Acidity (TTA) was measured on 10-g dough samples, which have been homogenized with 90 ml of distilled water for 3 min inside a Bag Mixer 400P (Interscience, St Nom, France), and is expressed because the quantity (in ml) of 0.1 N NaOH to attain pH 8.three. Lactic and acetic acids have been determined in the water-soluble extract on the sourdough. Ten grams of sourdough was homogenized with 90 ml of 50 mM Tris-HCl buffer, pH eight.8. Just after incubation (30 min at 25 with stirring), the suspension was centrifuged (12,857 g; 10 min; 4 ), and the supernatant was analyzed utilizing an ta Purifier Glyoxalase (GLO) Purity & Documentation Technique (GE Healthcare Bio-Sciences, Uppsala, Sweden) equipped using a refractive index detector (PerkinElmer Corp., Waltham, MA). The fermentation quotient (FQ) was defined because the molar ratio among lactic and acetic acids. The concentration of absolutely free amino acids (FAA) with the water-soluble extract was determined using the S1PR1 Source 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, along with cysteic acid, methionine sulfoxide, methionine sulfone, tryptophan, and ornithine, and used as the external common (24). PCR amplification and denaturing gradient gel electrophoresis (DGGE) evaluation. Ninety milliliters of 50 mM potassium phosphate, pH 7.0, buffer was added to 10 g of sourdough and homogenized for five min, and 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 of your 16S rRNA genes of the Lactobacillus group, such as 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 on the 16S rRNA genes, which developed amplicons of approximately 330 bp (27). Normal-aem.asm.orgApplied and Environmental MicrobiologyFirm- and Liquid-Sourdough Fermentationization of your gels was performed working with reference ladders of DNA from pure cultures of Acetobacter malorum DSM 14337 and Gluconobacter oxydans DSM 7145 mixed in equal volumes from the very same concentration. DNA from yeasts was amplified with primers NL1 (5=-GCCATATCA ATAAGCGGAGGAAAAG-3=) and LS2 (5=-ATTCCCAAACAACTCGAC TC-3=), corresponding towards the D1-D2 region with the 26S ribosomal DNA (rDNA) (28). The PCR core program was carried out as described previously (26?eight). Amplicons have been separated by DGGE making use of the Bio-Rad DCode Universal Mutation detection Technique (Bio-Rad Laboratories, Milan, Italy). Sybr green I-stained gels were photographed through the Gel Doc 2000 documentation method (Bio-Rad Laboratories). Profiles had been digitally normalized by way of comparison with the common reference (MassRuler Low Variety DNA Ladder, ready-to-use; 80 to 1,031 bp; Fermentas Molecular Biology Tools, Thermo Fisher Scientific Inc., Waltham, MA) and BioNumerics software program, version two.50 (Applied Maths, St. Martens-Latem, Belgium). The DGGE bands of yeasts have been cut out and eluted in 50 l of sterile water overnight at four . Two microliters in the eluted DNA was reamplified, along with the PCR products have been separated as described above. The amplicons had been eluted in the gel and purified with the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). DNA-sequencing reactions had been carried out by MWG Biotech AG (Ebersberg,.