*2.10. Antimicrobial Activity*

The antimicrobial activity of lacticaseicin 30 and its variants (500 μg/mL) was tested by the critical dilution micromethod using different target strains [44]. Samples were acidified to pH 5 and sterilized by filtration. Serial double dilutions of the filtrate samples were made in 200 μL volumes of BHI in a sterile 96-well Falcon microtiter plate (Corning, Tewksbury, MA, USA). Each well was inoculated with 2 μL of overnight culture of the target strain. Then, microplates were incubated at 37 ◦C for 24 h without agitation. Bacterial growth was estimated by measuring absorbance at 620 nm using a microtiter plate scanner (Biotech Instruments Inc., Winooski, VT, USA). The antibacterial activity was determined in arbitrary units per milliliter (AU/mL) according to the following formula: 2<sup>n</sup> × (1000 <sup>μ</sup>L/deposited volume), with *n* corresponding to the highest number of dilution at which growth inhibition of the sensitive strain is observed [45]. Therefore, the minimum inhibitory concentration (MIC) was directly determined from the bacteriocin activity and defined as the lowest concentration of bacteriocin resulting in no visible turbidity after 24 h of incubation.

### **3. Results**

#### *3.1. Heterologous Expression and Characterization of Lacticaseicin 30*

The pT7-6his-030 plasmid [30] was transferred to *E. coli* Rosetta strain. Production of lacticaseicin 30 was induced by addition of isopropylthio-β-galactoside (IPTG). Lacticaseicin 30 was purified by Ni-NTA chromatography, and the 6his-tag was removed using the TEV-protease. The purified and his-tag-depleted bacteriocin, produced by heterologous

expression, was characterized by mass spectrometry (MS) (Figure 1) to confirm the bacteriocin amino acid sequence. According to the plasmid construction and the Tev-protease manufacturer's instructions, the histidine-tag removal with Tev-protease leaves a serine residue at the N-terminal position (Ser-lacticaseicin 30), and therefore the calculated average theoretical molecular mass (MM) of the Ser-lacticaseicin is 12,339.2 Da (using ProtParam (https://web.expasy.org/protparam/, accessed on April 2022)). As shown in Figure 1A, the most intense mass signal (mass-to-charge ratio (*m*/*z*) of [M + H]+ bacteriocin ions = 12,350.2) and the second main mass signal (*m*/*z* = 6178.3) corresponding to [M + 2H]2+ bacteriocin ions matched well with the theoretical MM of the Ser-lacticaseicin 30. Consequently, the MALDI-TOF-MS measured MM was assessed as 12,349.2 Da for the heterologous expressed Ser-lacticaseicin 30. To accurately measure the molecular mass of Ser-lacticaseicin 30, this latter was concentrated by organic solvent precipitation, redissolved in ammonium bicarbonate buffer and subjected to reverse-phase high-performance liquid chromatography coupled to an electrospray ionization (ESI) high-resolution mass spectrometer. Figure 1B shows the ESI-MS spectrum and the corresponding deconvoluted mass spectrum, revealing an MM of 12,340 Da, confirming (i) that the purified and his-tag-depleted bacteriocin, produced by heterologous expression is the Ser-lacticaseicin 30, and (ii) that no further proteolytic maturation occurred. The entire amino acid sequence of the heterologously expressed lacticaseicin 30 was confirmed by peptide fingerprinting (Figure 1C). Indeed, after trypsin/Lys-C hydrolysis, the Ser-lacticaseicin-30-issued peptides were subjected to an RP-HPLC-MS/MS analysis, and the obtained LC-MS/MS data were submitted to PEAKS Studio Xpro for peptide identification against the UniProtKB/Swiss-Prot-TrEMBL databases restricted to *Lactobacillus* (accessed on April 2022). As shown in Figure 1C, PEAKS studio Xpro returned the identification of pre-bacteriocin from *Lactobacillus casei* with a protein identification score (−10logP) of 172.53, a sequence coverage of 88% for 20 identified peptides. Only three short-size amino acid sequences were not identified: (i) as expected due to the addition of a serine residue at the N-terminal extremity, the first five N-terminal amino acids; (ii) the six amino acid sequence AEPALR; and (iii) the dipeptide YR in the C-terminal region. Note that the methionine oxidations are due to the fingerprinting experimental procedure, and therefore the methionine residues of purified Ser-lacticaseicin are not oxidized. All together, these MS data evidenced that the amino acid sequence of the heterologously expressed, purified and His-tag-depleted bacteriocin corresponds to Ser-lacticaseicin 30.

The antimicrobial activity of the purified lacticaseicin 30 was tested at pH 5, with and without the 6×his-tag, and the MICs were determined using *E. coli* ATCC 8739 as the target strain. Lacticaseicin 30 displayed the same MIC value of 40 μg/mL, with and without the 6×his-tag.
