Proteinase k

Cells were harvested and DNA was extracted as previously described by Siebert et al. [24 (link)]. In brief, 30 mL bovine raw milk were treated with 1.8 mL 0.3 M EDTA. After the cell harvesting by centrifugation (20 min at 4 °C) and the removal of the milk fat and skimmed milk in the supernatant, the selective lysis of the somatic DNA was performed using proteinase K (20 mg/mL, AppliChem GmbH, Darmstadt, Germany) and DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The DNA extraction was performed with the PowerFood Microbial DNA isolation kit (Qiagen, Hilden, Germany), modified by an additional enzymatic lysis step. Towards this end, lysozyme (25 µg/mL, Carl Roth) and mutanolysin (100 U, Sigma-Aldrich, St. Louis, MO, USA) were added to the cell suspensions together with the MBL solution of the DNA isolation kit, followed by an incubation at 37 °C and 350 rpm for 30 min. After an additional treatment with proteinase K (12.5 mg/mL, AppliChem), the remaining bacterial cells were disrupted in tubes with silica beads using a FastPrep-24TM instrument (MP Biomedicals, LLC, Irvine, CA, USA). The subsequent DNA isolation followed the manufacturer’s protocol, i.e., that of the PowerFood Microbial DNA isolation kit. The DNA was finally eluted in 2 × 24 µL of PCR-grade water (preheated to 55 °C).

Anti-Listeria active metabolites of the LAB isolates were examined for their sensitivity to proteinase K (AppliChem GmbH, Darmstadt, Germany), papain (Carl Roth, Karlsruhe, Germany), and trypsine (Carl Roth, Karlsruhe, Germany), using a well diffusion assay, as described above. Three 6.5 mm wells were cut in line, ensuring a 6 to 8 mm distance between each. In total, 100 µL of a proteolytic enzyme solution, which contained 100 µL of the corresponding enzyme (1 mg/mL of a solution of 5 µL of calcium chloride solution 1 M and 895 µL of dH₂O), was placed into the middle well, and the outer wells were filled with 100 µL of CFS. Plates were incubated for 24 h at 37 °C. The enzyme degradation of anti-Listeria active metabolites in the CFS was shown by a characteristic crescent-shaped zone of inhibition, opened toward the proteolytic enzyme. Uninoculated MSM+ broth was used as a negative control. The well diffusion assay was performed with three independent CFSs of each LAB.

For PCR genotyping, tail tips or tissues were lysed overnight at 55°C in PBND buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.45% NP‐40, 0.45% Tween‐20) supplemented with 8 U/ml proteinase K (AppliChem) and afterward inactivated at 95°C for 10 min. For LOH and residual disease detection in Eμ‐Myc lymphoma samples, crude lysates were prepared as above and 50 ng of genomic DNA purified with peqGOLD Tissue DNA Mini Kit (PeqLab) was used as template for qPCR with primers specific for the EμMyc transgene and a control locus for normalization.
For reverse transcription–quantitative PCR (RT–qPCR), RNA was isolated from cells or tissue samples using the RNeasy Mini Kit (Qiagen) and cDNA was generated with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Gene expression was analyzed on a LightCycler 480 (Roche) using the ABsolute QPCR SYBR Green Mix (Thermo Scientific). Data were evaluated by the ΔΔCt method with β‐actin as a housekeeping gene for normalization.
For mtDNA content analysis, genomic DNA was analyzed by quantitative PCR for the mitochondrial genes mt‐Nd4, mt‐Co1, mt‐Cyb, and mt‐Nd2, and Ct values were normalized to the nuclear gene Trp53.
Primer sequences are provided in Table EV1.