Acidity (TTA) was measured on 10-g dough samples, which had been homogenized with 90 ml of distilled water for 3 min in a Bag Mixer 400P (Interscience, St Nom, France), and is expressed as the amount (in ml) of 0.1 N NaOH to achieve pH 8.3. Lactic and acetic acids have been determined in the water-soluble extract with the sourdough. Ten grams of sourdough was homogenized with 90 ml of 50 mM Tris-HCl buffer, pH eight.8. Right after incubation (30 min at 25 with stirring), the suspension was centrifuged (12,857 g; ten min; 4 ), as well as the supernatant was analyzed using an ta Purifier program (GE 5-HT Receptor Agonist site Healthcare Bio-Sciences, Uppsala, Sweden) equipped using a refractive index detector (PerkinElmer Corp., Waltham, MA). The fermentation quotient (FQ) was defined as the molar ratio involving lactic and acetic acids. The concentration of totally free amino acids (FAA) of the water-soluble extract was determined making use of the Biochrom 30 Amino Acid Analyser (Biochrom Ltd., Cambridge Science Park, Cambridge, England). A mixture of amino acids at known concentration (Sigma Chemical Co., Milan, Italy) was added, in addition to cysteic acid, methionine sulfoxide, methionine sulfone, tryptophan, and ornithine, and utilized as the external normal (24). PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. Ninety milliliters of 50 mM potassium phosphate, pH 7.0, buffer was added to ten g of sourdough and homogenized for 5 min, and also the DNA extraction was carried out as described by Minervini et al. (25). Bacterial DNA was amplified with primers Lac1 (5=-AGCAGTAGG GAATCTTCCA-3=) and Lac2 (5=-ATTYCACCGCTACACATG-3=), targeting a 340-bp region in the 16S rRNA genes on the Lactobacillus group, including the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella (26). DNA from acetic acid bacteria was amplified with primers WBAC1 (5=-GTCGTCAGCTCGTGTCGTGAGA-3=) and WBAC2 (5=-CCCGGG AACGTATTCACCGCG-3=) targeting the V7-V8 regions of the 16S rRNA genes, which made amplicons of around 330 bp (27). Normal-aem.asm.orgApplied and Environmental MicrobiologyFirm- and Liquid-Sourdough Fermentationization from the gels was performed making use of reference ladders of DNA from pure cultures of Acetobacter malorum DSM 14337 and Gluconobacter oxydans DSM 7145 mixed in equal volumes with the similar concentration. DNA from yeasts was amplified with primers NL1 (5=-GCCATATCA ATAAGCGGAGGAAAAG-3=) and LS2 (5=-ATTCCCAAACAACTCGAC TC-3=), corresponding for the D1-D2 region from the 26S ribosomal DNA (rDNA) (28). The PCR core program was carried out as described previously (26?8). Amplicons were separated by DGGE applying the Bio-Rad DCode Universal Mutation detection System (Bio-Rad Laboratories, Milan, Italy). Sybr green I-stained gels were photographed by means of the Gel Doc 2000 documentation system (Bio-Rad Laboratories). Profiles were digitally Dopamine Transporter drug normalized via comparison with the regular reference (MassRuler Low Range DNA Ladder, ready-to-use; 80 to 1,031 bp; Fermentas Molecular Biology Tools, Thermo Fisher Scientific Inc., Waltham, MA) and BioNumerics computer software, version 2.50 (Applied Maths, St. Martens-Latem, Belgium). The DGGE bands of yeasts have been reduce out and eluted in 50 l of sterile water overnight at 4 . Two microliters of the eluted DNA was reamplified, as well as the PCR goods were separated as described above. The amplicons had been eluted in the gel and purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). DNA-sequencing reactions were carried out by MWG Biotech AG (Ebersberg,.