Preliminary study by molecular methods on lactic acid
bacteria and yeasts of Calabrian sourdoughs
A. Martorana, A. Caridi and R. Sidari
Department of Agraria, Mediterranea University of Reggio Calabria, Loc. Feo di Vito, 89122 Reggio Calabria, Italy
MATERIALS AND METHODS
Four samples of sourdough were collected from bakeries located in Catanzaro (PF1, PF2, and PF4) and in Reggio Calabria (PF5), Calabria region, Italy (Figure 1). Chemical, microbiological, and molecular analyses were carried out.
Chemical analyses
The pH was determined by a spin electrode pH-meter (HI99161, Hanna Instruments) in different parts of the samples. Total titratable acidity (TTA) was determined by titration with 0.1 N NaOH and expressed as milliliters of NaOH.
Microbiological analyses
Each sample (10 g) was homogenized, serially diluted, and plated in MRS and SDB (30 °C for 48 h in anaerobic conditions) for LAB and in YPD (30 °C for 48 h) for yeasts. From each medium plated with the different sample, ten colonies (for LAB Gram-positive and catalase negative) with different morphologies were isolated, purified by successive sub-culturing, and stored at – 80 °C.
Molecular analyses
DNA was extracted by InstaGene matrix (Bio-Rad) both from LAB and yeasts and subjected to amplification using specific primers [6,7]. The LAB and the yeasts identification was performed by PCR-ARDRA [8] and by PCR-RFLP [7] comparing the profile isolates to those previously described in literature and to International Collection reference strains.
REFERENCES
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ABSTRACT
Sourdough is widely utilized in the production of traditional bread. Lactic acid bacteria (LAB) and yeasts are usually present in sourdough in the ratio of 1:100 [1]. LAB and yeasts co-exist establishing a dynamic equilibrium that determines the peculiarity of this product as well as its long-term storage. Lactobacillus is the genus mainly associated to the sourdough fermentation but it is possible to find other genus such as Enterococcus, Lactococcus, Leuconostoc, Pediococcus,
Streptococcus and Weissella [2]. Lactobacillus sanfrancisciensis is the typical lactic acid bacterium isolated from the sourdough and it has a significant role in the
sourdough production [3]. Yeasts such as Saccharomyces cerevisiae, Candida milleri, Kazachstania exigua, Candida krusei, and Pichia anomala can harboured the sourdough environment even if the most present is S. cerevisiae [4,5]. At present there is very few information about Calabrian sourdoughs, therefore the aim of the present study was to investigate LAB and yeasts population of sourdoughs from Calabria region in order to select the best autochthonous strains as starter in sourdough production.
RESULTS AND DISCUSSION
Table 1 shows pH, TTA values, and microbial loads of analyzed sourdoughs. Among them, PF1 showed the lowest pH (3.93) and the highest TTA (9.60 ml of NaOH 0.1 N) while the opposite was observed in PF2 (pH: 5.59 and TTA: 2 ml of NaOH 0.1 N). The highest count for LAB (109 CFU/g) was recorded in PF4 both in MRS and SDB while for yeasts in PF2 (108 CFU/g). In PF1 and PF5, a dominance of L. sanfrancisciensis was observed; in PF2 additionally was observed Pediococcus acidilactici while in PF4 the obtained profiles did not match neither reference strains profiles nor strains profiles present in literature. Concerning the yeasts, the specie S. cerevisiae was detected in all the sourdoughs. Figure 2 shows restriction patterns obtained with HaeIII, HinfI, and CfoI of some S. cerevisiae and obtained with HaeIII, AluI, and FokI of some L. sanfrancisciensis strains.
The values of pH, TTA, and microbial loads are in accordance with previous results on sourdoughs [5,9] except for PF2 (named Pitta) which is a sourdough with characteristics different from the other Calabrian sourdoughs (named pane tradizionale/pane di grano). As reported in literature [10], S. cerevisiae was dominant in all the samples probably due to the contamination by baker’s yeast [11].
Further analyses are necessary to test LAB and yeasts for technological features to select the best autochthonous strains to use as starter in sourdough production.
Figure 1 - Calabrian provinces where sourdoughs were sampled.
This work was supported by PROGETTO PON03PE_00090_1 Innovazione di prodotto e di processo nella filiera dei prodotti da forno e dolciari
Figure 2 - Representative restriction patterns obtained with HaeIII (a),
HinfI (b), and CfoI (c) for Saccharomyces cerevisiae strains and with HaeIII (d), AluI (f), and FokI (g) for Lactobacillus sanfrancisciensis. M:
100-bp plus molecular marker.
M M M M M M a b c d e f a Expressed as ml of 0.1N NaOH
Table 1 - pH, total titratable acidity (TTA), and microbial loads of the sourdoughs. MRS SDB YPD PF1 - pane tradizionale 3.93±0.06 9.60±0.28 7.9±7.1*108 8.8±7.1*107 4.3±7.1*107 PF2 - Pitta 5.59±0.03 2.00±0.14 3.2±0.7*106 3.9±2.1*106 1.9±19.8*108 PF4 - pane tradizionale 3.96±0.05 7.10±0.30 1.1±11.3*109 1.1±6.4*109 3.1±1.4*107 PF5 - pane tradizionale 4.59±0.27 6.10±0.26 4.5±25.5*108 6.8±24.0*108 2.6±4.2*105
