International Journal of Food Microbiology 163 (2013) 71–79 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro The lactic acid bacteria and yeast microbiota of eighteen sourdoughs used for the manufacture of traditional Italian sweet leavened baked goods Anna Lattanzi a, Fabio Minervini a,⁎, Raffaella Di Cagno a, Annamaria Diviccaro a, Livio Antonielli b, Gianluigi Cardinali b, Stefan Cappelle c, Maria De Angelis a, Marco Gobbetti a a Department of Scienze del Suolo, della Pianta e degli Alimenti, University of Bari Aldo Moro, via Amendola 165/a., Bari, 70126, Italy b Department of Applied Biology, Microbiology Division, University of Perugia, via Borgo 20 Giugno, Perugia, 74-06121, Italy c Beldem S.A., Rue Borrie 12, Andenne, Belgium article info abstract Article history: The lactic acid bacteria and yeast microbiota of eighteen sourdoughs used for the manufacture of some traditional Received 27 September 2012 Italian sweet leavened baked goods were studied through culture-dependent method and pyrosequencing. Received in revised form 7 February 2013 Flours used for back slopping and sourdoughs were also biochemically characterized. Principal component Accepted 22 February 2013 analysis was applied to explore eventual correlations between process parameters applied during back slopping, Available online 1 March 2013 some flour nutrients, profile of microbiota, and biochemical characteristics of sourdoughs. The median values of Keywords: the cell density of lactic acid bacteria and yeasts were 8.05 and 7.03 log CFU/g, respectively. As shown by fi Sourdough culture-dependent method, Lactobacillus sanfranciscensis was identi ed in all the sourdoughs, except for Lactic acid bacteria Panaredda and Torcolo di San Costanzo. For eleven sourdoughs, all the lactic acid bacteria isolates were allotted Yeasts to this species. For Buccellato di Lucca, Mbriagotto, Pandoro, and Nadalin sourdoughs, at least 80% of the isolates was allotted to this species. Other lactic acid bacteria isolated with a relatively high frequence were Lactobacillus plantarum and Leuconostoc citreum.Pyrosequencingconfirmed and complemented the culture-dependent approach, detecting L. sanfranciscensis also in Panaredda and Torcolo di San Costanzo sourdoughs. Saccharomyces cerevisiae was identified in all the sourdoughs, except for Mbriagotto, Ciambella di Mosto and Pandolce Genovese. These latter sourdoughs harbored strains of Candida humilis,whereasfive sourdoughs combined the presence of both yeast species. Positive correlations were found between time of back slopping and cell density and main metabolites of lactic acid bacteria. Percentage of sourdough used as inoculum was mainly correlated with the cell density of yeasts and the concentration of ethanol. This study provided a comprehensive and comparative approach to highlight the dominant microbiota of Italian sourdoughs, which could be exploited further to guarantee a highly reproducible quality of the Italian sweet goods studied, while preserving their traditional trait. © 2013 Elsevier B.V. All rights reserved. 1. Introduction (Gobbetti, 1998; Zannini et al., 2010). Sourdough is a preparation of flour and water, containing high numbers of metabolically active lactic The annual production of Italian sweet leavened baked goods is acid bacteria and yeasts (Gobbetti, 1998; Hammes and Gaenzle, estimated to be ca. 31033 t (ISTAT, 2011). Several sweet leavened 1998). According to traditional procedure, dominant microorganisms baked goods are consumed mainly in the occasion of some religious are kept metabolically active by back slopping (De Vuyst and Neysens, feasts (e.g., Panettone, Pandoro and Nadalin, and Pizza di Pasqua, 2005). More than 50 species of lactic acid bacteria, especially belonging Panaredda and Colomba for Christmas and Easter, respectively). to the genus Lactobacillus, and more than 20 species of yeasts, mostly Despite their geographical origin (e.g., Lumbardy for Panettone), belonging to the genera Saccharomyces and Candida, were found during sweet leavened baked goods have, nowadays, a national and interna- sourdough propagation for making traditional/typical leavened baked tional diffusion (ISTAT, 2011). goods (Corsetti et al., 2001; Minervini et al., 2012a; Palomba et al., Traditional sweet leavened baked goods share the use of a sour- 2011; Venturi et al., 2012). Whereas the sourdough microbiota of tradi- dough called “madre” (mother sponge) as the basis for their production tional breads was largely studied (Catzeddu et al., 2006; Minervini et al., 2012a; Reale et al., 2011; Valmorri et al., 2010; Zotta et al., 2008), that of ⁎ Corresponding author. Tel.: +39 080 5442948; fax: +39 080 5442911. sweet leavened baked goods received little attention. Although most E-mail address: [email protected] (F. Minervini). studies involved a limited number of products, a certain degree of 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.02.010 72 A. Lattanzi et al. / International Journal of Food Microbiology 163 (2013) 71–79 microbial diversity was found in the sourdoughs used for manufacturing 2. Material and methods sweet leavened baked goods (Foschino et al., 2004; Garofalo et al., 2008; Palomba et al., 2011; Venturi et al., 2012). 2.1. Sourdoughs The microbial composition of sourdough is influenced by several parameters. Specific technological process parameters include pH and Eighteen sourdoughs used for the manufacture of some traditional redox potential, dough hydration and yield, number of sourdough Italian sweet leavened baked goods, representative of several Italian back slopping steps, fermentation time between back slopping, leave- regions (Fig. S1), were studied. Except for sourdough used for making ning and storage temperature, the use of starters and/or baker's yeast, Cornetto di semola (T. durum flour), all sourdoughs were made with and the percentage of starter cultures, or sourdough, or baker's yeast T. aestivum flour. The only ingredients used for preparing the sour- used as inoculum. Compared to the above parameters, microbiological, doughs were: flour, sourdough coming from a previous fermentation, chemical and enzymatic composition of the flour and the house and tap water. The sourdoughs mainly differed in terms of the following microbiota may be regarded as parameters which are not fully under process parameters (Table 1): percentage (expressed on total dough the control whoever manages the sourdough (De Vuyst et al., 2009). weight) of sourdough used as inoculum; number of back slopping It has been recently reported that the type of flour (Triticum durum or steps necessary to get the “mature” sourdough; time of back slopping; Triticum aestivum), as well as the concentration of some flour nutrients and temperature of back slopping. Three batches of each sourdough (fermentable carbohydrates and free amino acids) required by the were collected in three consecutive days at local bakeries (Weckx microorganisms dominating the sourdough ecosystem, may have a et al., 2010). All samples were taken immediately at the end of the last key role for selecting the population of lactic acid bacteria (Minervini back slopping (mature sourdough), i.e. before being used as leavening et al., 2012a). Consequently, the microbiota structure distinguished agent of doughs containing sugar, eggs, and butter. Mature sourdoughs almost all the sourdoughs used for the manufacture of nineteen were stored at 4 °C for 12 h before analyses. Eighteen sourdoughs were traditional/typical Italian breads (Minervini et al., 2012a). Overall, the analyzed in three different days in duplicate. sourdoughs used for manufacturing sweet products mainly differ from those used for manufacturing breads in terms of: (i) higher inoculum ratio; (ii) shorter fermentation time; and (iii) higher number of back 2.2. Measurement of pH and determination of carbohydrates, organic slopping steps. acids, ethanol and free amino acids This study was aimed at describing the composition of the lactic acid bacteria and yeast microbiota of eighteen sourdoughs, which The values of pH were measured by a pH-meter. The water- are used for the manufacture of traditional Italian sweet leavened soluble extracts were obtained as previously described (Minervini baked goods. Dominating lactic acid bacteria and yeasts were et al., 2012a) and were used for determining the concentrations of monitored by culture-dependent and -independent methods. Possible carbohydrates, organic acids and ethanol through HPLC (Zeppa et al., correlations between some flour nutrients (soluble carbohydrates 2001) and the concentrations of individual free amino acids and free amino acids), process parameters and composition of the (FAA) through a Biochrom 30 Amino Acid Analyser (Biochrom LTD, sourdough microbiota, and between composition of the microbiota Cambridge Science Park, UK) (De Angelis et al., 2007). The quotient of and the biochemical characteristics of sourdoughs, were explored fermentation (QF) was calculated as the molar ratio between D,L-lactic through principal component analysis. and acetic acids. Table 1 Process parameters of sourdoughs used for the manufacture of traditional Italian sweet leavened baked goods. Sweet leavened Code Sourdough No. of back slopping Time (h) and temp (°C) of back sloppingb baked good (% on total dough weight)a steps before use Buondì SA 40 3 4.5, 26 Panettone (Puglia) SB 29