*3.3. Whey Valorization as Wound Repair Agent*

Buffalo milk whey has been shown to contain various proteins with immunomodulatory and antitumor activity, and a series of bioactive peptides with antimicrobial, antioxidant, antihypertensive and remineralizing properties [5]. For example, the effect of whey proteins in improving the inflammatory status during wound healing in diabetic rats, in particular by reducing the expression of specific cytokines involved in the reparation, was previously reported [27]. Kalinina and collaborators [28] identified whey proteins, and in particular WFDC12, as a specific marker for the last stage of keratinocytes differentiation, probably enhancing the occurrence of epidermal homeostasis.

Our research aimed to evaluate the ability of ultrafiltered and concentrated whey from buffalo milk, to prompt cell migration and regeneration of the human keratinocyte monolayers by using a scratch assay [29]. This assay is generally run to obtain a preliminary but robust evaluation of the regenerative potential of biomolecules.

The sprayed powder was diluted in the cell growth medium and tested by an in vitro wound healing assay, as reported in the material and methods section. Representative fields of view of wound closure reported in Figure 3a are clearly showing that the addition of UF\_Ret20Pow prompts keratinocytes migration, confirming the role of whey proteins in dermal repair. Quantitative analyses (Figure 3b) indicate that in the presence of the sprayed powder at a concentration of 4 g/L, 40% wound closure occurred within 15 h, as compared to the control that, at the same time, reached about 20% of repair. Moreover, the sample induced complete healing within 30 h, whereas the closure area of the control was lower than 40%. Data analysis by means of a two-tailed non homoscedastic Student *t*-test indicates that the wound closure rate significantly improves (*p* < 0.05) in the supplemented sample from 9 h onwards, as compared to the control (Figure 3b). The treatment with a less concentrated sample (2 g/L) did not improve significantly the natural occurrence of wound healing. Overall these data clearly establish a beneficial effect of diluted fractionated whey powder, suggesting a potentially promising effect in topical products for skin treatments.

diluted fractionated whey powder, suggesting a potentially promising effect in topical

products for skin treatments.

**Figure 3.** Wound healing assay (**a**) Representative field of view of HaCat scratch assays during time course of the experiments. At least five fields of view for each sample were analyzed; (**b**) Quantitative analyses of wound closure as percentage over time. Data significance was analyzed by two-tailed non homoscedastic Student *t*-tests. The vertical line in the graph indicates the time point from which a significant difference of wound closure % was observed between the CTR and the UF\_Ret20Pow 4 g/L treated sample. CTR, control. **Figure 3.** Wound healing assay (**a**) Representative field of view of HaCat scratch assays during time course of the experiments. At least five fields of view for each sample were analyzed; (**b**) Quantitative analyses of wound closure as percentage over time. Data significance was analyzed by two-tailed non homoscedastic Student *t*-tests. The vertical line in the graph indicates the time point from which a significant difference of wound closure % was observed between the CTR and the UF\_Ret20Pow 4 g/L treated sample. CTR, control.
