Dneasy powerfood microbial kit

The bacterial samples were collected and identified using conventional laboratory techniques, which involved the standard procedure for isolating bacteria from body fluids or anaerobic cultures. For anaerobic bacteria, the isolates were obtained by placing the samples on Lactobacillus de Man Rogosa and Sharpe (MRS) agar plates. Subsequently, these plates were kept in anaerobic conditions at 37 °C for 72 h [40 (link)].
Microbial DNA was extracted from 56 different bacterial strains from 30 breast milk samples using a modified protocol from the DNeasy Powerfood Microbial kit (Qiagen, UK). DNA templates were amplified by PCR using the universal primers amplifying a 1000-bp region of the 16S rRNA gene: 616 V: 5′-AGAGTTTGATYMTGGCTCAG-3′ and 699R: 5′-RGGGTTGCGCTCGTT-3′. The 616 V and 699R primers, Taq DNA polymerase, and dNTP mix were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Amplicons were purified using the commercial Metabion GmbH mi-PCR Purification Kit (Planegg, Germany), followed by sequencing reactions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA), with a premixed format. The resulting sequences were automatically aligned, visually inspected, and compared with the online tool BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, 5 June 2023). The strain was identified based on the highest scores.

E. coli isolates were cultivated in BHI broth (37°C, orbital shaker at 300 rpm), 18–24 h. Genomic DNA was extracted using DNeasy PowerFood Microbial Kit (QIAGEN) with minor modifications. Eight mL of culture liquid was centrifuged (15 min at 4°C, 20,000 × g) to pellet the bacteria. The pellet was resuspended in 450 μL lysis buffer and incubated for 10 min at 65°C. Thereafter, the whole component was transferred to the Powerbead tubes and secured horizontally to a vortex adapter (Vortex-2 Genie®) and vortexed at a maximum speed for 10 min. After washing steps to remove protein and other inhibitors, purified DNA was eluted and the cconcentration and purity of the isolated genomic DNA evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). A sample of DNA was considered acceptable if the A260/280 ratio was ~1.8.

DNA of all strains and reconstituted starters were extracted using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer’s instructions. The DNA of the fermented milks was extracted with DNeasy PowerFood Microbial Kit (Qiagen) according to the manufacturer’s instructions with the following modifications: first, the cell pellet was resuspended in the MBL solution and incubated at 70°C for 10 min. The genomic DNA concentration (C_ng/μl) was obtained by measuring absorbance at 260 nm on a Nanodrop ND2000. For the sake of convenience, C_ng/μl was converted into C_copies/μl according to the following equation: C_copies/μl = Na × C_ng/μl × 10^–9/(L × 660), where Na is the Avogadro constant (6.02 × 10²³/mol, and L is the average size of a lactococcal genome (2.5 × 10⁶ bp). This calculation provides only an estimated value in copies/μl since A260 measurement depends on various factors and does not distinguish between intact and fragmented targets.
DNA purity was evaluated by the ratio of absorbance 260 nm/280 nm. Extracted DNA was diluted and used for the ddPCR assays at appropriate dilutions.

For direct DNA extraction from PBMA samples the DNeasy^® PowerFood^® Microbial Kit (Qiagen) was used. For that purpose, the cell pellets stored in PBS (−80 °C) were thawed on ice, centrifuged (3000 × rcf, 30 min) and resuspended in 450 μl MBL buffer. Deviating from the DNeasy^® PowerFood^® Microbial Kit Handbook⁸⁷ , the lysis step was proceeded in Lysing Matrix A, 2 ml tubes (MP Biomedicals Germany GmbH, Eschwege, Germany). 16S rRNA gene amplicon library generation and sequencing was performed at the Vienna Biocenter Core Facilities NGS Unit (Vienna, Austria, www.vbcf.ac.at). Sequencing libraries of the 16S rRNA gene (V3/4 region) were prepared based on Illumina 16S rRNA Gene Amplicon Sequencing Library Preparation recommendations. Primers 341F (5’-CCT ACG GGN GGC WGC AG-3’) and 805R (5’-GAC TAC HVG GGT ATC TAA TCC-3’) (Klindworth et al. 2013) were used together with Illumina adapter sequences (5’-CGT CGG CAG CGT CAG ATG TGT ATA AGA GAC AG-3’ and 5’-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3’, respectively) for amplification. Libraries were constructed by ligating sequencing adapters and indices onto purified PCR products using the Nextera XT Sample Preparation Kit (Illumina). Equimolar amounts of each of the purified amplicons were pooled and sequenced on an Illumina MiSeq Sequencer with a 300 bp paired-end read protocol, yielding a median of 96,965 sequences per sample.

Trying to avoid the rind, a fixed amount of 1 g of cheese belonging to the central portion was homogenized with 9 mL of phosphate-buffered saline (PBS; pH 6.5). Subsequently, 1.5 mL of each resuspended cheese sample was subjected to bacterial DNA extraction using a DNeasy PowerFood microbial kit according to the manufacturer’s instructions (Qiagen, Germany). Then, each cheese sample’s DNA concentration and purity was investigated by employing a Picodrop microtiter Spectrophotometer (Picodrop, Hinxton, UK). The extracted DNA was prepared using the Illumina Nextera XT DNA library preparation kit. Briefly, the DNA samples were enzymatically fragmented to 550 to 650 bp using a BioRuptor machine (Diagenode, Belgium), barcoded, and purified involving the Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany). Then, samples were quantified using the fluorometric Qubit quantification system (Life Technologies, USA), loaded on a 2200 TapeStation instrument (Agilent Technologies, USA), and normalized to 4 nM. Sequencing was performed using an Illumina NextSeq 500 sequencer with NextSeq high output v2 kit chemicals (150 cycles) (Illumina Inc., San Diego, CA 92122, USA). All sequencing data were uploaded with BioProject PRJNA865096 and SRA study SRP389312.

Total genomic DNA was extracted directly from goji berries (G1–G4) using a DNeasy PowerFood Microbial Kit (Qiagen, Venlo, the Netherlands), according to the manufacturer’s protocol with some modifications. The goji berries were weighed (250 mg of each sample) and ground with a lysing matrix (1.4 mm ceramic spheres, 0.1 mm silica spheres, 4 mm glass bead). The quantity and quality of the isolated DNA were measured using a Qubit 2.0 Fluorometer (Invitrogen/Life Technologies, Carlsbad, CA, USA).

We used DNeasy^® PowerFood^® Microbial Kit (Qiagen, Hilden, Germany) from here on PF_alone, the Milk Bacterial DNA Isolation kit (Norgen Biotek Corp., Ontario, Canada) from here on NG_alone, and the MolYsis™ Plus kit (Molzym, Bremen, Germany), from here on Mol Plus_alone, according to manufacturers’ protocol. We followed some of the manufacturers’ recommendations for extra DNA yield for the PF Kit, including heating the cell lysate at 70°C for 10 min.