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Food Microbiology 27 (2010) 897e908

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Food Microbiology

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Robustness of Lactobacillus plantarum starters during dailypropagation of wheat flour sourdough type I

Fabio Minervini*, Maria De Angelis, Raffaella Di Cagno, Daniela Pinto, Sonya Siragusa,Carlo Giuseppe Rizzello, Marco GobbettiDipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, via Amendola 165/a, 70126 Bari, Italy

a r t i c l e i n f o

Article history:Received 30 September 2009Received in revised form19 May 2010Accepted 20 May 2010Available online 1 June 2010

Keywords:SourdoughLactobacillus plantarumRobustness

* Corresponding author. Tel.: þ39 080 5442948; faE-mail address: f.minervini@agr.uniba.it (F. Minerv

0740-0020/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.fm.2010.05.021

a b s t r a c t

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillus plantarum.Seven strains were singly used as sourdough type I starters under daily back-slopping propagation (tendays) using wheat flour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of9.13e9.46 log cfu g�1) between and within started sourdoughs, as well as the acidifying activity (medianvalues of 1.24e1.33). After three days also the control sourdough (unstarted) had the same values. Asshown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintainedelevated cell numbers (ca. 9 log cfu g�1) throughout ten days. The other two strains progressivelydecreased to less than 5 log cfu g�1. As identified by partial sequencing of 16S rRNA and recA genes,L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated theflour microbiota. Monitoring of lactic acid bacteria during sourdough propagation was carried out byculture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except forthe sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain ofL. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiaeand Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1.The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughscontaining persistent starters behaved similarly and their profiles were clearly differentiated from theothers. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previouslyshown to be persistent (Siragusa et al., 2009), were used as the mixed starter to produce a wheat floursourdough. Both strains cohabited and dominated during ten days of propagation.

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1. Introduction

The current renaissance of sourdough application in breadmaking is motivated by the beneficial effects of sourdough on theflavor, texture, shelf life and nutritional properties of bread andother baked goods (Gänzle et al., 2007). Three types (I, II and III) ofsourdough are distinguished based on the protocol of propagationand metabolic activities of the main lactic acid bacteria. Type IIsourdough is propagated at high temperatures (>30 �C) with longfermentation time (up to 5 days) and high water content, and it ismainly used as acidifying and aroma carrier. Type III mainlycorresponds to a dried sourdough used as flavoring agent (Hammesand Gänzle, 1998). Type I sourdough has the largest application andresembles the traditional processes. It is characterized by contin-uous (daily) propagation to keep the microorganisms in an active

x: þ39 080 5442911.ini).

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state, as indicated by high metabolic activity. Propagation of sour-dough type I is achieved by daily back-slopping, using the mothersponge taken from the preceding fermentation, mixing it withwater and flour, and allowing to ferment for at least 6 h at ca. 30 �C(De Vuyst and Neysens, 2005). For industrial bread making, back-slopping of sourdough type I should occur less frequently (weeklyor even monthly), but in this case starter cultures have to be used.This option is preferred under industrial conditions since lesslaborious, rarely subjected to variation of the microbial composi-tion and leading to a more standardized quality of baked goods.Nevertheless, only a few studies have considered the use of startercultures during propagation of sourdough type I (Corsetti et al.,2007; Siragusa et al., 2009; Vogelmann et al., 2009).

To prepare starters for bread making, functional selection ofstrains is the primary requisite (Stoltz and Böcker, 1996). Design ofstarters is mainly based on acidification and proteolysis properties,and synthesis of volatile compounds during sourdough fermenta-tion (Collar, 1996; Corsetti et al., 1998; Hammes and Gänzle, 1998;

F. Minervini et al. / Food Microbiology 27 (2010) 897e908898

Gobbetti et al., 2005). Nevertheless, functional sourdough startershave also to well adapt to sourdough environment and process, inorder to guarantee their stable persistence in the dough (De Vuyst,2000; De Vuyst and Neysens, 2005). Although species belonging toLeuconostoc, Pediococcus orWeissella genera had been isolated fromsourdough, the majority of the autochthonous sourdough strainsbelong to the Lactobacillus genus. Among over 150 species belongingto the genus Lactobacillus, a large number (ca. 40) was first isolatedfrom sourdoughs or from spontaneous fermentation of cereals(De Vuyst and Neysens, 2005; Ehrmann and Vogel, 2005; Hammeset al., 2005). Therefore, selection of potential starters for sour-dough fermentation is more convenient within Lactobacillus. Inparticular, traditional type I sourdoughs frequently select for Lacto-bacillus sanfranciscensis, Lactobacillus pontis, Lactobacillus brevis,Lactobacillus fermentum, Lactobacillus paralimentarius and Lactoba-cillus plantarum (De Vuyst and Vancanneyt, 2007). In a recent study(Siragusa et al., 2009), the structure and stability of the dominantlactic acid bacteria populationwere investigated during propagationof sourdough type I. Nine selected strains of L. sanfranciscensis weresingly used as starters. Only three out of nine starter strains domi-nated throughout ten days of propagation, regardless of the type offlour. These results highlighted the need for selecting startersnot only based on functional features but also on the robustness ofstrains, and suggested that L. sanfranciscensis could not be the bestcandidate to be used as starter for sourdough type I. L. plantarum isan ubiquitous species, found in several food ecosystems (Corsettiand Gobbetti, 2002), including sourdoughs (Gobbetti et al., 1994a,b), due to its versatile metabolism, capacity of large adaptation todifferent environmental conditions and large spectrum antimicro-bial activity (Kleerebezem et al., 2003; Di Cagno et al., 2009a, 2010a).To the best of our knowledge, no studies considered the use, prop-agation andmonitoring of L. plantarum as starter for sourdough typeI. This species should harbor interesting metabolic traits to becompetitive towards other wheat flour autochthonous speciesand/or biotypes of the same species.

Following the recent report on L. sanfranciscensis (Siragusa et al.,2009), this study aimed at exploring the robustness of selectedL. plantarum during propagation of wheat flour sourdough typeI. Isolation, identification, monitoring and technology performanceof the dominant lactic acid bacteria population during fermentationof sourdough type I were described.

2. Material and methods

2.1. Microorganisms and growth conditions

L. plantarum DB200, 3DM, CF1, 2MF8, G10C3, 12H1 and LP20isolated from Italian sourdoughs and belonging to the Culture

Table 1Maximum acidification rate (mmax), synthesis of diacetyl and peptidase activities of Lacto

Strain mmax (DpH 10 min�1)a Diacetyl (mM)b Aminopeptidase ty

DB200 0.34 � 0.03 1.5 � 0.3 38.2 � 0.53DM 0.41 � 0.05 1.7 � 0.1 43.3 � 0.7CF1 0.25 � 0.02 2.2 � 0.1 22.0 � 0.42MF8 0.36 � 0.02 0.8 � 0.3 35.6 � 0.4G10C3 0.33 � 0.06 1.6 � 0.2 70.1 � 0.212H1 0.38 � 0.05 1.9 � 0.2 33.5 � 0.5LP20 0.28 � 0.04 1.3 � 0.1 47.0 � 0.2

a Maximum acidification rate was determined during incubation (at 30 �C, for 24 h) oAngelis et al., 2007).

b Concentration was determined by HPLC analysis in spent culture mMRS broth inoculafor 24 h (Zeppa et al., 2001).

c Peptidase activities were determined by using cell suspensions (cell density of 9.0 logwere carried on relatively specific p-nitroanilide substrates. One unit (U) of activity wasminute (Gobbetti et al., 1999).

Collection of the Department of Plant Protection and AppliedMicrobiology of Bari University, Italy, were used in this study. Strainswere previously selectedwithin a large number of isolates belongingto the same species. As shown in Table 1, the main selection traitsregarded acidification rate, synthesis of diacetyl and peptidaseactivities. Cell cultureswere routinely propagated at 30 �C for 24 h inmodified MRS (mMRS) broth (Oxoid, Basingstoke, Hampshire,England)with the addition of freshyeast extract (5%, v/v) and28mMmaltose at a final pH of 5.6. Growth was monitored directly byplating onmMRS agar during 24h of incubation at 30 �C or indirectlybymeasuring the OD620 nm.When used for sourdough fermentation,cells of lactobacilli were incubated until the exponential phase ofgrowthwas reached (ca.12 h) (cells diluted 1:10 gave anO.D.620 nm ofca. 0.25), harvested by centrifugation (9000�g for 10 min at 4 �C),washed once with 50 mM phosphate buffer, pH 7.0 and re-sus-pended in sterile distilled water at cell density of about9 log cfu mL�1 (cells diluted 1:10, gave an O.D.620 nm of ca. 0.25).

2.2. Laboratory sourdough production and propagation

Triticum aestivum type 0 flour F175was kindly supplied byMoliniTandoi Pellegrino (Corato, BT, Italy). Moisture was determinedaccording to the standard AACC method (AACC, 1983). Nitrogencontent was measured by the semimicro-Kieldahl method. Proteinwas calculated bymultiplying the nitrogen concentration (N)� 5.70(AACC, 2000). The total concentration of starch was determinedusing the Megazyme starch assay kit (Megazyme Int., Wicklow,Ireland) official method 76-13 (AACC, 2000). Carbohydrates weredetermined by HPLC (Wang et al., 2002). Damaged starch in flourwas determined according to the Megazyme AACC method (AACC,2000) using kit Megazyme Starch Damage (Megazyme Interna-tional Ireland Ltd., Bray, Ireland). The gross composition was asfollows:moisture,14.70%� 0.13%; protein (N� 5.70),13.70%� 0.03%of dry matter (d m); starch, 61.29% � 0.20% of d m; total solublecarbohydrates, 4.15% � 0.13% of d m; and damaged starch,5.76% � 0.26% of d m.

Sourdough production and propagation was based on a tradi-tional protocol widely used for bread making in the Centre andSouth of Italy. Wheat flour F175 (187.5 g), tap water (82.5 mL) andcell suspension (30 mL), containing about 9 log cfu mL�1 of theabove individual L. plantarum strains (final cell number in thedough of ca. 8 log cfu g�1 of dough), were used to prepare 300 g ofdough with a continuous high-speed mixer (60 � g, dough mixingtime 5 min) (Chopin & Co., Boulogne, Seine, France). A controlsourdough, without starter, was also produced under the sameconditions. The value of dough yield (dough weight � 100/flourweight) was 160 for all doughs. The eight sourdoughs (seven withindividual L. plantarum strains and the control) were incubated in

bacillus plantarum strains used as sourdough starters.

pe Nc Proline iminopeptidasec X-Prolyl dipeptidyl aminopeptidasec

27.5 � 0.2 41.0 � 0.322.1 � 0.1 58.6 � 0.519.7 � 0.5 38.6 � 0.525.4 � 0.4 44.9 � 0.218.8 � 0.3 52.4 � 0.326.0 � 0.4 39.8 � 0.321.4 � 0.2 53.5 � 0.2

f a dough inoculated with a single strain (initial cell density of 7.0 log cfu g�1) (De

ted with a single strain (initial cell density of 7.0 log cfu ml�1) and incubated at 30 �C

cfu ml�1) obtained from cells grown at 30 �C, 24 h on mMRS broth. Enzyme assaysdefined as the amount of enzyme required to liberate 1 mmol of p-nitroaniline per

F. Minervini et al. / Food Microbiology 27 (2010) 897e908 899

sterile plastic beakers at 30 �C for 8 h. After fermentation, sour-doughs were stored at 10 �C for about 16 h and further used forpropagation.

Sourdoughs were propagated according to the back-sloppingprotocol. Each of the above sourdoughs was individually used asthe starter (inoculum rate 10%, w/w) to ferment a mixture of flourF175 (168.75 g) and tap water (101.25 mL) having dough yield of160. Sourdoughs were fermented at 30 �C for 6 h and stored at 10 �Cfor about 16 h between back-slopping. Sourdoughs were propa-gated daily for ten days.

2.3. Lactic acid bacteria and yeasts enumeration and isolation

Ten grams of sourdough were homogenized with 90 mL ofsterile sodium chloride (0.9%, w/v) solution using a Classic Blender(PBI International, Milan, Italy). Serial dilutions were plated onmMRS agar for determination of presumptive lactic acid bacteria.After incubation at 30 �C for 48 h, at least ten colonies wererandomly selected from plates containing the two highest sampledilutions. Gram-positive, catalase negative, non motile rod andcocci isolates were cultivated in mMRS broth at 30 �C for 24 h andre-streaked into mMRS agar. All isolates considered for furtheranalyses could acidify the culture medium, as determined bymeasuring the pH of the MRS broth after incubation at 30 �C for24 h with single isolates. Stock cultures were stored at �20 �C in10% (v/v) glycerol.

The number of yeasts in sourdoughs was estimated by usingMalt Agar (Difco, Detroit, USA) at 30 �C for 72 h.

2.4. DNA extraction and molecular identificationby 16S rRNA and recA genes sequencing

Total DNA from presumptive lactic acid bacteria isolates wasextracted from 2mL samples of overnight cultures grown at 30 �C inmMRS broth. Total DNAswere obtained as reported by De Los Reyes-Gavilàn et al. (1992). The concentration and purity of DNA wasassessed by a biophotometer (Eppendorf, Hamburg, Germany). Aprimer pair (Invitrogen Life Technologies, Milan, Italy), LpigF/LpigR(50-TACGGGAGGCAGCAGTAG-30 and 50-CATGGTGTGACGGGCGGT-30) (De Angelis et al., 2006), (corresponding to the position 369e386,and 1424e1441, respectively, of the 16S rRNA gene sequence ofLactobacillus mucosae, accession number AF126738) was used toamplify the 16S rRNA gene fragment of presumptive lactic acidbacteria. Fifty microliters of each PCR mixture contained 200 mM ofeach 20-deoxynucleoside 50-triphosphate, 1 mM of both forward andreverse primer, 2mMMgCl2, 2 U of TaqDNApolymerase (Invitrogen)in the supplied buffer, and approximately 50 ng of DNA. PCR (Poly-merase Chain Reaction) amplification was performed using theGeneAmp PCR System 9700 thermal cycler (Applied Biosystems,USA). Amplification consisted of initial denaturation step at 94 �C for4min followedby30 cycles at 94 �C for 45 s, at 49 �C for 45 s andfinalextension at 72 �C for 1 min. Additional final extension at 72 �C for7 minwas used. PCR products were separated by electrophoresis on1.5% (w/v) agarose (GE Healthcare Bio-Sciences, Uppsala, Sweden)gel stained with ethidium bromide (0.5 mg mL�1). The ampliconswere eluted from gel and purified by the GFX� PCR DNA and GelBand Purification Kit (GE Healthcare Bio-Sciences). DNA sequencingreactions were performed by MWG Biotech AG (Ebersberg,Germany) using both, forward and reverse, primers. Taxonomicidentification of strains was performed by comparing the sequencesof each isolate with those reported in the Basic BLAST database(Altschul et al., 1997) (http://www.ncbi.nlm.nih.gov).

Primers designed on recA gene were also used to discriminatebetween L. plantarum, Lactobacillus pentosus, Lactobacillus para-plantarum and Lactobacillus delbrueckii subsp. bulgaricus species

(Torriani et al., 2001). Part of the recA gene was amplified using thedegenerate primer pair (MWG Biotech AG) recALb1F/recALb1R (50-CRRTBATGCGBATGGGYG-30 and 50-CGRCCYTGWCCAATSCGRTC-30)derived from the homologous regions of the recA gene sequencesfrom L. plantarum subsp. plantarum (accession no. AJ621668).PCR reactions were performed as described for 16S rRNA gene andrelated amplicons were separated, purified and sequenced asdescribed above.

2.5. DNA extraction from sourdoughs

Total DNA was extracted after sourdough production (8 h offermentation) and after daily sourdough propagation (6 h offermentation). Ninety millilitres of peptone-physiological solution(0.1%,w/v bacteriological peptone [Oxoid] and0.85%,w/vNaCl)wereadded to 10 g of sourdough and homogenized for 5 min. Fifty mil-lilitres of this suspension were centrifuged at 1000�g for 5 min at4 �C. To harvest the cells, the supernatant was centrifuged fora second time (5000�g for 15 min at 4 �C) and the cell pellet wasstored at �20 �C. An aliquot of the pellet of ca. 500 mg was used forDNA extraction (Bornay-Llinares et al., 1999) using the FastDNA� ProSoil-Direct Kit (MP Biomedicals, CA., United States) and FastPrep�

instrument (BIO 101), according to the manufacturer’s instructions.Fifty microliters of application-ready DNA were recovered. Qualityand concentration of DNA extracts were assayed both on 0.7%agaroseeTBE 0.5� gels, stained with ethidium bromide, and byspectrophotometric measurements using a biophotometer (Eppen-dorf). In details, Bionumerics software version 4.00 (Applied Maths,Kortrijk, Belgium) was used to estimate the relative amount of DNAloaded into the agarose gel. The band intensity was normalized tothe proportion to the average value if necessary (relative bandintensity). When the relative band intensities were within 0.9e1.1,the bandswere considered to have the same amount of DNA in them.This was based on the analysis of several identical samples electro-phoresed in an agarose gel.

2.6. Polymerase chain reaction (PCR) amplification and denaturinggradient gel electrophoresis (DGGE) analysis

Primers Lac1 (50-AGCAGTAGGGAATCTTCCA-30) and Lac2 (50-ATTYCACCGCTACACATG-30) were used for DGGE analysis (Pitcheret al., 1989), primer Lac2 being extended with the 40 mer GC-clamp at the50 end to separate the corresponding PCRproducts in thegradient gel (Sheffield et al., 1989). Lac1 and Lac2 targeted a 340 bpregion of the 16S rDNA of Lactobacillus group, including Lactobacillus,Leuconostoc, Pediococcus and Weissella genera. The specificity of theprimer pair was experimentally tested by using DNA extracted fromthe following bacterial species: L. plantarum ATCC 14917, Weissellaconfusa DSM20196, Pediococcus pentosaceus DSM 20336, and Leuco-nostoc lactis DSM 20202. The primer set gave positive PCR results forthe corresponding microbial target and did not cross-react with anyother of the non target microorganisms. DGGE protocols and condi-tions were previously described by Di Cagno et al. (2009b). Dena-turing gradient was from 45 to 55%. Gradient gels were preparedusing a gradient mixer (Gradiente forming devices, Sigma ChemicalCo., Milan, Italy) and a peristaltic pump (Pump P-1, GE HealthcareBio-Sciences).

2.7. Genotypic characterization by Randomly AmplifiedPolymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis

Genomic DNA from each isolates was extracted as describedabove. Three oligonucleotides, P1 50-GCGGCGTCGCTAATACATGC-30

(Klijn et al., 1991), P4 50-CCGCAGCGTT-30, (Corsetti et al., 2003)and M13 50-GAGGGTGGCGGTTCT-30 (Stendid et al., 1994), with

F. Minervini et al. / Food Microbiology 27 (2010) 897e908900

arbitrarily chosen sequences, were used for isolates biotyping.The reaction mixture and PCR conditions reported by Corsetti et al.(2003) were used for primers P1 and P4, whereas those reported byZapparoli et al. (1998) were used for primer M13. The PCR products(15 mL) were separated by electrophoresis at 100 V for 200 min on1.5% (w/v) agarose gel and the DNA was detected by UV trans-illumination after staining with ethidium bromide (0.5 mg mL�1).The molecular sizes of the amplified DNA fragments were esti-mated by comparison with the 1-kb DNA molecular size markers(Invitrogen Life Technologies). RAPD-PCR profiles were acquired byGel Doc EQ System (Bio-Rad, Hercules, CA) and compared usingFingerprinting II Informatix� Software (Bio-Rad). The similarity ofthe electrophoretics profiles was evaluated by determining the Dicecoefficients of similarity and by using the unweighted pair groupmethod with average linkage (UPGMA). The presence or absence offragments was recorded as 1 or 0, respectively. Only reproduciblewell-marked amplified fragments were scored, faint bands beingignored. The reproducibility of the RAPD fingerprints was assessedby comparing the PCR products obtained with primers P1, P4, andM13 and DNA prepared from three separate cultures of the samestrain. Seven strains were studied, and the patterns for the samestrain were 95% similar, indicating that the reproducibility of thetechnique under the conditions used was high.

2.8. Acidification during sourdough propagation

The acidification during sourdough propagation was monitoredon-line by a pH-meter (Model 507, Crison, Milan, Italy) with a foodpenetration probe. Concentrations of organic acids were deter-mined by High Performance Liquid Chromatography (HPLC) usingan ÄKTApurifier� system (GE Healthcare Bio-Sciences). The anal-yses were carried out isocratically at 0.6 mL min�1 and 60 �C witha 300 � 7.8 mm i.d. cation exange column (Aminex HPX-87H)(Bio-Rad Laboratories). Mobile phase was 10 mM H2SO4 preparedby diluting reagent grade sulfuric acid with distilled water, filteringthrough a 0.22 mm Durapore� membrane filter (Millipore Co.,Bedford, MA, USA), and degassing under vacuum. A mixture oforganic acids at known concentration (Sigma Chemical Co.) wasadded and used as standard (Zeppa et al., 2001).

2.9. Determination of free amino acids

The concentration of free amino acids (FAA) in the waterextracts of sourdoughs was determined by using the Amino AcidAnalyser (Biochrom LTD, Cambridge Science Park, England) usinga Na-cation exchange column (20 � 0.46 cm i.d.). A mixture ofamino acids at known concentration (Sigma Chemical Co.) wasadded with cysteic acid, methionine sulphoxide, methionine sul-phone, tryptophan and ornithine, and used as standard. Proteinsand peptides in the samples were precipitated by addition of 5%(v/v) cold solid sulfosalicylic acid, holding at 4 �C for 1 h andcentrifuging at 15,000�g for 15 min. The supernatant was filteredthrough a 0.22 mmpore size filter and diluted, when necessary, withsodium citrate (0.2 M, pH 2.2) loading buffer. Amino acids werepost-column derivatised with ninhydrin reagent and detected byabsorbance at 440 (proline and hydroxyproline) or 570 nm (all theother amino acids) (De Angelis et al., 2007).

2.10. Community Level Catabolic Profiles (CLCPs)

Carbon source utilization patterns of sourdough microbialcommunities during propagationwere assessed by using BIOLOG96-well Eco-Microplates (Biolog, Inc., Hayward, CA, USA) (Crecchio et al.,2004). It contained 31 different carbon sources (carbohydrates,carboxylic acids, polymers, amino acids, amines and miscellaneous

substrates) in triplicate. Ten grams of sourdough were homogenizedwith 90 mL of sterile sodium chloride (0.9%, w/v) solution (ClassicBlender) and centrifuged at 10,000�g for 15 min at 4 �C. The pelletwaswashedwith 50mM TriseHCl (pH 8.8), thenwith sterile sodiumchloride solution and centrifuged as described above. The cellularsuspension was diluted (1:100) in sterile sodium chloride solutionand subsequently dispensed (150 mL) into each of 96 wells of theBIOLOG Eco-Microplates. The microplates were incubated at 30 �C inthe dark and the color development was measured at 590 nm every24 h thereafter up to 120 h with a microplate reader (BIOLOGMicrostation). Three indices were determined (Shannon, 1948a, b;Zak et al., 1994). Shannon’s diversity (H0), indicating the substrateutilization pattern: H0 ¼ �P

pi ln (pi), where pi is the ratio of theactivity of a particular substrate to the sums of activities of allsubstrate activity at 120 h. Substrate richness (S), measuring thenumber of different substrates used, was calculated as the number ofwells with a corrected absorbance greater than 0.25. SubstrateEvenness (E) was defined as the equitability of activities across allutilized substrates: E ¼ H0/log S.

2.11. Production and propagation of a mixed-started sourdough

L. plantarum DB200 and also L. sanfranciscensis LS44, previouslyreported to dominate during ten days of sourdough propagation(Siragusa et al., 2009), were used as the mixed starter to producea sourdough, made with the same flour type 0 F175 and underthe process parameters (dough yield, initial cell density) describedabove. The sourdough fermented by the mixed starter wasconsecutively propagated for ten days. After the tenth propagation,sourdough was subjected to plate count on mMRS agar. Isolates oflactobacilli were identified through partial sequencing of 16S rRNAgene and, if necessary, of recA gene, and characterized throughRAPD-PCR analyses.

2.12. Statistical analysis

Data from microbial growth, acidification, organic acids andFAA of three independent fermentation trials, twice analyzed,were subjected to one-way ANOVA (SAS Institute, 1985), and pair-comparison of treatment means was achieved by Tukey’s proce-dure at P < 0.05 (Tukey, 1994), using the statistical softwareStatistica for Windows (Statistica 7.0 per Windows). In addition,some data were further subjected to non-parametric statisticalanalysis by determining the median value of aggregate strain data.

3. Results

3.1. Lactic acid bacteria enumeration and isolation

Preliminarily, seven selected strains of L. plantarum (see Table 1 forthe main features) were individually used to produce as many sour-doughs made of wheat flour type 0 F175. After 8 h of fermentation at30 �C, all sourdoughs had values of pH in the range of 4.00� 4.17 andcontained9.1�0.02 to 9.5� 0.13 log cfu g�1 of presumptive lactic acidbacteria. After 8 h of fermentation, the control (unstarted) sourdoughcontained a number of presumptive lactic acid bacteria about 10,000times lower than the started sourdoughs (data not shown). Eachsourdough was then propagated for ten days by daily back-slopping.Cell densities after each sourdough fermentation are shown in Fig. 1.The median value of presumptive lactic acid bacteria in the controlwas 9.32 log cfu g�1, and the values corresponding to 5th and 95thpercentiles of the data were 8.52 and 9.40 log cfu g�1, respectively.The lowest value (ca. 4.85� 0.08 log cfu g�1) was found after the firstday of propagation. Cell numbers rapidly increased (P < 0.05) to9.35 � 0.11 log cfu g�1 during further 3 days, and remained almost

4

5

6

7

8

9

10

11

Control S-DB200 S-3DM S-CF1 S-2MF8 S-G10C3 S-12H1 S-LP20

gUF

CgoL

-1

Fig. 1. Cell densities (cfu g�1) after sourdough propagation at 30 �C for 6 h during ten subsequent days by using wheat flour type 0 F175. Data are the means of three independentexperiments (n ¼ 3). The center line of each box represents the median (,), the top and bottom of the box represent the 75th and 25th percentile of the data, respectively. The topand bottom of the error bars represent the 5th and 95th percentile of the data, respectively. The circles in each box plot extend to the outliers of the data (B), and very extremepoints are represented as individual data points (*). Unstarted sourdough (Control); sourdoughs (S) started with strains Lactobacillus plantarum DB200; 3DM; CF1; 2MF8; G10C3;12H1; or LP20.

F. Minervini et al. / Food Microbiology 27 (2010) 897e908 901

constant. Except for the sourdough started with L. plantarum 12H1(S-12H1) after eight days of propagation (10.08 � 0.08 log cfu g�1),cell densities of presumptive lactic acid bacteria ranged from9.00� 0.04 to 9.82� 0.02 log cfu g�1 in all other started sourdoughs.Median values were almost similar (9.32e9.46) for all sourdoughs,with the exception of S-CF1 and S-LP20 which showed slightly lowermedian values (9.19 and 9.13, respectively). Apart from control sour-dough, thewidest range of the 5th and 95th percentileswas found forS-12H1 (9.12e9.82 log cfu g�1).

Independently from the type of sourdough, yeasts reached celldensities of about 7 log cfu g�1 since the fourth day of propagationand remained almost constant.

4

5

6

7

8

9

10

0 1 2 3 4

gU

FC

goL

1-

Ste

Fig. 2. Kinetics of growth of Lactobacillus plantarum DB200 (A), 3DM (:), CF1 (-), 2MF8 (–6 h during ten subsequent days by using wheat flour type 0 F175. Data are the means of th

3.2. Dynamics of the lactic acid bacteria population duringsourdough propagation

Isolates of presumptive lactic acid bacteria from each dailysourdough propagation were subjected to RAPD-PCR analysis byusing single primers P1, P4 or M13. The reproducibility of RAPDfingerprints was assessed by comparing the PCR products obtainedfrom three separate cultures of the same strain. The patterns showedabout 92e95% similarity, indicating the reproducibility of the tech-nique under the conditions used (data not shown). The use of primerM13 clearly differentiated the RAPD patterns of strains used asstarters from those of all the other isolates either fromsourdoughs or

5 6 7 8 9 10p of propagation

A–), G10C3 (–:–), 12H1 (C) and LP20 (–-–) after sourdough propagation at 30 �C forree independent experiments � standard deviations (n ¼ 3).

Table 2Identification by 16S rRNA gene sequencing of sourdough lactic acid bacteria iso-lated after ten days of daily sourdough propagation at 30 �C for 6 h by using wheatflour type 0 F175.

Sourdough (S)a Isolates (I)b Closestrelative

Closest relative Accessionnumber (Identity � 98%)

Control I (1e6, 8) L. plantarum FJ785723.1I (7, 9e10) L. rossiae FJ429980.1

S-DB200 I (11e15) L. plantarum EF439680.1I (16e20) L. plantarum EF439683.1

S-3DM I (21e25, 30) L. plantarum FJ785723.1I (26e29) L. plantarum AJ621668.1

S-CF1 I (31e40) L. plantarum FJ785723.1S- I (41, 44) L. plantarum FJ785723.12MF8 I (45e50) L. plantarum FJ785723.1S-G10C3 I (51e53, 55�60) L. plantarum EF439682.1

I (54) L. plantarum EF439683.1S-12H1 I (61e62, 64e65) P. pentosaceus FM179610.1

I (63, 66e70) L. plantarum FJ785723.1S-LP20 I (71e80) L. plantarum FJ785723.1

L., Lactobacillus; P., Pediococcus.a S, sourdoughs started with Lactobacillus plantarum DB200, (S-DB200); 3DM, (S-

3DM); CF1 (S-CF1); 2MF8, (S-2MF8); G10C3, (S-G10C3); 12H1, (S-12H1); or LP20,(S-LP20).

b Isolates with different RAPD-PCR profiles were grouped in separate rows.

Fig. 3. Denaturing gradient gel electrophoresis (DGGE) by primers Lac1 and Lac2 targeting tday of production; lane 2, control sourdough after ten days of propagation; lane 3, sourdouproduction); lane 4, Lactobacillus plantarum DB200; lane 5, sourdough S-DB200 after ten dapropagation; lane 8, L. plantarum 2MF8; lane 9, sourdough S-2MF8 after ten days of propa

F. Minervini et al. / Food Microbiology 27 (2010) 897e908902

the control (data not shown). The cell number of each starter wascalculated as the number of colonies with the specific RAPD pattern.As expected, strains of L. plantarum dominated at the beginning ofpropagation (Fig. 2). Strains DB200, 3DM, G10C3, 12H1 and LP20maintained elevated cell numbers (ca. 9 log cfu g�1) throughoutten days of propagation. On the contrary, strains CF1 and 2MF8progressively decreased (especially after the fifth day) and reacheda cell density lower than 5 log cfu g�1 at ten days of propagation.

Lactic acid bacteria dominating the microbiota of raw wheatflour type 0 F175 were identified by sequence analysis of at least700 bp of the 50 region of the 16S rRNA gene. The partial sequenceof the recA gene was also used to discriminate between L. planta-rum, L. pentosus, L. paraplantarum and L. delbrueckii subsp. bulgar-icus. The following species were found: L. plantarum (11 isolates);P. pentosaceus (4), Pediococcus spp. (3), Lactobacillus casei (3) andLactobacillus rossiae (2). The same analyses were carried out toidentify isolates dominating the microbiota of the control andstarted sourdoughs during propagation. The species identified afterten days are shown in Table 2. L. plantarum (one or two isolates)was found in all sourdoughs and it was the lactic acid bacteriumfound alone (other lactic acid bacteria species were at cell densitylower than 7.0 log cfu g�1) in six out of eight sourdoughs. Apartfrom L. plantarum, the control contained also L. rossiae, as well asthe sourdough started with L. plantarum 12H1 contained also P.pentosaceus. Both the species L. rossiae and P. pentosaceus had been

he region of 16S rRNA gene of the Lactobacillus group. Lane 1, control sourdough on thegh S-DB200 on the day of production (representative of all sourdoughs on the day ofys of propagation; lane 6, L. plantarum 3DM; lane 7, sourdough S-3DM after ten days ofgation; M, Low Range DNA Ladder.

F. Minervini et al. / Food Microbiology 27 (2010) 897e908 903

isolated from the flour type 0 F175. L. casei was the only speciesfrom the microbiota of the wheat flour that did not becomedominant during propagation.

The DNAs directly extracted from sourdoughs on the day ofproduction and after ten days of propagationwere subjected to PCRusing primers Lac1 and Lac2 targeting for Lactobacillus group(Pitcher et al., 1989). All sourdoughs on the day of production hadan identical DGGE profile. In particular, the control showed at leastfour bands, two of them (a and b, Fig. 3) corresponding to L. plan-tarum ATCC 14917 and L. rossiae DSM 15814, respectively (data notshown). This was in partial agreement with the identificationof isolates from wheat flour. Indeed, no bands corresponding toPediococcus spp. and L. casei were found. One band was present insourdoughs S-DB200, S-3DM, S-2MF8 after ten days of propagation,having the same mobility as the DNA from the correspondingstarter strains of L. plantarum (Fig. 3). The same was found for theother started sourdoughs (S-CF1, S-G10C3, S-12H1 and S-LP20, datanot shown). This finding seemed to be in agreement with the

Fig. 4. Representative Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (Rpropagation at 30 �C for 6 h by using wheat flour type 0 F175. (A) DNA molecular size standsourdough (S) started with L. plantarum DB200 (S-DB200); L. plantarum 3DM; sourdough stL. plantarum LP20 (S-LP20). (B) L. plantarum 12H1; sourdough started with L. plantarum 12HL. plantarum CF1; sourdough started with L. plantarum CF1 (S-CF1); L. plantarum G10C3; andPCR analysis. Lactic acid bacteria isolates (I) are coded based on partial 16S rRNA and recA

identification of isolates through 16S rRNA and recA gene partialsequencing (Table 2). After ten days of propagation, at least oneisolate of L. plantarum was always present in all sourdoughs.Although sourdough S-12H1 harbored P. pentosaceus together withL. plantarum, no band corresponding to Pediococcus spp. was found.

Fig. 4A and B show the RAPD-PCR analysis (primer M13) of thedifferent isolates from sourdoughs after ten days of propagation. Theisolates of sourdoughs S-CF1 and S-LP20 had a unique and differentRAPD profile. While the profile of the ten isolates from S-LP20 cor-responded to the profile of strain L. plantarum LP20, that of isolatesfrom S-CF1 did not correspond to the respective starter strain. Theisolates harbored by the other five sourdoughs (S-DB200, S-3DM,S-2MF8, S-G10C3 and S-12H1) gave two distinct RAPD profiles. Oneof the RAPD profiles of the sourdoughs S-DB200 (5 isolates), S-3DM(4 isolates), S-G10C3 (1 isolate) and S-12H1 (6 isolates) correspondedto profiles of strains L. plantarum DB200, 3DM, G10C3 and 12H1,respectively. Isolates having the RAPD profile different from that ofstarter strains corresponded to autochthonous strains of L. plantarum

APD-PCR) patterns of starters and isolates of lactic acid bacteria after ten days of dailyards (12000e100 bp) (st); control sourdough (Control); Lactobacillus plantarum DB200;arted with L. plantarum 3DM (S-3DM); L. plantarum LP20; and sourdough started with1 (S-12H1); L. plantarum 2MF8; sourdough started with L. plantarum 2MF8 (S-2MF8);sourdough started with L. plantarum G10C3 (S- G10C3). Primer M13 was used for RAPD-gene sequence comparisons and correspond to those of Table 1.

Fig. 4. (continued).

F. Minervini et al. / Food Microbiology 27 (2010) 897e908904

or of P. pentosaceus (sourdough S-12H1). Isolates from control sour-dough had two RAPD profiles, one corresponding to autochthonousL. plantarum, and the other one corresponding to autochthonousL. rossiae. Each sourdough was populated by strains (most of thembelonging to L. plantarum) that exclusively characterized the differentsourdoughs. Cluster analysis by using RAPD-PCR profiles fromprimers P1, P4 and M13 of different isolates after ten days of propa-gation confirmed the above results (Fig. 5).

3.3. Acidification during sourdough propagation

The acidifying activity was rather homogeneous between theseven started sourdoughs. Indeed, during sourdough propagation,the median values of ΔpH showed quite a narrow range (Fig. 6).Sourdoughs S-2MF8, S-G10C3, S-12H1, S-LP20 and control showedsimilar median values (1.29e1.33). A slightly lower median value ofΔpH (1.24) was found for the sourdough started with L. plantarumCF1, whereas sourdoughs started with strains DB200 and 3DMshowed median values of 1.47 and 1.46, respectively. The acidifyingactivity of the control increased during propagation; it reached thevalue of ΔpH of 1.26 � 0.02 after the fourth day of propagation.Since then until the tenth day, the value of ΔpH of the control wasalmost constant. On the day of production, the concentration of D,L-lactic acid ranged from ca. 3 � 0.71 (control) to 72 � 1.02 mM(sourdough S-3DM). During propagation of sourdoughs S-DB200,S-CF1 and S-LP20, D,L-lactic acid was constant and ranged from ca.45 � 0.45 to 59 � 0.78 mM. This agreed with the low variation of

the ΔpH which characterized those sourdoughs. The concentrationof D,L-lactic acid significantly (P < 0.05) increased during propa-gation of control and sourdough S-12H1 (ca. 45 � 0.45 and58 � 0.92 mM at ten days). Compared to values at five days, a slightand progressive decrease (ca. 10 mM) of the concentration of D,L-lactic acid was found in sourdoughs S-3DM, S-2MF8 and S-G10C3during further propagation. Traces of acetic acid were found just inthe control.

3.4. Proteolysis during sourdough propagation

The total concentration of free amino acids (FAA) was deter-mined throughout propagation. Control showed the largest varia-tion by comparing the day of production (0.55 � 0.01 g kg�1) to thetenth day of propagation (1.08 � 0.01 g kg�1). Except for sour-doughs S-2MF8 and S-LP20, which were characterized by an almostconstant concentration of FAA (0.78 � 0.01e0.89 � 0.01 g kg�1),the other sourdoughs showed an increased (P < 0.05) level of FAAduring propagation (range of values from 0.80 � 0.01 to1.11 � 0.01 g kg�1). Apart from the day of propagation, Asp and Leuwere the FAA found at the highest concentrations (average values:ca. 0.12 g kg�1 and 0.11 g kg�1, respectively).

3.5. Community Level Catabolic Profiles (CLCPs)

The catabolic profiles of the microbiota of sourdoughs duringpropagation were determined by calculating the indices H0, S and E

Fig. 5. Dendrogram obtained by combined Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) patterns of the isolates from ten days of daily sourdoughpropagation at 30 �C for 6 h by using wheat flour type 0 F175. Primers P1, P4 and M13 were used for RAPD-PCR analysis. Lactic acid bacteria isolates I (1e100) correspond to those ofTable 1. Cluster analysis was based on the simple matching coefficient and unweighted pair grouped method with arithmetic average.

F. Minervini et al. / Food Microbiology 27 (2010) 897e908 905

(Table 3). According to the utilization pattern substrate (H0 index),sourdoughs were significantly (P < 0.05) distinguished in twogroups: (i) control, S-CF1 and S-2MF8 showing a constant valuethroughout propagation; and (ii) S-G10C3, S-DB200, S-12H1 andS-LP20, showing an increase until the fifth day of propagation anda constant value thereafter. S-3DM was not included in any of theabove groups, as it showed a decrease until the fifth day of prop-agation was reached. S-3DM was also the only sourdough showingthe lowest value of H0 after ten days of propagation. The S index hadthe same trend of H0 and grouped sourdoughs similarly. The E indexgave a measure of the statistical significance (equitability) of thevalues expressed by H0 and S indices.

3.6. Persistence of L. plantarum and L. sanfranciscensisstrains during sourdough propagation

L. plantarum DB200, one of the persistent strain selected in thisstudy, and L. sanfranciscensis LS44, previously reported to dominateduring ten days of sourdough propagation (Siragusa et al., 2009),were used as the mixed starter to produce a sourdough made withflour type 0 F175. After production, sourdough was daily propa-gated for ten days. Gram-positive, catalase negative, non motile rodisolates from the highest dilution mMRS plates were first charac-terized through RAPD-PCR. Sixteen and fourteen out of thirtyisolates had the same RAPD pattern as the starters L. plantarum

Fig. 6. Values of DpH (difference in pH units between the initial pH0 and final pHt) after sourdough propagation at 30 �C for 6 h during ten subsequent days by using wheat flourtype 0 F175. Data are the means of three independent experiments (n ¼ 3). The center line of each box represents the median (,), the top and bottom of the box represent the 75thand 25th percentile of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentile of the data, respectively. The circles in each box plot extendto the outliers of the data (B). Unstarted sourdough (Control); sourdoughs (S) started with Lactobacillus plantarum DB200; 3DM; CF1; 2MF8; G10C3; 12H1; or LP20.

Table 3Community Level Catabolic Profiles (CLCPs) of the sourdough microbiota after one(T1), five (T5) and ten (T10) days of daily sourdough propagation at 30 �C for 6 h byusing wheat flour type 0 F175.

Sourdougha H0 S E

Control (T1) 3.11 � 0.10 14.00 � 2.64 2.73 � 0.10T5 3.15 � 0.09 14.33 � 2.08 2.55 � 0.04T10 3.09 � 0.06 13.93 � 0.58 2.40 � 0.02

S-G10C3 (T1) 2.05 � 0.47 5.33 � 2.08 2.43 � 0.02T5 3.19 � 0.14 16.0 � 1.00 2.65 � 0.13T10 3.05 � 0.15 14.66 � 2.31 2.45 � 0.07

S-2MF8 (T1) 2.17 � 0.21 7.66 � 2.08 2.49 � 0.13T5 2.00 � 0.17 7.00 � 1.00 2.38 � 0.03T10 2.0.06 � 0.20 7.66 � 1.53 2.35 � 0.05

S-CF1 (T1) 3.04 � 0.09 13.00 � 2.00 2.75 � 0.20T5 3.24 � 0.04 15.66 � 0.58 2.71 � 0.04T10 3.18 � 0.07 14.00 � 1.00 2.32 � 0.02

S-3DM (T1) 2.15 � 0.78 9.33 � 1.15 2.56 � 1.04T5 1.08 � 0.11 2.56 � 1.05 1.52 � 0.77T10 1.10 � 0.29 2.33 � 0.57 2.43 � 0.24

S-DB200 (T1) 2.58 � 0.29 10.66 � 2.88 2.53 � 0.19T5 3.05 � 0.18 15.25 � 1.91 2.69 � 0.10T10 3.03 � 0.03 15.00 � 1.00 2.58 � 0.06

S-12H1 (T1) 2.05 � 0.54 8.66 � 3.05 2.71 � 0.38T5 2.90 � 0.06 15.66 � 1.15 2.45 � 0.11T10 2.80 � 0.34 15.33 � 3.05 2.45 � 0.15

S-LP20 (T1) 2.03 � 0.22 6.66 � 2.08 2.52 � 0.16T5 3.23 � 0.10 18.00 � 2.64 2.58 � 0.09T10 3.22 � 0.04 18.33 � 2.51 2.55 � 0.09

Values are means � standard deviation (n ¼ 3).H0 , Shannon’s diversity index; S, substrate richness; and E, substrate evenness.

a Unstarted sourdough, Control; sourdoughs (S) started with Lactobacillus plan-tarum DB200, S-DB200; 3DM, S-3DM; CF1, S-CF1; 2MF8, S-2MF8; G10C3, S-G10C3;12H1, S-12H1; and LP20 S-LP20.

F. Minervini et al. / Food Microbiology 27 (2010) 897e908906

DB200 and L. sanfranciscensis LS44, respectively. According tothe RAPD pattern, isolates were identified as L. plantarum andL. sanfranciscensis.

4. Discussion

To enlarge the use of sourdough type I under industrial condi-tions, starter cultures have to be employed and criteria for selectionshould combine functional features and robustness. While most ofthe literature (for reviews see De Vuyst, 2000; Gobbetti et al., 2005)was previously focused on functional selection of starters, onlya few studies characterized the sourdough microbiota when startercultures had been used, with the aim to show their real persistence.A recent study (Siragusa et al., 2009) reported that only three out ofnine functional starter strains of L. sanfranciscensis were persistentduring ten days of daily propagation of sourdough type I. This studyaimed at exploring the suitability of functional L. plantarum strainsto be competitive during propagation of sourdough type I.

Since the first day of propagation, all started sourdoughs reachedandmaintained cell numbers of presumptive lactic acid bacteria thatare usually found in mature sourdoughs (Gobbetti, 1998). Acidifi-cation was also constant during propagation. After three days ofpropagation, the same was found in the sourdough without starteradded (control). Apparently, all sourdoughs were standardized andbehaved similarly but the microbiota changed in most of the cases.

As determined by RAPD-PCR analyses, five out of the sevenstarters dominated throughout ten days of propagation. The othertwo starters were outcompeted by the flour autochthonouspopulation. Bakery environment (Scheirlink et al., 2009) and,especially, the flour are the main sources for autochthonous lacticacid bacteria. Partial sequencing of the 16S rRNA gene, in somecases coupledwith partial sequencing of the recA gene, showed thatthe wheat flour used in this study harbored strains of L. plantarum,pediococci, L. rossiae and L. casei. During sourdough propagation,the culture dependent approach and PCR-DGGE analysis werecombined. PCR-DGGE analysis did not reveal the presence of

F. Minervini et al. / Food Microbiology 27 (2010) 897e908 907

P. pentosaceus, probably due to differences in the efficiency of DNAextraction or by preferred amplification of certain templates in PCR(Meroth et al., 2003; Settanni et al., 2006). Except for the sourdoughstarted with L. plantarum LP20, at least one autochthonous strain ofthis species emerged in all the other sourdoughs. None of theautochthonous strains of L. rossiae and L. caseiwere found in startedsourdoughs. A strain of P. pentosaceus was only found in the sour-dough started with L. plantarum 12H1. L. plantarum was found inmany mature sourdoughs (Van der Meulen et al., 2007; Scheirlincket al., 2008, 2009; Vogelmann et al., 2009). None of these sour-doughs was made using selected starters and, therefore, dominantstrains of L. plantarum mainly originated from flour. This could bea further problem when functional strains of this species are usedas starters, since they have to compete not only with other speciesbut especially with other strains of L. plantarum. As previouslyshown for other ecosystems (Zak et al., 1994; Di Cagno et al., 2010b),the Community Level Catabolic Profiles (CLCPs) were successfulused in this study to estimate the global metabolic activities of thesourdough communities. CLCPs of carbohydrates, carboxylic acids,polymers, amino acids, amines and miscellaneous substrates werelinked to the microbial diversity of sourdoughs. Except for S-3DM,the CLCPs of sourdoughs containing persistent starters of L. plan-tarum behaved similarly and differed from the other sourdoughs.

Robustness of L. plantarum strains is attributed to severalfactors: competition for nutrients, fermentation of hexose andpentose carbohydrates, adaptation to environmental stresses, rapidacidification, synthesis of diacetyl and hydrogen peroxide, andespecially synthesis of bacteriocins (Gobbetti et al., 2005). Plan-taricins were shown to be active also during sourdough fermen-tation (Settanni et al., 2005) and their activity was effective towardsother L. plantarum strains (Todorov et al., 1999). Recently (Di Cagnoet al., 2010a), it was shown that plantaricin A (plnA) synthesized byL. plantarum DC400 acted as a pheromone in the mechanism ofinter-species cellecell communication. Under conditions whichmimicked the sourdough fermentation, the level of plnA dependedon the microbial partner and it acted as inhibitory compound,especially towards some strains of L. sanfranciscensis.

Overall, associations between homo- and hetero-fermentativespecies are preferred for sourdough starters because of bettersensory properties (Corsetti et al., 2001). Combining the results ofthis and the previous study (Siragusa et al., 2009), the persistentstrains L. plantarum DB200 and L. sanfranciscensis LS44 were shownto cohabit and dominate the sourdough ecosystem during ten daysof propagation.

Compared to other processes (e.g., cheese or wine making), theuse of starter cultures is relatively new in sourdough fermentationand selection was mainly or only based on functional features.The results of this study showed that functional selection is only thefirst step to get efficient starter cultures. Indeed, two out of sevenselected L. plantarum strains markedly decreased from sourdoughsduring propagation. Robustness of sourdough starters is indis-pensible, especially because autochthonous strains may frequentlyoutcompete starters.

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