Fermentation of soy juice by Lactiplantibacillus plantarum confers a yogurt-like texture, flavours and health-promoting metabolites
Fermentation of soy juice by Lactiplantibacillus plantarum confers a yogurt-like texture, flavours and health-promoting metabolites
The experimentations were performed in five from independent subcultures. Soy juice (Sojade, Triballat) was inoculated with Lactobacillus plantarum CIRM-BIA777 at 1% v/v. Incubation was conducted at 37°C and without anoxic conditions to mimic industrial conditions. Samples were collected throughout fermentation when the targeted pH 6.5, 6.0, 5.0 were reached. Cultures were then sampled for RNA extraction.  RNA was extracted from 1 mL of FSJ directly treated with 2 mL of RNA protect cell reagent (Qiagen, Hilden, Germany). This sample was centrifuged 5 min at 4°C, 10,000 g to discard the supernatant. The pellet was frozen at - 80°C with 2 mL of RNA protect. After defrost, the samples were centrifuged again for 5 min at 4°C, 10,000 g to discard the supernatant. Cell pellets were suspended in 200 μL lysis buffer (50 mM Tris–HCl, 1 mM EDTA; pH 8.0) 20 mg/mL lysozyme (MP Biomedicals, Illkirch, France) and 50 U/mL mutanolysin (Sigma, Saint Quentin Fallavier, France) and incubated for 30 min at 24°C. Suspensions were then transferred to 2 mL tubes containing 50 mg zirconium beads (diameter: 0.1 mm; BioSpec Products, Bartlesville) and 50 µL SDS (20 %). Samples were then shaken in a Precellys Evolution (Bertin, Montigny-le-Bretonneux, France) for two cycles of 40 s at 6500 rpm.  RNA was then extracted from the cell lysate using the RNeasy Mini kit (Qiagen) and a subsequent DNase treatment (Dnase Rnase free, Ambion) according to the manufacturer's instructions. Some extractions (S60b2110 and S50e1510) were realized in duplicates with phenol-chloroform since a unique method was not optimal to extract RNA in every sample. An additional step was realized before RNeasy Mini kit: lysates were transferred in Phase Lock Gel Heavy with 900 µL QIAzol in which 200 µL chloroform was added, left 2 min and centrifuged 15 min at 12,000 g.   Total RNA samples are quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity is checked with TapeStation (Agilent Technologies, Palo Alto, CA, USA). RNA library preparations, sequencing reactions, and initial bioinformatics analysis is conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). Whole transcriptome RNA enrichment is performed using NEBNext rRNA Depletion Kit (Bacteria) and NEBNext Ultra II RNA Library Prep Kit for Illumina by following manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA).