*2.8. In Vitro Scratch Test and Time-Lapse Video Microscopy (TLVM)*

Briefly, HaCaT cells were seeded in 12-wells until complete cellular confluence was reached. Successively the confluent monolayer was scratched with a sterile tip (Ø = 0.1 mm) and the diluted UF\_Ret20 samples were added to the medium.

The in vitro cell migration was analyzed by a video microscopy time-lapse station (TLVM) (OKOLAB, Pozzuoli, Italy), assembled with an inverted microscope (AxioVision200, Zeiss, Germany), a CCD-gray-camera (ORCA ER, Hamamatsu Photonics, Hamamatsu City, Japan) that records the images, a motorized stage incubator that logs the position and maintains the in vitro condition of the cell culture (37 ◦C, 5% CO<sup>2</sup> in humidified air) and the custom-tailored software OKO-Vision 4.3 software that follows the overall process and allows image analysis. The TVLM tracks the wound repair process in real-time for 48–72 h, due to the selection and recording of representative images of the experiments. The quantitative analysis of wound closure rates is calculated as [(Area t0 − Area t)/Area t0] × 100 directly by the software or alternatively by manual mode tracking for each image of the wound area over time. For each well a minimum of 5 fields of view was used for deriving the overall averaged curves of wound closures (%) as a function of the time, thus ensuring the statistical significance of the experiment. the position and maintains the in vitro condition of the cell culture (37 °C, 5% CO<sup>2</sup> in humidified air) and the custom-tailored software OKO-Vision 4.3 software that follows the overall process and allows image analysis. The TVLM tracks the wound repair process in real-time for 48–72 h, due to the selection and recording of representative images of the experiments. The quantitative analysis of wound closure rates is calculated as [(Area t0 − Area t)/Area t0] × 100 directly by the software or alternatively by manual mode tracking for each image of the wound area over time. For each well a minimum of 5 fields of view was used for deriving the overall averaged curves of wound closures (%) as a function of the time, thus ensuring the statistical significance of the experiment. *2.9. Statistical Analysis* All data were analyzed by means of two-tailed non homoscedastic Student's *t*-test,

wound healing assay, the concentration of FBS in the medium was reduced to 1% *v*/*v* to

Briefly, HaCaT cells were seeded in 12-wells until complete cellular confluence was reached. Successively the confluent monolayer was scratched with a sterile tip (Ø = 0.1

The in vitro cell migration was analyzed by a video microscopy time-lapse station (TLVM) (OKOLAB, Pozzuoli, Italy), assembled with an inverted microscope (AxioVision200, Zeiss, Germany), a CCD-gray-camera (ORCA ER, Hamamatsu Photonics, Hamamatsu City, Japan) that records the images, a motorized stage incubator that logs

slow down the migration phenomena that could then be better evaluated.

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 5 of 11

*2.8. In Vitro Scratch Test and Time-lapse Video Microscopy (TLVM)*

mm) and the diluted UF\_Ret20 samples were added to the medium.

#### *2.9. Statistical Analysis* and *p* < 0.05 was considered as statistically significant.

All data were analyzed by means of two-tailed non homoscedastic Student's *t*-test, and *p* < 0.05 was considered as statistically significant. **3. Results and Discussion**

#### **3. Results and Discussion** In line with current circular economy approaches, a waste material from one of the

In line with current circular economy approaches, a waste material from one of the production sectors of excellence of the Campania region, namely the dairy industry, was processed and recovered to obtain on the one side purified water for cleaning use within the manufacturing facility, and on the other a spray-dried concentrate that could be tested as substrate for the growth of *L. fermentum* and as bioactive compound useful in promoting tissue regeneration. An overview of the process approach is presented in Figure 1. production sectors of excellence of the Campania region, namely the dairy industry, was processed and recovered to obtain on the one side purified water for cleaning use within the manufacturing facility, and on the other a spray-dried concentrate that could be tested as substrate for the growth of *L. fermentum* and as bioactive compound useful in promoting tissue regeneration. An overview of the process approach is presented in Figure 1.

**Figure 1. Figure 1.** Overall downstream process of discarded whey and obtainment of Overall downstream process of discarded whey and obtainment of added value fractions. added value fractions.
