic yoghurts were high pressurized at 400 MPa, (in the absence or presence of conventional homogenization step), a greater decrease in starter culture total counts was observed which were well below the WHO/FAO acceptable thresholds.

The remaining probiotic counts of probiotic dairy beverages after HP treatment and during storage are shown in Table 4 for *Bifidobacterium lactis* BB12 and in Figure 3 for *Lactobacillus acidophilus* LA5.


**Table 4.** Viability of *Bifidobacterium lactis* BB12 in different treated plain and cherry-flavored yoghurt during storage.

\* Control samples were not HP-treated but only homogenized at 10 bar after the break of the coagulum. Letter D indicates the day of production. N.D. indicates microbial load below the acceptable limit of 7.0 log10 CFU/g of the total microbial counts for the starter culture at the end of shelf life. Different letters indicate significant difference (*p* < 0.05) between tested samples according to Duncan's mean values post hoc comparison test.

In agreement with the probiotics survival findings in the model system, pressure increase resulted in significantly higher sub-lethality of Bifidobacteria cells compared to Lactobacilli. Bifidobacteria cell population underwent a decrease of about 0.1 to 1.4 log10 CFU/g or 0.1 to 1.1 log10 CFU/g for HP and Homo-HP-treated samples, respectively, when the products treated in the pressure range of 100−300 MPa. The corresponding decrease in Lactobacilli population was 0.1−0.9 log10 CFU/g and 0.0−0.6 log10 CFU/g for HP and Homo-HP-treated samples, respectively. Similar results for probiotics viability were obtained during storage, where a further slight decrease in probiotic populations of ca. 0.2−0.7 log10 CFU/g were observed for all tested samples. Based on these observations it can be hypothesized that after HP treatment there are potentially more injured cells unable to recover in the case of Bifidobacteria than of Lactobacilli. Despite the observed decrease, when products were pressurized in the range of 100−300 MPa, the remaining probiotic population was above the recommended level of 10 <sup>6</sup> CFU/g for both probiotic strains and both treated products (HP and Homo-HP samples) [1]. In the HP-treated products the decrease in probiotic cells was greater than the viability loss observed for Homo-HP-treated products. However, during storage at 5 ◦C for 28 days, Homo-HP samples presented higher viability loss of probiotic cultures as compared to that of HP samples. Similar to the case of starter culture microorganisms, when products were pressurized at 400 MPa, counts of both probiotic strains decreased below the legislative acceptable levels in all alternatively treated products (depicted as N.D. in Table 4 and, \* symbol in Figure 3).

**Figure 3.** Viability of *Lactobacillus acidophilus* LA5 during 28 days of storage at 5◦C in different treated (**a**) plain and, (**b**) cherry-flavored probiotic yoghurt (Letter D indicates the day of production. Different letters among bars indicate significant difference (*p* < 0.05) between tested samples according to Duncan's mean values post hoc comparison test.).

In the case of cherry-flavored yoghurt samples, a similar reduction of about 0.2−0.4 log10 CFU/g was observed for total starter culture population when yoghurt samples were subjected to HP processing (both HP and Homo-HP samples), followed by a further decrease of 0.2−0.4 log10 CFU/g during 28 days of storage. Probiotic populations in cherry-flavored yoghurt samples subjected to HP processing underwent a reduction of approximately 0.2−1.8 log10 CFU/g, followed by a further decrease of 0.2 and 0.1−0.5 log10 CFU/g for BB12 and LA5 populations, respectively, during 28 days of storage, indicating a significant lower reduction at the first day of production and during storage to that observed for plain yoghurt samples. Addition of syrup resulted in an increase in the amount of polysaccharides and sugars in these samples, a growth factor for probiotics survival, indicating that the use of substances for the enhancement of taste and flavor in dairy products could also improve the viability of microbial populations [33].
