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.     

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.     

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Induction of barotolerance by heat shock treatment in yeast

In Saccharomyces cerevisiae, heat shock treatment provides protection against subsequent hydrostatic pressure damage. Such an induced hydrostatic pressure resistance (barotolerance) closely resembles the thermotolerance similarly induced by heat shock treatment. The parallel induction of barotolerance and thermotolerance by heat shock suggests that hydrostatic pressure and high temperature effects in yeast may be tightly linked physiologically.     

In vitro stability and expression of green fluorescent protein under high pressure conditions

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.     

Evidence for Contribution of Neutral Trehalase in Barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.     

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.     

A role for proline synthesis and transport in Listeria monocytogenes barotolerance

Aims: To assess the contribution of proline biosynthesis to listerial barotolerance. Methods and Results: Using a Listeria monocytogenes proBA deletion mutant, incapable of synthesizing proline, together with a proline‐overproducing strain, the contribution of proline synthesis to listerial barotolerance was determined. The ΔproBA strain does not survive as well as the wild type when subjected to treatment of 500 MPa in rich media and 400 MPa in minimal media (c. 1 log lower survival in both conditions). Betaine and carnitine decrease the ability of the wild type to survive at low pressures (300 MPa), but confer normal or slightly increased levels of protection at higher pressures (350 and 400 MPa). Conclusions: A functional proline synthesis system is required for optimal survival of Listeria following treatment at high‐pressure (HP) levels (500 MPa in brain heart infusion and 400 MPa in defined medium), particularly where other compatible solutes are absent or limiting. Significance and Impact of the Study: Given the potential of HP processing as an effective food processing/safety strategy, understanding how pathogens such as Listeria have evolved to cope with such stresses is an important food safety consideration. In this context, the work presented here may help to develop safer and more effective processing regimens.     

Effect of culture conditions on lactic acid production of Tetragenococcus species

AIMS: To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T. halophilus. METHOD AND RESULTS: The growth, lactic acid production and pH reduction ability of five strains of T. muriaticus and T. halophilus in MRS broth medium under various culture conditions such as salt concentration (3, 7, 15 and 23% NaCl), temperature (20, 30 and 40 degrees C), and initial medium pH (5.8, 6.5 and 7.5) were investigated. Those of T. halophilus were seriously affected by a high salinity (23% NaCl); in contrast, those of T. muriaticus were affected by a low initial pH (5.8). CONCLUSIONS: The results indicate that high saline concentrations and low pH values have significant impact on the growth, lactic acid production and pH reduction ability of T. halophilus and T. muriaticus, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study appears to be important in biopreservation during the manufacture of fermented food products. Both T. muriaticus and T. halophilus may support each other in reducing pH in hypersaline or low pH environment. To our knowledge, this is the first report on the fermentation ability of T. muriaticus.     

High pressure effects step-wise altered protein expression in Lactobacillus sanfranciscensis

In this study we investigated the cellular response to the application of high hydrostatic pressure. High pressure is increasingly used for food preservation. With high resolution 2-D electrophoresis we compared the protein patterns of atmospherically grown Lactobacillus sanfranciscensis with those pressure treated up to 200 MPa. We performed the comparative study by using overlapping immobilized pH gradients covering the pH range from 2.5 up to 12 in order to maximize the resolution for the detection of stress relevant proteins. For improved quantitative analysis, staining with SyproRuby was used in addition to silver staining. By computer aided image analysis we detected more than a dozen spots within the pH range from 3.5 to 9 that were more than two-fold increased or 50% decreased in their intensity upon high pressure treatment. Two of them (approx. values: pI 4.0 and 4.2, respectively; M(r) approximately 15 000) have almost identical matrix-assisted laser desorption/ionization-time of flight mass spectrometry spectra and were identified by liquid chromatography-tandem mass spectrometry as putative homologs/paralogs to cold shock proteins of Lactococcus lactis. Their expression is opposed (i.e. the more acidic one is repressed, while the other one is induced); this effect is maximal at 1 h, 150 MPa. It was further remarkable that by monitoring the barosensitivity of the cells within 25 MPa steps, we observed a differential pressure induction or repression of the detected proteins as well. For example one protein (approx. values: pI 4.2, M(r) approximately 15 000) shows a maximum induction after 1 h, 150 MPa while another one (pI 7.5, M(r) approximately 25 000) is maximally induced after 1 h, 50/75 MPa. This indicates a successive cell response and different signalling pathways for these responses.     

Hydrostatic pressure is like high temperature and oxidative stress in the damage it causes to yeast

Abstract A comparison of barotolerance, thermotolerance and oxygen tolerance was made under different physiological conditions, such as heat shocked and recovered state, different growth phases and changes of physiological conditions by mutations. The three kinds of tolerance showed similar features under different physiological conditions. We suggest that the damage caused by hydrostatic pressure may be essentially the same as that due to high temperature and oxidative stress in yeast.     

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^TΔgshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451^TΔgshR 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 μM to 10.5 μM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451^TΔgshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 μM 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^TΔgshR 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.     

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.     

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.     

Role for compatible solutes glycine betaine and L-carnitine in listerial barotolerance

Increased listerial barotolerance at elevated osmolarity is attributed, in part, to the presence of accumulated betaine and L-carnitine. The percentage of listerial survival following exposure to 400 MPa for 5 min increased from 0.008 to 0.02% with added L-carnitine (5 mM) and to 0.05% with added betaine (5 mM). Furthermore, listerial cells incapable of transporting compatible solutes fail to adapt to high pressure at elevated osmolarity.     

Magnesium and calcium ions enhance barotolerance of streptococci

Summary Magnesium and calcium ions were found to enhance barotolerance of Streptococcus faecalis ATCC strain 9790 growing in a complex, glucose-containing medium. Enhancement was indicated both by higher growth rates and yields at 408 atm, and also by an increase in the maximum pressure permitting growth from 550 to 700 atm. The optimum concentration of either ion was ca. 50 mM, and both ions appeared to be equipotent in affecting the same processes by chemically specific interactions. Sodium, potassium, strontium, manganous, chloride, bromide or sulfate ions were all ineffective or only marginally effective in enhancing barotolerance. Mg⁺⁺ and Ca⁺⁺ also enhanced growth of compressed, ribose-degrading cultures. Pressure increased the sensitivity of streptococcal growth to low pH, and there appeared to be two distinct effects of Mg⁺⁺ and Ca⁺⁺ on barotolerance. First, the rate of exponential growth was enhanced prior to the time at which culture acidity began to limit growth. Second, growth was possible in more acid conditions under pressure when the ions were present, and enhanced yields from compressed cultures were related to this partial reversal of the potentiating effect of high pressure on acid inhibition of growth.Barotolerance of Escherichia coli or Saccharomyces cerevisiae was not enhanced by these ions; while tolerance of two types of chain-forming cocci freshly isolated from a rotting mussel was enhanced.     

Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.     

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.