Ing for the method of De Los Reyes-Gavil et al. (31). Three
Ing for the process of De Los Reyes-Gavil et al. (31). Three oligonucleotides, P4 (5=-CCGCAGCGT T-3=), P7 (5=-AGCAGCGTGG-3=) (32), and M13 (5=-GAGGGTGGCGG TTCT-3=) (33), with arbitrarily selected sequences were made use of for biotyping of lactic acid and DP Agonist custom synthesis acetic acid bacterial isolates. The reaction mixture and PCR conditions for primers P4 and P7 have been those described by Corsetti et al. (32), whereas those reported by Zapparoli et al. (34) were made use of for primer M13. Genomic DNA of yeast was extracted utilizing a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s guidelines. Two oligonucleotides, M13m (5=-GAGGGTGGCGGTTC-3=) and Rp 11 (5=-GAAACTCGCCAAG-3=) (35), were employed singly in two series of amplifications for biotyping of yeast isolates. RAPD-PCR profiles were acquired by the Gel Doc 2000 Documentation System and compared usingFingerprinting II Informatix application (Bio-Rad Laboratories). We evaluated the similarity with the electrophoretic profiles by determining the Dice coefficients of similarity and making use of the unweighted-pair group system utilizing typical linkages (UPGMA) algorithm. Because RAPD profiles with the isolates from one batch of every type of sourdough have been confirmed by analyzing isolates from two other batches, strains isolated from a single batch have been further analyzed. Genotypic identification of lactic acid and acetic acid bacteria and yeasts. To identify presumptive lactic acid bacterial strains, two primer pairs, LacbF/LacbR and LpCoF/LpCoR (Invitrogen Life Technologies, Milan, Italy), had been utilized for amplifying the 16S rRNA genes (36). Primers created for the recA gene had been also utilised to distinguish Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum species (37). Primers made for the pheS gene were used for identifications towards the species level inside the genera Leuconostoc and Weissella (38). Sequencing evaluation for acetic acid bacteria was carried out utilizing primers 5=-CGTGTCGTGAGATGTTGG-3= (positions 1071 to 1087 on 16S rRNA genes; Escherichia coli numbering) and 5=-CGGGGTGCTTTTCACCTTT CC-3= (positions 488 to 468 on 23S rRNA genes; E. coli numbering), as outlined by 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=), have been utilized for amplifying the divergent D1-D2 domain in the 26S rDNA (40). Electrophoresis was carried out on an agarose gel at 1.five (wt/vol) (Gellyphor; EuroClone), and amplicons have been purified with GFX PCR DNA and also a Gel Band Purification Kit (GE Healthcare). Sequencing electrophoregram data were processed with Geneious. rRNA ERK1 Activator review sequence alignments were carried out utilizing the multiple-sequence alignment approach (41), and identification queries were fulfilled by a BLAST search (29) in GenBank (ncbi.nlm.nih.gov/GenBank/). Determinations of VOC and VFFA. VOC have been extracted through purge and trap coupled with gas chromatography-mass spectrometry (PT C-MS) in line with the method of Di Cagno et al. (42). Volatile no cost fatty acids (VFFA) were extracted by solid-phase microextraction coupled with GC-MS (SPME C-MS). Prior to PT and SPME analyses, a suspension of 10 (wt/wol) sourdough in UHQ water deodorized by boiling for 15 min was homogenized with Ultra-Turrax (IKA Staufen, Germany). For extraction of VOC, 10 ml of this suspension was poured into a glass extractor connected towards the PT apparatus (Tekmar LSC 3000; Agilent Technologies, Les Ulis, France). Extract.
