*2.3. In Vitro Cytotoxicity Assay*

The most interesting, least described, and perhaps most important activity of PJ is probably its cytotoxicity against cancer cells [22,23]. On the basis of the obtained results presented in Table 5, it was found that the hydrolysate of PJ protein has a cytotoxic effect, which can be increased with concentration using membrane separation. Moreover, the use of a nanofiltration membrane to concentrate the obtained hydrolysate additionally increased the biological activity of cPJPH. By comparing the IC<sup>50</sup> values, it was found that the concentration process caused a 1.4–3.3-fold increase in the cytotoxicity of PJPH depending on the cell line tested. The highest cytotoxicity of cPJPH was found in the Caco-2 colon cancer cells. In contrast, the weakest cytotoxic effects were observed in the culture of normal colon mucosa CCD 841 CoN cells. The first cytotoxic dose (IC10) of cPJPH to Caco-2 cells was 7.3-fold lower than the IC<sup>10</sup> to CCD 841 CoN cells. Moreover, the half-maximal inhibitory concentration (IC50) and the lethal concentration (IC90) of cPJPH determined for Caco-2 cells were significantly lower (3.4- and 1.6-fold, respectively) than those obtained for CCD 841 CoN cells. The high cytotoxic activity of both PJPH and cPJPH was also observed in the colon cancer HT-29 cell culture. As a result of PJPH concentration, a significant increase in cytotoxic potential was found in the stomach cancer Hs746T cell cultures (Table 5). Commonly used anti-cancer drugs cause significant damage to the body of patients because of their non-selective action [67–69]. Therefore, there is a need for substances that will act in a more targeted manner. The results of the cytotoxic activity of cPJPH indicated significantly lower IC<sup>50</sup> doses for neoplastic cells compared to normal cells, which may be of particular interest in the context of further research into the use of PJ ingredients for the treatment of gastrointestinal cancer.


**Table 5.** Cytotoxic doses for stomach cancer cells (Hs746T line), colon cancer cells (HT-29 and Caco-2 lines), and colon normal cells (CCD 841 CoN line) [mgdm/mL].

Mean values denoted by different letters (a–c) in columns differ statistically significantly (*p* < 0.05).

The comparison of the various methods used to process PJ suggests some conclusions regarding the question of which substances in PJ are actually responsible for its cytotoxic effect on cancer cells. While concentration by ultrafiltration using a 5 kDa cut-off membrane resulted in a product with a high content of nutritious protein [23], the membrane-assisted enzymatic hydrolysis presented in this study resulted in a product with significantly increased cytotoxic activity against cancer cells. Moreover, the thermal deproteination of PJ also resulted in products with higher cytotoxic activity against cancer cells compared to the raw materials [22]. These results prove that the anti-proliferative effect of PJ on cancer cells is not related to protein fraction but to other molecules of rather low molecular mass. Surely, this hypothesis requires confirmation with further analyses, but the phenomena observed so far provide important evidence that may direct further studies.

normal cells (CCD 841 CoN line) [mgdm/mL].

#### **3. Materials and Methods 3. Materials and Methods**

Mean values denoted by different letters (<sup>a</sup>–<sup>c</sup>

that may direct further studies.

#### *3.1. Enzymatic Hydrolysis of Potato Juice Proteins 3.1. Enzymatic Hydrolysis of Potato Juice Proteins*

*Molecules* **2021**, *26*, x FOR PEER REVIEW 6 of 11

**Table 5.** Cytotoxic doses for stomach cancer cells (Hs746T line), colon cancer cells (HT-29 and Caco-2 lines), and colon

Hs 746T 4.43 ± 0.21 <sup>b</sup> 1.80 ± 0.18 <sup>b</sup> 6.30 ± 0.09 <sup>b</sup> 2.95 ± 0.13 <sup>b</sup> 8.96 ± 0.61 <sup>b</sup> 4.84 ± 0.07 <sup>b</sup> Caco-2 2.58 ± 1.13 <sup>c</sup> 0.55 ± 0.17 <sup>c</sup> 5.26 ± 0.78 b,c 1.62 ± 0.24 <sup>c</sup> 11.35 ± 2.00 <sup>a</sup> 4.88 ± 0.46 <sup>b</sup>

**PJPH cPJPH PJPH cPJPH PJPH cPJPH**

**Cell Line IC<sup>10</sup> IC<sup>50</sup> IC<sup>90</sup>**

membrane-assisted enzymatic hydrolysis presented in this study resulted in a product with significantly increased cytotoxic activity against cancer cells. Moreover, the thermal deproteination of PJ also resulted in products with higher cytotoxic activity against cancer cells compared to the raw materials [22]. These results prove that the anti-proliferative effect of PJ on cancer cells is not related to protein fraction but to other molecules of rather low molecular mass. Surely, this hypothesis requires confirmation with further analyses, but the phenomena observed so far provide important evidence

The experimental material, potato juice (PJ), was collected during the starch production season from the production line of "Trzemeszno" Sp. z o.o. Potato Industry Company (Trzemeszno, Poland). The enzyme Savinase® (Sigma-Aldrich, Saint Louis, MO, USA), isolated from the *Bacillus* species, was used as the proteolytic preparation. On the basis of previous preliminary studies (data not shown), an enzyme dose of 4 µL/g of potato protein was adopted. The enzyme was added at the start of the hydrolysis process, according to the initial volume of PJ used in the experiment, and further portions were added every 60 min because of the continuous process, in which the finished hydrolysis product was removed and a new portion of PJ was added in its place. A polyethersulfone spiral-wound ultrafiltration membrane with a molecular weight of 1 kDa cut-off and an area of 3.5 m<sup>2</sup> (type 3838, SUEZ Water Technologies & Solutions, Budapest, Hungary) was used to perform the enzymatic hydrolysis and, consequently, to obtain a PJ protein hydrolysate (PJPH). The non-hydrolyzed PJ was returned to the initial tank of the system (recirculation). The PJPH was then concentrated on a polyamide thin film composite nanofiltration membrane, with a molecular weight of 300–500 Da cut-off and an area of 4.0 m<sup>2</sup> (type 3838, SUEZ Water Technologies & Solutions, Budapest, Hungary), to obtain a concentrated fraction of hydrolyzed, soluble potato proteins (retentate, denoted in the text as cPJPH) and a non-protein low molecular weight fraction (filtrate). A flowchart of the process used for these products is presented in Figure 1. The experimental material, potato juice (PJ), was collected during the starch production season from the production line of "Trzemeszno" Sp. z o.o. Potato Industry Company (Trzemeszno, Poland). The enzyme Savinase® (Sigma-Aldrich, Saint Louis, MO, USA), isolated from the *Bacillus* species, was used as the proteolytic preparation. On the basis of previous preliminary studies (data not shown), an enzyme dose of 4 μL/g of potato protein was adopted. The enzyme was added at the start of the hydrolysis process, according to the initial volume of PJ used in the experiment, and further portions were added every 60 min because of the continuous process, in which the finished hydrolysis product was removed and a new portion of PJ was added in its place. A polyethersulfone spiral-wound ultrafiltration membrane with a molecular weight of 1 kDa cut-off and an area of 3.5 m<sup>2</sup> (type 3838, SUEZ Water Technologies & Solutions, Budapest, Hungary) was used to perform the enzymatic hydrolysis and, consequently, to obtain a PJ protein hydrolysate (PJPH). The non-hydrolyzed PJ was returned to the initial tank of the system (recirculation). The PJPH was then concentrated on a polyamide thin film composite nanofiltration membrane, with a molecular weight of 300–500 Da cut-off and an area of 4.0 m<sup>2</sup> (type 3838, SUEZ Water Technologies & Solutions, Budapest, Hungary), to obtain a concentrated fraction of hydrolyzed, soluble potato proteins (retentate, denoted in the text as cPJPH) and a non-protein low molecular weight fraction (filtrate). A flowchart of the process used for these products is presented in Figure 1.

) in columns differ statistically significantly (*p* < 0.05).

**Figure 1. Figure 1.** Schematic diagr Schematic diagram of the applied membrane separation system for ultrafiltration. am of the applied membrane separation system for ultrafiltration.
