Biodiversity of Lactobacillus sanfranciscensis strains isolated from five sourdoughs

AIMS: Five different sourdoughs were investigated for the composition of lactic acid bacteria (LAB) and the biodiversity of Lactobacillus sanfranciscensis strains. METHODS AND RESULTS: A total of 57 strains were isolated from five sourdoughs. Isolated strains were all identified by the 16S rDNA sequence and species-specific primers for L. sanfranciscensis. Results of identification showed that LAB strains were L. sanfranciscensis, Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus fermentum, Lactobacillus pontis, Lactobacillus casei, Weisella confusa and Pediococcus pentosaceus. A total of 21 strains were identified as L. sanfranciscensis and these isolates were detected in all five sourdoughs. Ribotyping was applied to investigate the relationship between intraspecies diversity of L. sanfranciscensis and sourdough. A total of 22 strains of L. sanfranciscensis including L. sanfranciscensis JCM 5668T were compared by ribotyping. The dendrogram of 21 ribotyping patterns showed four clusters, and L. sanfranciscensis JCM 5668T was independent of the others. The different biotypes of L. sanfranciscensis were present in two sourdoughs compared with other three sourdoughs. CONCLUSIONS: The LAB compositions of five sourdoughs were different and the relationship between intraspecies diversity of L. sanfranciscensis strains and five sourdoughs was shown by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that ribotyping was useful for distinguishing L. sanfranciscensis strains. A further important result is that the intra-species diversity of L. sanfranciscensis strains seems to be related to the sourdough preparation.     

Functional characterization of the proteolytic system of Lactobacillus sanfranciscensis DSM 20451T during growth in sourdough

Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.     

Metabolism by bifidobacteria and lactic acid bacteria of polysaccharides from wheat and rye, and exopolysaccharides produced by Lactobacillus sanfranciscensis

AIMS: The metabolism by bifidobacteria of exopolysaccharide (EPS) produced by Lactobacillus sanfranciscensis was investigated. To evaluate the significance of the EPS produced by Lact. sanfranciscensis during dough fermentation on the overall prebiotic properties of bread, metabolism by bifidobacteria of water-soluble polysaccharides (WSP) from wheat and rye was investigated. METHODS AND RESULTS: Polyglucose and polyfructan contained in WSP from wheat and rye were metabolized by bifidobacteria. In contrast, WSP isolated from fermented doughs were not metabolized by bifidobacteria. The arabioxylan fraction of WSP was metabolized neither by bifidobacteria nor by lactobacilli. All the bifidobacteria tested were able to metabolize fructan from Lact. sanfranciscensis. The kinetics of EPS metabolism by various bifidobacteria were characterized by diauxic utilization of fructose and EPS. CONCLUSIONS: Bifidobacteria metabolize fructan from Lact. sanfranciscensis. Polyfructan and the starch fractions from wheat and rye, which possess a bifidogenic effect, were degraded by cereal enzymes during dough fermentation, while the EPS were retained. SIGNIFICANCE AND IMPACT OF THE STUDY: EPS produced by sourdough lactic acid bacteria will improve the nutritional properties of sourdough fermented products.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.     

Cell-cell communication in sourdough lactic acid bacteria: a proteomic study in Lactobacillus sanfranciscensis CB1

The mechanisms of cell-cell communication in Lactobacillus sanfranciscensis CB1 were studied. The highest number of dead/damaged cells of L. sanfranciscensis CB1 was found in cocultures with Lactobacillus plantarum DC400 or Lactobacillus brevis CR13 when the late stationary phase of growth (18 h) was reached. 2-DE analysis was carried out. Almost the same proteins were induced in all three cocultures at the mid-exponential phase of growth (7 h). The number of induced proteins markedly increased at 18 h, especially when L. sanfranciscensis CB1 was cocultured with L. plantarum DC400 or L. brevis CR13. Nineteen overexpressed proteins were identified. These proteins had a central role in stress response mechanisms and LuxS-mediated signalling was involved in the regulation of most of them. The luxS and metF genes were partially sequenced in L. sanfranciscensis CB1. RT-PCR showed that the expression of luxS gene decreased from 7 to 12 h. It was highest in cocultures with L. plantarum DC400 and L. brevis CR13. 2(3H)dihydrofuranone-5ethyl and 2(3H)dihydrofuranone-5pentyl were identified as presumptive signalling molecules when L. sanfranciscensis CB1 was cocultured with L. brevis CR13 and, especially, L. plantarum DC400. The synthesis of other volatile compounds and peptidase activities were also influenced by the type of microbial cocultures.     

Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.     

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.     

Glutamine deamidation by cereal-associated lactic acid bacteria

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.     

Differentiation of Lactobacillus sanfranciscensis strains by randomly amplified polymorphic DNA and pulsed-field gel electrophoresis

Genetic diversity of Lactobacillus sanfranciscensis strains isolated from naturally fermented sourdoughs of different origin was evaluated by using randomly amplified polymorphic DNA (RAPD). Computer-assisted comparison of the RAPD patterns revealed a clear separation of L. sanfranciscensis from other obligately heterofermentative Lactobacillus species closely related or normally present in sourdough. Six clusters, five of them constituted by strains of the same origin, were recognized at a similarity level of 63%. Pulsed-field gel electrophoresis (PFGE) results on strains chosen as representative were generally in good agreement with the grouping obtained by RAPD. Both techniques showed a high degree of discriminatory power and indicated the existence of a remarkable genetic polymorphism within the species. Furthermore, the chromosome size of L. sanfranciscensis was estimated by PFGE to be about 1.4 Mb.     

Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.     

Exopolysaccharide and kestose production by Lactobacillus sanfranciscensis LTH2590

The effect was investigated of sucrose concentration on sucrose metabolism and on the formation of exopolysaccharide (EPS) by Lactobacillus sanfranciscensis LTH2590 in pH-controlled fermentations with sucrose concentrations ranging from 20 to 160 g liter(-1). The EPS production increased and the relative sucrose hydrolysis activity decreased by increasing the sucrose concentration in the medium. The carbon recovery decreased from 95% at a sucrose concentration of 30 g liter(-1) to 58% at a sucrose concentration of 160 g liter(-1) because of the production of an unknown metabolite by L. sanfranciscensis. This metabolite was characterized as a fructo-oligosaccharide. The oligosaccharide produced by L. sanfranciscensis was purified and characterized as a trisaccharide with a glucose/fructose ratio of 1:2. The comparison of the retention time of this oligosaccharide and that of pure oligosaccharide standards using two different chromatography methods revealed that the oligosaccharide produced by L. sanfranciscensis LTH2590 is 1-kestose. Kestose production increased concomitantly with the initial sucrose concentration in the medium.     

Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.     

Genomic analysis reveals <Emphasis Type="Italic">Lactobacillus sanfranciscensis</Emphasis> as stable element in traditional sourdoughs

Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis is the predominant key bacterium in traditionally fermented sourdoughs.The genome of L. sanfranciscensis TMW 1.1304 isolated from an industrial sourdough fermentation was sequenced with a combined Sanger/454-pyrosequencing approach followed by gap closing by walking on fosmids. The sequencing data revealed a circular chromosomal sequence of 1,298,316 bp and two additional plasmids, pLS1 and pLS2, with sizes of 58,739 bp and 18,715 bp, which are predicted to encode 1,437, 63 and 19 orfs, respectively. The overall GC content of the chromosome is 34.71%. Several specific features appear to contribute to the ability of L. sanfranciscensis to outcompete other bacteria in the fermentation. L. sanfranciscensis contains the smallest genome within the lactobacilli and the highest density of ribosomal RNA operons per Mbp genome among all known genomes of free-living bacteria, which is important for the rapid growth characteristics of the organism. A high frequency of gene inactivation and elimination indicates a process of reductive evolution. The biosynthetic capacity for amino acids scarcely availably in cereals and exopolysaccharides reveal the molecular basis for an autochtonous sourdough organism with potential for further exploitation in functional foods. The presence of two CRISPR/cas loci versus a high number of transposable elements suggests recalcitrance to gene intrusion and high intrinsic genome plasticity.     

Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T

AIMS: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451T under various sublethal stress conditions. METHODS AND RESULTS: Stress conditions which reduce the growth rate of L. sanfranciscensis DSM 20451T to 10% of its maximum were determined. These conditions were met at 43, 12.5 degrees C, a pH value of 3.7, 1.9% NaCl, or 80 MPa respectively. In contrast to heat preincubation, other prestresses, including salt, cold and pressure led to an increase of barotolerance by hydrostatic pressure of 300 MPa for 30 min. Stationary-phase cells also showed an increased barotolerance. Sublethal pressure leads to enhanced heat tolerance. CONCLUSIONS: Stress response to salt, low temperature and acidic pH as well as starvation overlap with that one to high pressure by inducing barotolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation of bacteria by high pressure treatment is influenced by their history which modulates barotolerance. Mechanisms of barotolerance appear different from heat shock defence.     

Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.     

Bacterial population in traditional sourdough evaluated by molecular methods

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.     

Influence of temperature and backslopping time on the microbiota of a type I propagated laboratory wheat sourdough fermentation

Sourdough fermentation is a cereal fermentation that is characterized by the formation of stable yeast/lactic acid bacteria (LAB) associations. It is a unique process among food fermentations in that the LAB that mostly dominate these fermentations are heterofermentative. In the present study, four wheat sourdough fermentations were carried out under different conditions of temperature and backslopping time to determine their effect on the composition of the microbiota of the final sourdoughs. A substantial effect of temperature was observed. A fermentation with 10 backsloppings (once every 24 h) at 23°C resulted in a microbiota composed of Leuconostoc citreum as the dominant species, whereas fermentations at 30 and 37°C with backslopping every 24 h resulted in ecosystems dominated by Lactobacillus fermentum. Longer backslopping times (every 48 h at 30°C) resulted in a combination of Lactobacillus fermentum and Lactobacillus plantarum. Residual maltose remained present in all fermentations, except those with longer backslopping times, and ornithine was found in almost all fermentations, indicating enhanced sourdough-typical LAB activity. The sourdough-typical species Lactobacillus sanfranciscensis was not found. Finally, a nonflour origin for this species was hypothesized.     

Molecular source tracking of predominant lactic acid bacteria in traditional Belgian sourdoughs and their production environments

Aims:  To investigate the circulation of predominant sourdough lactic acid bacteria (LAB) species in the production environment of two Belgian artisan sourdough bakeries.
Methods and Results:  Isolates were collected from sourdoughs, flour, hands of the baker and air in the bakery setting and taxonomically characterized using repetitive element sequence-based PCR fingerprinting, pheS and/or 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. In parallel, PCR-DGGE (denaturing gradient gel electrophoresis) analysis of V3-16S rDNA amplicons was applied to visualize the predominant bacterial population in the sourdoughs and the corresponding bakery environment (flour, hands of the baker, air and bakery equipment). Both approaches revealed that sourdoughs produced at D01 and D10 were mainly dominated by Lactobacillus spicheri and L. plantarum and by L. sanfranciscensis , respectively, and that these LAB species also circulated in the corresponding bakery environment. Furthermore, AFLP fingerprinting demonstrated that sourdough and bakery environment isolates of these species were genetically indistinguishable. For more sensitive source-tracking, SYBR Green-based real-time PCR assays were developed using species-specific primers targeting the pheS gene of L. plantarum and L. sanfranciscensis, detected in air samples from D01 and D10, respectively.
Conclusions:  The results obtained in this study indicate that specific strains of LAB persist in artisan doughs over years and circulate in the bakery environment. Furthermore, the importance of air as a potential carrier of LAB in artisan bakery environments was demonstrated.
Significance and Impact of the Study:  PheS -based real-time PCR can be used to detect, quantify and/or monitor specific LAB species (e.g. starter cultures) in sourdough and bakery environment samples.