Diversity and Stability of Lactic Acid Bacteria in Rye Sourdoughs of Four Bakeries with Different Propagation Parameters.
ABSTRACT: We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5-28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected.
Project description:Silage making has become a significant method of forage conservation worldwide. To determine how tomato pomace (TP) may be used effectively as animal feed, it was ensilaged for 90 days and microbiology counts, fermentation characteristics and chemical composition of tomato pomace silage (TPS) were evaluated at the 30th, 60th, and 90th days, respectively. In addition, 103 lactic acid bacteria were isolated from TPS. Based on the phenotypic and chemotaxonomic characteristics, 16S rDNA sequence and carbohydrate fermentation tests, the isolates were identified as 17 species namely: Lactobacillus coryniformis subsp. torquens (0.97%), Lactobacillus pontis (0.97%), Lactobacillus hilgardii (0.97%), Lactobacillus pantheris (0.97%), Lactobacillus amylovorus (1.9%), Lactobacillus panis (1.9%), Lactobacillus vaginalis (1.9%), Lactobacillus rapi (1.9%), Lactobacillus buchneri (2.9%), Lactobacillus parafarraginis (2.9%), Lactobacillus helveticus (3.9%), Lactobacillus camelliae (3.9%), Lactobacillus fermentum (5.8%), Lactobacillus manihotivorans (6.8%), Lactobacillus plantarum (10.7%), Lactobacillus harbinensis (16.5%) and Lactobacillus paracasei subsp. paracasei (35.0%). This study has shown that TP can be well preserved for 90 days by ensilaging and that TPS is not only rich in essential nutrients, but that physiological and biochemical properties of the isolates could provide a platform for future design of lactic acid bacteria (LAB) inoculants aimed at improving the fermentation quality of silage.
Project description:Seven mature type I sourdoughs were comparatively back-slopped (80 days) at artisan bakery and laboratory levels under constant technology parameters. The cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation. On the contrary, the number of yeasts markedly decreased from artisan bakery to laboratory propagation. During late laboratory propagation, denaturing gradient gel electrophoresis (DGGE) showed that the DNA band corresponding to Saccharomyces cerevisiae was no longer detectable in several sourdoughs. Twelve species of lactic acid bacteria were variously identified through a culture-dependent approach. All sourdoughs harbored a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation. As shown by statistical permutation analysis, the lactic acid bacterium populations differed among sourdoughs propagated at artisan bakery and laboratory levels. Lactobacillus plantarum, Lactobacillus sakei, and Weissella cibaria dominated in only some sourdoughs back-slopped at artisan bakeries, and Leuconostoc citreum seemed to be more persistent under laboratory conditions. Strains of Lactobacillus sanfranciscensis were indifferently found in some sourdoughs. Together with the other stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs and largely differed between artisan bakery and laboratory levels. The environment of propagation has an undoubted influence on the composition of sourdough yeast and lactic acid bacterium microbiotas.
Project description:A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
Project description:Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50 degrees C, with L. amylolyticus being able to grow at temperatures as high as 54 degrees C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.
Project description: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.
Project description:A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
Project description:The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g(-1). Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.
Project description:Four traditional type I sourdoughs were comparatively propagated (28 days) under firm (dough yield, 160) and liquid (dough yield, 280) conditions to mimic the alternative technology options frequently used for making baked goods. After 28 days of propagation, liquid sourdoughs had the lowest pH and total titratable acidity (TTA), the lowest concentrations of lactic and acetic acids and free amino acids, and the most stable density of presumptive lactic acid bacteria. The cell density of yeasts was the highest in liquid sourdoughs. Liquid sourdoughs showed simplified microbial diversity and harbored a low number of strains, which were persistent. Lactobacillus plantarum dominated firm sourdoughs over time. Leuconostoc lactis and Lactobacillus brevis dominated only some firm sourdoughs, and Lactobacillus sanfranciscensis persisted for some time only in some firm sourdoughs. Leuconostoc citreum persisted in all firm and liquid sourdoughs, and it was the only species detected in liquid sourdoughs at all times; it was flanked by Leuconostoc mesenteroides in some sourdoughs. Saccharomyces cerevisiae, Candida humilis, Saccharomyces servazzii, Saccharomyces bayanus-Kazachstania sp., and Torulaspora delbrueckii were variously identified in firm and liquid sourdoughs. A total of 197 volatile components were identified through purge and trap-/solid-phase microextraction-gas chromatography-mass spectrometry (PT-/SPME-GC-MS). Aldehydes, several alcohols, and some esters were at the highest levels in liquid sourdoughs. Firm sourdoughs mainly contained ethyl acetate, acetic acid, some sulfur compounds, and terpenes. The use of liquid fermentation would change the main microbial and biochemical features of traditional baked goods, which have been manufactured under firm conditions for a long time.
Project description:This study determined the effects of specific metabolic traits of Lactobacillus reuteri on its competitiveness in sourdoughs. The competitiveness of lactobacilli in sourdough generally depends on their growth rate; acid resistance additionally contributes to competitiveness in sourdoughs with long fermentation times. Glycerol metabolism via glycerol dehydratase (gupCDE) accelerates growth by the regeneration of reduced cofactors; glutamate metabolism via glutamate decarboxylase (gadB) increases acid resistance by generating a proton motive force. Glycerol and glutamate metabolisms are lineage-specific traits in L. reuteri; therefore, this study employed glycerol dehydratase-positive sourdough isolates of human-adapted L. reuteri lineage I, glutamate decarboxylase-positive strains of rodent-adapted L. reuteri lineage II, as well as mutants with deletions in gadB or gupCDE. The competitivenesses of the strains were quantified by inoculation of wheat and sorghum sourdoughs with defined strains, followed by propagation of doughs with a 10% inoculum and 12-h or 72-h fermentation cycles. Lineage I L. reuteri strains dominated sourdoughs propagated with 12-h fermentation cycles; lineage II L. reuteri strains dominated sourdoughs propagated with 72-h fermentation cycles. L. reuteri 100-23ΔgadB was outcompeted by its wild-type strain in sourdoughs fermented with 72-h fermentation cycles; L. reuteri FUA3400ΔgupCDE was outcompeted by its wild-type strain in sourdoughs fermented with both 12-h and 72-h fermentation cycles. Competition experiments with isogenic pairs of strains resulted in a constant rate of strain displacement of the less competitive mutant strain. In conclusion, lineage-specific traits of L. reuteri determine the competitiveness of this species in sourdough fermentations.
Project description: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.