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, 75 (4), 1099-109

Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria During Wheat Flour Sourdough Type I Propagation Using Lactobacillus Sanfranciscensis Starters

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Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria During Wheat Flour Sourdough Type I Propagation Using Lactobacillus Sanfranciscensis Starters

Sonya Siragusa et al. Appl Environ Microbiol.

Abstract

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.

Figures

FIG. 1.
FIG. 1.
Cell densities (CFU g−1) after sourdough propagation at 30°C for 6 h in 10 subsequent days with use of wheat flour type 0 F114. Data are the means from three independent experiments (n = 3). The center line of each box (□) represents the median; the top and bottom of the box represent the 75th and 25th percentiles of the data, respectively. The tops and bottoms of the error bars represent the 5th and 95th percentiles of the data, respectively. The circles (○) in each box plot extend to the outliers of the data, and very extreme points are represented as individual data points (*). Control, unstarted sourdough. Sourdough (S) started with strain Lactobacillus sanfranciscensis LS3, LS6, LS8, LS12, LS14, LS40, LS41, LS44, or LS48 is indicated.
FIG. 2.
FIG. 2.
Kinetics of growth of Lactobacillus sanfranciscensis LS3 (▪), LS6 (⧫), LS8 (•), LS12 (*), LS14 (□), LS40 (▴), LS41 (…), LS44 (○), and LS48 (⋄) after sourdough propagation at 30°C for 6 h during 10 subsequent days with use of wheat flour type 0 F114. The data are the means from three independent experiments ± standard deviations (n = 3).
FIG. 3.
FIG. 3.
(A) Representative RAPD-PCR patterns of lactic acid bacteria isolated after 10 days of daily sourdough propagation at 30°C for 6 h with use of wheat flour type 0 F114. Primer P7 was used for RAPD-PCR analysis. st, DNA molecular size standards (12,000 to 100 bp); Control, unstarted sourdough. Sourdough started with Lactobacillus sanfranciscensis LS3 (S-LS3), LS6 (S-LS6), LS8 (S-LS8), LS12 (S-LS12), LS14 (S-LS14), LS40 (S-LS40), LS41 (S-LS41), LS44 (S-LS44), or LS48 (S-LS48) is indicated. Lactic acid bacterium isolates (I) are coded based on partial 16S rRNA and recA gene sequence comparisons and correspond to those of Table 1. Lb., Lactobacillus; Lc., Lactococcus. (B) Dendrogram obtained by combined random amplification of polymorphic DNA patterns of the isolates from 10 days of daily sourdough propagation at 30°C for 6 h by using wheat flour type 0 F114. Primers P4, P7, and M13 were used for RAPD-PCR analysis. Lactic acid bacterium isolates I(1) to I(100) correspond to those of Table 1. Cluster analysis was based on the simple matching coefficient and unweighted-pair group method with arithmetic average.
FIG. 4.
FIG. 4.
ΔpH values (difference in pH units between the initial pH and final pH after sourdough propagation at 30°C for 6 h during 10 subsequent days by using wheat flour type 0 F114. Data are the means from three independent experiments (n = 3). The center line of each box represents the median (□); the top and bottom of the box represent the 75th and 25th percentiles of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentiles of the data, respectively. The circles in each box plot extend to the outliers of the data (○). Control, unstarted sourdough. Sourdough started with Lactobacillus sanfranciscensis LS3, LS6, LS8, LS12, LS14, LS40, LS41, LS44, or LS48 is indicated.
FIG. 5.
FIG. 5.
(A) Representative RAPD-PCR patterns of lactic acid bacteria isolated after 10 days of daily sourdough propagation at 30°C for 6 h by using wheat flour type 0 F164. Primer P7 was used for RAPD-PCR analysis. st, DNA molecular size standards (12,000 to 100 bp); Control, unstarted sourdough. Sourdoughs started with Lactobacillus sanfranciscensis LS8 (S-LS8), LS14 (S-LS14), or LS44 (S-LS44) are shown. Lactic acid bacterium isolates (I) are coded based on partial 16S rRNA and recA gene sequence comparisons and correspond to those of Table 3. Lb., Lactobacillus. (B) Dendrogram obtained by combined random amplification of polymorphic DNA patterns for the isolates from 10 days of daily sourdough propagation at 30°C for 6 h by using wheat flour type 0 F164. Primers P4, P7, and M13 were used for RAPD-PCR analysis. Lactic acid bacterium isolates I(101) to I(140) correspond to those of Table 3. Cluster analysis was based on the simple matching coefficient and unweighted-pair group method with arithmetic average.

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