Amplifying the 16S rRNA genes (36). Primers designed for the recA gene had been also employed to distinguish Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum species (37). Primers developed for the pheS gene had been utilized for identifications to the species level within the genera Leuconostoc and Weissella (38). Sequencing BRD4 Protein Synonyms evaluation for acetic acid bacteria was carried out using primers 5=-CGTGTCGTGAGATGTTGG-3= (positions 1071 to 1087 on 16S rRNA genes; Escherichia coli Prostatic acid phosphatase/ACPP Protein Storage & Stability numbering) and 5=-CGGGGTGCTTTTCACCTTT CC-3= (positions 488 to 468 on 23S rRNA genes; E. coli numbering), according to the procedure described by Trcekl and Teuber (39). To iden tify presumptive yeasts, two primers, NL-1 (5=-GCATATCAATAAGCGG AGGAAAAG-3=) and NL-4 (5=-GGTCCGTGTTTCAAGACGG-3=), had been employed for amplifying the divergent D1-D2 domain from the 26S rDNA (40). Electrophoresis was carried out on an agarose gel at 1.five (wt/vol) (Gellyphor; EuroClone), and amplicons were purified with GFX PCR DNA in addition to a Gel Band Purification Kit (GE Healthcare). Sequencing electrophoregram information were processed with Geneious. rRNA sequence alignments were carried out working with the multiple-sequence alignment technique (41), and identification queries had been fulfilled by a BLAST search (29) in GenBank ( Determinations of VOC and VFFA. VOC had been extracted by way of purge and trap coupled with gas chromatography-mass spectrometry (PT C-MS) based on the approach of Di Cagno et al. (42). Volatile totally free fatty acids (VFFA) have been extracted by solid-phase microextraction coupled with GC-MS (SPME C-MS). Before PT and SPME analyses, a suspension of ten (wt/wol) sourdough in UHQ water deodorized by boiling for 15 min was homogenized with Ultra-Turrax (IKA Staufen, Germany). For extraction of VOC, ten ml of this suspension was poured into a glass extractor connected towards the PT apparatus (Tekmar LSC 3000; Agilent Technologies, Les Ulis, France). Extraction was carried out at 45 for 45 min with helium at a flow rate of 40 ml/min on a Tenax trap (Agilent Technologies) at 37 . Trap desorption was at 225 , and injection into the chromatograph was performed directly into the column using a cryo-cooldown injector at 150 . The chromatograph (6890; Agilent Technologies) was equipped with a DB5-like (apolar) capillary column (RTX5; Restek, Lisses, France; 60-m length, 0.32- m inside diameter [i.d.], and 1- m thickness). The helium flow rate was 2 ml/min; the oven temperature was 40 for the duration of the very first 6 min, and after that it was enhanced at three /min to 230 . The mass detector (MSD5973; Agilent Technologies) was applied in electronic effect at 70 eV in scan mode from 29 to 206 atomic mass. Identification of volatile compounds was carried out by comparison of experimental mass spectra with spectra on the NIST/EPA/MSDC Mass Spectral Database (Royal Society of Chemistry, Cambridge, Uk). Semiquantification was accomplished by integration of one particular ion characteristic of every compound, allowing comparison of your location of each and every eluted compound involving samples. Measurements are given in arbitrary area units of characteristic ions. Analyses have been duplicated. For SPME extraction of VFFA, each and every sample was analyzed 3 instances at 3 unique dilutions; 200 l, 400 l, or 1 ml from the 10 suspension of sourdough was poured into a 10-ml flask with one hundred l of 2 N sulfuric acid and 900, 700, or 100 l, respectively, of UHQ water. The flask was sealed and placed into a bath at 60 for 15 min. An SPME carboxen/polydimethyl.