2.4. Evaluation of the microbial population in doughs
At appropriate leavening intervals, 5–8 g of dough were diluted in
45–72 mL sterile peptone water (10 g/L Bacto-peptone in distilled
water, pH 6.8) and homogenized in a Stomacher 400 Circulator (Seward, Worthing, UK) for 5 min at 260 rpm. After decimal dilutions in
the same solution, suspensions were plated in appropriate media. L.
sanfranciscensis population was plated onto MRSm agar (MRSm broth
added of 15 g/L agar) while Z. mobilis onto DSM agar (DSM broth
added of 15 g/L agar). Plates were then incubated at 30 °C for 3 days
in anaerobic conditions. Total bacterial count (TBC) was determined
pour plating in Tryptic Soy Agar (TSA, Scharlab, Barcelona, Spain) after
incubation at 30 °C for 48–72 h. Yeasts and moulds were determined
pour plating in Yeast Glucose Chloramphenicol Agar (YGC-Scharlab)
and incubated at 25 °C for 3–5 days. Counts were reported as logarithms
of the number of colony forming units (Log CFU/g of dough), and means
and standard deviations were calculated (n = 3).
2.5. Analytical determinations
Sugars (maltose and glucose) consumption, as well as ethanol, lactic
and acetic acid produced during leavening were determined through an
HPLC system (L 7000, Merck Hitachi) equipped with RI and UV
(210 nm) detectors serially connected, using a (300–8 mm) SH1821
(Shodex, München, Germany) column, maintained at 50 °C and eluted
with 5 mM H2SO4 at 0.5 mL/min. Aliquots of 2–4 mL of homogenized
and appropriately diluted dough samples were centrifuged (Eppendorf
5804, 10,600 ×g, 10 min) and the obtained supernatants were filtered
through a 0.45 μm syringe filter (VWR International, USA) before
HPLC analysis. Date were referred to 1 g dough (mg/g dough).
Dough pH was monitored at different intervals on the integral undiluted dough sample (pH-meter Eutech Instruments pH 510).
2.6. Statistical analysis
Data were submitted to t-test and one-way analysis of variance
(ANOVA) performed with SPSS software, version 21.0 (SPSS Inc., Chicago, IL, USA). When the effect was significant (p b 0.05), differences between means were assessed by Tukey-b test of multiple comparisons.