*3.1. E*ff*ect of HPP on TPC, AC, and Microbial Counts of Chokeberry Milkshakes*

Effects of HPP treatments on the TPC, AC, and microbiological survival fraction are shown in Table 2. The analyses were conducted on untreated and treated samples to observe differences with HPP treatment. Results for molds and yeasts are not shown because there were no changes in any treatments.

TPC concentration in untreated samples with 2.5%, 6.25%, and 10% (*w*/*v*) of chokeberry pomace is 53.02 ± 0.14, 73.92 ± 3.17, and 121.16 ± 0.31 mg GAE/100 mL, respectively. Furthermore, the AC results for samples 2.5, 6.25, and 10% (*w*/*v*) of chokeberry pomace, are 6.06 ± 0.14, 9.27 ± 0.20, and 14.89 ± 0.30 μmol Trolox/mL, respectively. As expected, the TPC and AC results are higher with higher pomace concentrations.

In treated samples, the highest result for TPC is 155.28 ± 2.07 mg GAE/100 mL with 500 MPa during 1 min and 10% (*w*/*v*) pomace addition; the lowest is 42.45 ± 2.89 mg GAE/100 mL with 350 MPa during 5.5 min and 2.5% (*w*/*v*) pomace addition. Yet, the highest AC is 17.3 ± 1.08 μmol Trolox/mL for the treatment of 200 MPa during 1 min with 10% (*w*/*v*) pomace addition. The treatment that obtained the lower AC matches the one that obtained the lower TPC (350 MPa during 5.5 min with 2.5% (*w*/*v*) pomace addition).

The lowest results for TPC and AC are obtained for the intermediate pressure and not for the lowest as expected, yet these low results are similar to other treatments at different processing conditions but with the same pomace concentration (2.5%). In contrast, the higher value results for TPC and AC are obtained for milkshakes with 10% of chokeberry. When comparing the results of the treatments at each concentration, values were higher as the pomace concentration increased. In addition, comparing the results of treatments at each concentration with its untreated counterpart, samples 6.25 and 10% show an increase in TPC and AC, influenced by the pressures and times studied. However, for samples at 2.5%, this effect is less acute, affected only by high pressures (500 MPa).


**Table 2.** Effect of HPP and chokeberry pomace on TPC, AC, and the microbial survival fraction.

<sup>a</sup> Central point; HPP: High Pressure Processing; TPC: Total Phenolic Content; AC: Antioxidant Capacity; N: final cell concentration; N0: initial cell concentration.

Thus, the results are affected by all the factors in this study (pressure, time, and concentration), but mainly the pomace concentration. The HPP treated foods are either unaffected or have increased TPC and/or extractability following treatments with high pressures [30]. Andrés et al. [31] found increases of 6.6% and 4.2% in TPC values for fruit smoothies treated at 450 and 600 MPa, respectively. Corrales et al. [32] showed that treating at 600 MPa enhanced the anthocyanin extraction and its AC in grape by-products than with conventional extraction methods. Liu et al. [33] found treatments at 200 MPa, for 5 and 10 min, led to an increased TPC of 14.24% and 14.35% in wild *Lonicera caerulea* berry, respectively, however, for treatments at 500 and 600 MPa there was a significant decrease of TPC. In contrast, other authors found HPP had little effect on phenolic content. Barba et al. [34] observed TPC to be relatively resistant to the effect of processing in tomato purées. Hurtado et al. [35] did not observe differences in AC values between untreated and treated red fruit-based smoothies for treatments at 350 MPa, 10 ◦C, and 5 min. Patras et al. [36] found that phenol content in HPP treated strawberry purées was relatively resistant to the effect of processing at 400 and 500 MPa, only showing an increase in treatments at 600 MPa for TPC and AC. Therefore, the results obtained with HPP depend of several conditions, such as the matrix in which they are applied, and the severity of the treatment and it is necessary to study the behavior of different samples with these treatments.

In fluorescence microscopy, the intensity value of a pixel is related to the number of fluorophores present at the corresponding area in the particle. Thus, the digital images can be used to extract the intensity values to determine the local concentration of fluorophores in a specimen [37]. In our case of study, the images in Figure 1 show the pomace particles dispersed into the milk matrix.

To analyze the florescence intensity the images corresponding to the lower (200 MPa, 1 min, 2.5%), central (350 MPa, 5.5 min, 6.25%), and higher (500 MPa, 10 min, 10%) treatments were selected. The particle with greater intensity was selected to generate intensity profiles (Figure 1a–c). A line (shown in yellow) was drawn across the particle, and a plot (graph) was generated to show the intensity values of the pixels along the line (Figure 1d–i). In addition, Figure 1j–l shows the relation between the percentages of particles at each fluorescence intensity interval.

**Figure 1.** Fluorescence intensity of: 200 MPa, 1 min, 2.5% (**a**,**d**,**g**,**j**); 350 MPa, 5.5 min, 6.25% (**b**,**e**,**h**,**k**); and 500 MPa, 10 min, 10% (**c**,**f**,**i**,**l**).

The fluorescence intensity for the isolated particles is higher in the medium (Figure 1k) and high treatments (Figure 1l) than in the low treatment (Figure 1j). Comparing the background intensity, corresponding to the liquid phase of the sample, fluorescence increases as the severity of the treatment increases. Several authors [16,38,39] have described the cell wall degradation and breakage in plant tissue after HPP, leading to a leaching of contents from the pomace cells (included polyphenols) to the milk acting as a liquid medium [32,38]. In addition, Gonzalez and Barrett [40] described that treatments at pressures above 220 MPa were responsible for the breakage of the membrane structure because of protein unfolding and interface separation. Therefore, as higher pressures are applied, the phenolic contents are being released to the medium due to the membrane breakage, giving as a result higher values of fluorescence intensity. The particle frequency plots show that the frequency of particles at high intensities rises with the severity of the treatment. These results agree with the results of TPC and AC, i.e., higher fluorescence intensities correspond to the treatments that obtained the higher TPC and AC results. Therefore, measurement of fluorescence intensity can be an indicator for TPC and AC in this type of sample. Further research is needed to prove if the analysis is usable in other matrices.

Besides the effect of HPP on the polyphenols, there could be a microbial inactivation because of changes induced in the microbial cells. These changes include alterations in the cell membrane, effects on proteins, and effects on the genetic mechanism of microorganisms [18,41,42]. Seen in microbiological inactivation results in Table 2, treatments at 200 MPa during 1 min with 2.5% (*w*/*v*) of pomace and at 200 MPa during 10 min with 2.5% of pomace do not show microbial inactivation. At 2.5% pomace concentration and low pressure (200 MPa), longer treatment time is not enough for microbial inactivation. Muñoz-Cuevas et al. [43] also observed this behavior. Still, it is necessary to reach a minimum treatment intensity (500 MPa, 10 min) to obtain significant *L. monocytogenes* inactivation. At this condition, an increase in chokeberry pomace concentration from 2.5% to 10% (*w*/*v*) increases microbial inactivation from 3.63 to 4.02 Log reductions.

Thus, increasing the pressure and treatment time results in an increase in the lethal effect of HPP treatment. This effect relates to food composition, technological parameters, and the factors acting in synergy [44,45].

Besides the effect of treatment conditions, several authors have described the high antimicrobial capacity of berry fruits and their by-products [46–48] and the synergistic effect between natural substances and high pressure treatments [28,49,50]. Despite the evidence found in the literature, except the treatments of 500 MPa, 10 min with 2.5% and 10% (*w*/*v*) pomace, the inactivation values are lower than similar treatments with other products and microorganisms. For example, Evrendilek & Balasubramaniam [49] concluded that samples of ayran (yogurt drink) treated at 600 MPa during 5 min had reduced in the levels of *L. monocytogenes* and *L. innocua* by more than five log units (*p* < 0.05) at ambient temperature. Nevertheless, Gervilla et al. [51] and Black et al. [52] have described a baroprotective effect that milk has on the cells. Thus, this effect could counteract the antimicrobial effect of chokeberry pomace, explaining the low inactivation levels found for *L. monocytogenes* in this study. To prove this effect, an experiment was conducted where the central point of the design (350 MPa, 5.5 min in milk with 6.25% (*w*/*v*) of chokeberry pomace) was used as a treatment to compare the inactivation reached in four different matrices: (i) peptone water with *L. monocytogenes*, (ii) milk with *L. monocytogenes*, (iii) peptone water with *L. monocytogenes* and chokeberry pomace, and (iv) milk with *L. monocytogenes* and chokeberry pomace. Results tested the baroprotective effect of milk and are shown in Figure 2.

**Figure 2.** Inactivation level of ingredients' different combinations: peptone water (W), milk (M), inoculated *Listeria monocytogenes* (LM), and pomace (P).

In samples without milk (W + LM and W + LM + P), the number of surviving cells is lower than with milk samples (M + LM and M + LM + P), and more prominent when pomace is added. Apart from the protective effect of milk, an increase is seen in the efficacy of HPP against *L. monocytogenes* when pomace is present in the sample. Thus, these results can describe the synergistic effect of pomace, HPP, and the protective effect of milk on *L. monocytogenes*. Still, there is microbial inactivation with some treatments, even with the protective effect of milk on the microbiological cells.
