Dneasy mericon food kit

Sample preparation preceding metabarcoding analysis was performed as previously described [13 (link)]. DNA extraction was performed using a DNeasy mericon Food Kit (Qiagen, Antwerp, Belgium), following the manufacturer’s instructions. The V3-V4 region of the 16 S rRNA gene was targeted to obtain the fragment libraries, which were amplified as described by the Illumina protocol and with primers of Klindworth et al. [19 (link)] on an Illumina MiSeq sequencer with 2 × 300 bp chemistry by a provider known as Admera Health (South Plainfield, NJ, USA). The following sample codes were used: V symbolizes vegetarian product, B indicates the batch number while L represents when a vegetarian product log was sliced (i.e. L1 and L3 represent vegetarian product logs sliced one and three weeks after production, respectively). D0 describes that the product was analyzed on the day of slicing, while D28 represent the product which was analyzed after 28 d of storage.

The DNA isolation, genome sequencing and genome annotation of L. paracasei KF1, L. paracasei ABK, L. paracasei MA2 and L. paracasei MA3 were performed as described by Savinova et al. [37 (link)]. In brief, total DNA was extracted from liquid MRS cultures using a DNeasy mericon Food Kit (Qiagen) according to the manufacturer’s protocol. The DNA library was prepared using the Ion AmpliSeq library kit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) and indexed with an Ion Xpress barcode adapters 1–16 kit (Thermo Fisher Scientific, MA, USA). Whole-genome sequencing was carried out using the Ion Torrent Personal Genome Machine (PGM) (Thermo Fisher Scientific). The obtained reads were pre-processed and assembled with CLC Genomics Workbench 11.0 (Qiagen). Upon submission, genome annotations were performed using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [40 (link)]. Additionally, annotation with ISfinder [41 (link)], PHASTER [42 (link)], PlasmidFinder [43 (link)] and BAGEL4 [44 (link)] was performed on the web. The comparative genome analysis was performed using the Anvi’o suite of programs [45 (link),46 (link)].

After the Douchi sample was ground evenly, 5 g was added to 45 ml normal saline. After beating for 10 min with a beater and centrifugation at 400 r/min for 5 min, the supernatant was again centrifuged at 10,000 r/min for 10 min. The macrogenomic DNA was extracted from the precipitate by QIAGEN DNeasy Mericon Food Kit, and the DNA samples were stored at −20°C (Wang et al., 2018).
The extracted macrogenomic DNA was amplified by PCR using the forward primer SSU0817F (5'‐TTAGCATGGAATAATRRAATAGGA‐3') and the reverse primer SSU1196R (5'‐TTAGCATGGAATARAATAGGAMAULY‐3'). Furthermore, seven nucleotide tags were added to the forward primer (Lin, 2019). The PCR amplification system was as follows: 0.8 μL forward primer (5 μmol/L), 0.8 μl reverse primer (5 μmol/L), 2 μl dNTPs mix (2.5 mmol/L), 4 μl 5× PCR buffer, 10 ng DNA template, and 0.4 μl DNA polymerase (5 U/μl). The mix was supplemented with ddH₂O to 20 μl. The PCR amplification conditions were as follows: 95°C 3 min, 95°C 30 s, 55°C 30 s, 72°C 45 s, 30 cycles; final extension at 72°C for 10 min (Tall & Meyling, 2018). The DNA products were sequenced by Illumina MiSeq high‐throughput sequencing platform.

Two independent extractions per sample were performed using the DNeasy mericon Food Kit (Qiagen, Hilden, Germany) recommended for this sample type^{35 (link),36 (link)}. A subset of the extractions was tested for inhibitors with quantitative real-time PCR (qPCR) applying different dilutions in triplicates. qPCR reagents and conditions were the same as in DNA metabarcoding PCR reactions (see below), with the addition of 10,000 fold diluted SybrGreen (Thermo Fisher Scientific, USA). Following these analyses, all samples were diluted fivefold before PCR amplification. All extractions were performed in a laboratory restricted to forensic or low DNA-content analyses.

The seeds were milled using a Cyclotec (FOSS Italia S.r.l., Padova, Italy) at a grid diameter of 0.2 mm, avoiding any contamination between samples. DNA was extracted from three biological replicates of milled seeds using the DNeasy mericon Food Kit (Qiagen, Milan, Italy), according to manufacturer’s instructions. The evaluation of quality and quantity of the extracted DNA was performed using a Qubit™ fluorometer in combination with the Qubit™ dsDNA BR assay kit (Invitrogen by Thermo Fisher Scientific, Monza, Italy).
The same procedure was applied for the DNA extraction from flour and food samples, starting from 2 g. The evaluation of quality and quantity of extracted DNA was performed as described above.

An aliquot of 200 mg of either homogenized wet pet food or ground dry pet food was sampled under sterile conditions in duplicate for each product. The samples were processed for DNA extraction using the DNeasy mericon food kit (QIAGEN, Hilden, Germany) according to manufacturer instructions. DNA quality and concentration were assessed after extraction by spectrophotometer (SmartSpec™ Plus spectrophotometer, Bio-Rad, USA).

DNA extraction was performed after careful selection of an appropriate DNA extraction procedure (see DNA Extraction section). Out of the analyses considered, the DNeasy mericon Food Kit (Qiagen GmbH, Hilden, Germany) was selected. Thawed tuna muscle tissue samples were processed according to the instructions of the kit manufacturer. Raw muscle analysis was performed in triplicate. Regarding commercial products (n = 70), preliminary sample processing was necessary. Analysis of each sample was performed in duplicate. In the case of canned tuna, the muscle tissue was drained in paper towels and freed from possible undesirable ingredients (fat, vegetables, etc.). Pastes, pâtés, spreadable pastes, and samples with thicker sauces were pre-centrifuged. Granules were crushed in a mortar. Total weight taken into the analysis was 200 mg with the addition of lysis buffer and proteinase K under specified conditions (60 °C, 1000 rpm, overnight).