3.3.2. Physicochemical/Rheological Characteristics After HP Treatment and During Storage

The physicochemical (pH, acidity, and WHC) and rheological parameter (consistency coefficient, rheological behavior index and apparent viscosity) data matrices for plain and cherry-flavored yoghurts were averaged, standardized and subjected into PCA (Figure 4).

**Figure 4.** Principal components analysis (a1,b1: loadings plot, a2,b2: scores plot) for the classification of plain (**a**) and cherry-flavored (**b**) yoghurt samples stored at 5 ◦C for 28 d, based on their physicochemical characteristics (principal components 1 and 2 accounted together the 72.1% and 84.3% of the total variance explained for plain and cherry-flavored yoghurts, respectively).

As seen in Figure 4, the titratable acidity was reduced proportionally to the intensity of the pressurization process for both plain and cherry-flavored products, which implies that the metabolic activity of the probiotic microorganisms and yoghurt starter is significantly slowed down [34,35]. The post-acidification was found to be pronouncedly higher in the case of cherry-flavored formulations for the entity of the tested processing conditions (HP and Homo-HP) and thus, it is assumed that the presence of readily available nutrient sources in the fruit syrup (e.g., sugar, glucose syrup, natural presence of sugars and fibers in cherry juice) stimulated the growth of the microbiota throughout the chilling storage.

Expectedly the cherry syrup addition suppressed the total color index from 84.5 to 78 for the control systems. A similar trend regarding the impact of the HP processing on total color i.e., from 79.6−85.9 to 75.1−78.9, was observed. However, no significant differences between the HP and Homo-HP samples were identified. Interestingly, the increase in the intensity of pressurization process resulted in a reduction of the total color of cherry-flavored yoghurts, most probably due to the degradation or isomerization of naturally occurring pigmenting compounds such as anthocyanins and carotenoids [36,37].

The WHC is a measure of the ability of acid gel to retain unbound water (loosely hold in the interspaces of the protein gel network) on the application of mechanical stress. It has been demonstrated that the more uniform association between denatured beta-lactoglobulin (as it is more sensitive than alpha-lactalbumin to high-pressure treatment) and the dissociation and re-aggregation of the micelle fragments occurring during the HP processing reduce the proneness of the acid protein gels to syneresis [13]. According to our findings, the WHC in the HP-treated plain yoghurts was significantly higher than that of the Homo-HP-treated ones. On the other hand, a reversed behavior was observed in the case of cherry-flavored yoghurts, where the Homo-HP exerted the highest WHC values. Although there is no conclusive explanation, it is presumed that the breakdown of the acid gel during the homogenization step, allowed the stabilizing agents (e.g., pectins) found in the syrup base, to occupy a higher hydrodynamic volume due to the reduction of steric hindrances, and thus it improved the ability of the overall acid protein/stabilizer protein network to retain more water via hydrogen bond bridging.

The implementation of the HP treatment at the final stage of the production process appeared to improve the rheological properties of the end product, either when it is applied individually or subsequently to conventional homogenization.

The values of consistency coefficient (*K*) of plain yoghurt beverages were significantly (*p* < 0.001) affected by the applied treatment, the level of the applied pressure and storage time. HP treatment of samples in a pressure range of 100−300 MPa led to an increase in consistency coefficient values compared to the controls (homogenized at 10 bar after the break down of the coagulum). At 400 MPa, consistency coefficient values decreased, although it was still higher than control samples. Flow behavior index values of plain yoghurt beverages were also significantly (*p* < 0.001) affected by the applied treatment and storage time, showing a significant decrease with increasing HP, either applied individually (HP samples) or subsequently to conventional homogenization (Homo-HP samples). This trend was reversed at 400 MPa. The apparent viscosity of plain yoghurt samples increased by 162, 188, 190 and 68% at pressures of 100, 200, 300 and 400 MPa, and 26, 46, 65 and 29% when HP was applied subsequently to conventional homogenization at the same pressures.

The degree of whey protein denaturation is a very important factor that affects the rheological behavior of the coagulum and can be related to the intensity of the pressure applied. It is also responsible for the protein-protein interactions, and the retention of whey proteins in the network gel. HP processing lead to enhanced whey protein hydrophobicity, resulting in an increase in the binding affinity of whey proteins and thus, alterations on their structure and improvement of their functional properties [38,39]. Moreover, it has been reported that when the applied pressure exceeds 200 MPa, a partial disintegration of casein micelles occurs, which results in an increased number of fragments of casein solution and in greater solvation of the protein. The partial fragmentation of casein micelles is accompanied by the solubilization of colloidal calcium phosphate, suggesting that the structure of the gels obtained is dominated by casein-casein interactions instead of the interactions of whey protein-casein, forming small particles that are often shaped into clumps and chains, and therefore into compact and strengthened protein networks [11]. The increase in yoghurt viscosity, with pressure, have been previously related to modifications of beta-lactoglobulin structure, since significant molecular unfolding and further protein aggregation occurs, especially when pressures above 100 MPa are applied and with a threshold at 400 MPa, where usually the coagulum becomes more coarse and an increase in syneresis is observed [16].

The parameters of consistency coefficient and flow behavior index of cherry-flavored yoghurt beverages were affected by the applied treatment similarly to plain yoghurt beverages. However, storage time did not seem to significantly affect these parameters, probably due to the increase in total

solids and the presence of small amounts of pectin in the syrup that could have stabilized the structure of the coagulum.
