Acidity (TTA) was measured on 10-g dough samples, which have been homogenized with 90 ml of distilled water for three min in a Bag Mixer 400P (Interscience, St Nom, France), and is expressed because the amount (in ml) of 0.1 N NaOH to attain pH eight.three. CDK11 manufacturer Lactic and acetic acids have been determined inside 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. Immediately after incubation (30 min at 25 with stirring), the suspension was centrifuged (12,857 g; ten min; 4 ), and also the supernatant was analyzed working with an ta Purifier system (GE Healthcare Bio-Sciences, Uppsala, Sweden) equipped having a refractive index detector (PerkinElmer Corp., Waltham, MA). The fermentation quotient (FQ) was defined because the molar ratio in between lactic and acetic acids. The concentration of absolutely free amino acids (FAA) in 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 identified concentration (Sigma Chemical Co., Milan, Italy) was added, as well as cysteic acid, methionine sulfoxide, methionine sulfone, tryptophan, and ornithine, and applied because the external typical (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 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 from the 16S rRNA genes in the Lactobacillus group, which includes the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella (26). DNA from acetic acid bacteria was amplified with primers WBAC1 (5=-GTCGTCAGCTCGTGTCGTGAGA-3=) and WBAC2 (5=-5-LOX Compound CCCGGG AACGTATTCACCGCG-3=) targeting the V7-V8 regions with the 16S rRNA genes, which produced amplicons of roughly 330 bp (27). Normal-aem.asm.orgApplied and Environmental MicrobiologyFirm- and Liquid-Sourdough Fermentationization in the gels was performed employing reference ladders of DNA from pure cultures of Acetobacter malorum DSM 14337 and Gluconobacter oxydans DSM 7145 mixed in equal volumes on the 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 area of the 26S ribosomal DNA (rDNA) (28). The PCR core plan was carried out as described previously (26?8). Amplicons were separated by DGGE working with the Bio-Rad DCode Universal Mutation detection Technique (Bio-Rad Laboratories, Milan, Italy). Sybr green I-stained gels were photographed by way of the Gel Doc 2000 documentation system (Bio-Rad Laboratories). Profiles have been digitally normalized by means of comparison together with the regular 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 computer software, 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 items were separated as described above. The amplicons have been eluted in the gel and purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). DNA-sequencing reactions have been carried out by MWG Biotech AG (Ebersberg,.
