**3. Results**

Aqueous extracts from fermented milk, obtained with three different microbial strains *Lactobacillus acidophilus*, *Lactobacillus delbrueckii subs. bulgaricus* and *Streptococcus thermophilus,* were used. Then, the samples underwent two different types of purification. To obtain peptide enriched fractions, the first one was a discontinuous step gradient of ACN (5–30% and 30–50%). The major amount of peptides was found in the 5–30% ACN step, while in the 30–50% fraction only a small amount of peptides was present. Subsequently, to provide a higher resolution in peptide separation, the fractions were further purified with RP-HPLC using a continuous gradient. The collected samples were tested for their antioxidant properties in vitro and in Caco-2 cells. The peptides present in the most active fraction were identified.

#### *3.1. Analysis of 5–30% ACN Fraction in Caco-2 Cells: Evaluation of Cell Viability*

To estimate the effects of 5–30% and 30–50% ACN fractions on Caco-2 cells, the MTT test was performed. Caco-2 cells (1 × 104) were treated with the peptide fractions (0.125 mg/mL) and, as shown in Table 1, both fractions were not cytotoxic. Moreover, when cells (1 × 104) were subjected to oxidative stress induced by 200 μM TbOOH we observed a decrease of viability. However, when cells were pretreated with the 5–30% ACN fraction a significant protective effect from oxidative stress was observed. Therefore, the further analysis was conducted only on this fraction.

**Table 1.** Percentage of viability (MTT test) in Caco-2 cells in the presence of the isolated 5–30% and 30–50% acetonitrile (ACN) fractions. Means of at least three experiments (eight replicates for each experiment) were compared with the treated control. (\* *p* < 0.05).


#### *3.2. HPLC Analysis, Antioxidant Properties In Vitro and In a Cellular Model of the Purified Fractions Obtained From the 5–30% ACN Pool*

Fraction 5–30% ACN, containing a large amount of peptides, was further purified, in order to isolate and identify the most active peptides. To this purpose, 35 μg of the 5–30% ACN fraction was subjected to RP-HPLC (PrepNova-Pak ® HR C18) employing a linear gradient from 5% to 40% ACN with a flow rate of 12 mL/min. Collecting the eluted solution every two minutes, fifteen fractions were obtained as described in Figure 1A. In particular, fractions from 0 to 5 and from 11 to 15 were discarded, because the amount of obtained peptides was negligible and insu fficient to perform further experiments, indicating a low peptide content at the beginning and at the end of the gradient. On the other hand, each fraction from 6 to 10 was analyzed for its antioxidant properties in vitro. As reported in Figure 1B, all the fractions showed an antioxidant capacity in vitro, as they exhibited moderate TEAC and DPPH scavenging values. Moreover, the e ffects on Caco-2 cells of the purified fractions were analyzed and the peptides did not show cytotoxicity (Figure 1C). In addition, all the fractions were evaluated for their protection from oxidative stress in cells pretreated with them and subsequently incubated with 200 μM TbOOH. As shown in Figure 1C, some fractions, in particular 6, rescued the viability of Caco-2 cells treated with the oxidative agent. For this reason, this fraction was selected for the further analysis. In order to identify the peptides, present in the most active fraction (6) and able to cross the intestinal barrier, the Transwell ® insert model was used. Caco-2 cells were grown on the Transwell ® insert for 21 days to reach the di fferentiated epithelium formation and peptide fractions were added in the apical compartment. After 10 and 120 min, apical and basolateral solutions were collected and analyzed by HPLC and mass spectrometry. As shown in Figure S1, some peptides present in the fraction 6 can cross Caco-2 monolayer.

**Figure 1. (A**)Purification of the 5–30% ACN fraction with RP-HPLC. Fractions were collected every 2 min. (**B**) Analysis of antioxidant capacity of the purified fractions in vitro with 2,2-azinobis(3- ethylbenzo-thiazoline 6-sulfonate) (ABTS) (green) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) (red)

scavenging tests. ( **C**) E ffects of the purified fractions on cell viability in the presence and absence of TbOOH. Caco-2 cells were treated with the indicated fractions for 24 h and oxidative stress was induced by 200 μM TbOOH. Means of at least three experiments (eight replicates for each experiment) were compared with the treated control. (\*\*\* *p* < 0.001, \*\* *p* < 0.01).

#### *3.3. Identification of the Peptides with Mass Spectrometry Analysis*

Then, peptides included in fraction **6** were analyzed with mass spectrometry in order to identify their sequence. The investigation with the Proteome Discoverer and Mascot gave a list of peptides reported in the Table S1. The list of peptides for each protein given by the proteomic identification was aligned with the sequences of the reference proteins (Figure S2) and the candidates for the solid-phase peptide synthesis were chosen in order to obtain the maximum protein sequences coverage. Some criteria were considered as fundamental for the choice of candidate peptides, such as the maximum protein sequences coverage, the best match score between the peptides given by the Proteome Discoverer Software and the reference proteins expressed by Peptide Spectral Match (#PSM), and the peptide length. When more than one peptide covered a region, only the peptide that has the best match score, expressed by the highest value of #PSM was chosen. Of note, the same analysis were performed for fraction 7, but the identified peptides were mostly the same as those identified in fraction 6. Due to the more significant activity in cellular model of fraction 6, the further experimentation was performed with the latter. The sequences and properties of the synthesized peptides were reported in Table 2.





\*: peptide N-15-M was identified by Proteome Discoverer Software as NTVPAKSCQAQPTTm, with an oxidized Methionine at position 15, but it was synthesized with not oxidized Methionine; \*\* Sodium-dependent phosphate transport protein 2B.
