Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log10 to 7.6 log10CFU/g while LAB counts varied from 7.2 log10 to 9.6 log10CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log10CFU/g, except for one sample at 4.4 log10CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic sourdough.

Genotypic diversity of Lactobacillus sanfranciscensis strains isolated from French organic sourdoughs.

Lactobacillus sanfranciscensis is the predominant key lactic acid bacterium in traditionally fermented sourdoughs. Despite its prevalence, sourdough and their related breads could be different regarding their physicochemical and sensorial characteristics. The intraspecific diversity of L. sanfranciscensis might explain these observations. Fifty-nine strains isolated from French sourdoughs were typed by a polyphasic approach including Multilocus Sequence Typing (MLST) and Pulsed-field Gel Electrophoresis (PFGE), in order to study their genotypic diversity. MLST scheme can be reduced from six to four gene fragments (gdh, gyrA, nox and pta) without a major loss of discrimination between strains. The genes mapA and pgmA are not good candidates for inclusion in an MLST scheme to type L. sanfranciscensis strains, as they could not be amplified for a set of 18 strains among the 59 studied. This method revealed 20 sequence types (STs). Of these, 19 STs were grouped in one clonal complex, showing a strong relatedness between these strains. PFGE using SmaI discriminated 41 pulsotypes and so distinguished isolates better than the MLST scheme. Both genotypic methods indicate a low diversity between strains isolated from the same sourdough and a higher diversity between strains isolated from different sourdoughs, suggesting an influence of baker practices and/or environmental conditions on the selection of strains. The use of these two methods targeting genetic variations gives an optimal genotypic characterization of L.sanfranciscensis strains.

Sourdough microbial community dynamics: An analysis during French organic bread-making processes.

Natural sourdoughs are commonly used in bread-making processes, especially for organic bread. Despite its role in bread flavor and dough rise, the stability of the sourdough microbial community during and between bread-making processes is debated. We investigated the dynamics of lactic acid bacteria (LAB) and yeast communities in traditional organic sourdoughs of five French bakeries during the bread-making process and several months apart using classical and molecular microbiology techniques. Sourdoughs were sampled at four steps of the bread-making process with repetition. The analysis of microbial density over 68 sourdough/dough samples revealed that both LAB and yeast counts changed along the bread-making process and between bread-making runs. The species composition was less variable. A total of six LAB and nine yeast species was identified from 520 and 1675 isolates, respectively. The dominant LAB species was Lactobacillus sanfranciscensis, found for all bakeries and each bread-making run. The dominant yeast species changed only once between bread-making processes but differed between bakeries. They mostly belonged to the Kazachstania clade. Overall, this study highlights the change of population density within the bread-making process and between bread-making runs and the relative stability of the sourdough species community during bread-making process.

Lactic acid bacterium and yeast microbiotas of sixteen French traditional sourdoughs.

Sixteen sourdoughs (FS1-FS16) used for the manufacture of traditional French breads were characterized by strongly acid conditions (median value of pH 3.5). The concentration of free amino acids (FAA) was highly variable, due to different proteolytic activity of flour used for back slopping and of dominant microorganisms. Median value of cell density of lactic acid bacteria (LAB) was 9.2 log CFU/g. The ratio between LAB and yeasts ranged from 10,000:1 to 10:1. According to the culture-dependent method and 16S metagenetics, Lactobacillus sanfranciscensis was the dominant species in French sourdoughs. FS5 and FS15, propagated according to protocols including one back slopping step at 14 °C, were the only exceptions. High positive correlations were found between L. sanfranciscensis, temperature of back slopping and FAA. The results of this study highlighted the broad adaptability of L. sanfranciscensis to very acid sourdough. Besides species frequently encountered (e.g., Lactobacillus parabrevis/Lactobacillus hammesii, Lactobacillus plantarum and Leuconostoc mesenteroides), first Lactobacillus xiangfangensis (FS5) and Lactobacillus diolivorans (FS15) were found in sourdough. As determined by RAPD-PCR analyses, the sourdough samples showed a different number of strains, ranging from 5 (FS9, FS11 and FS15) to 12 (FS1 and FS13), meaning a highly variable bacterial diversity. Cluster analysis showed that different sourdoughs, especially when propagated in the same bakery, may harbor similar strains. Except for L. plantarum (FS5) and Ln. mesenteroides (FS3), all the dominant species were detected by both 16S metagenetics and culture-dependent method. Yeast diversity was lower than LAB. Except for FS4 (solely dominated by Kazachstania servazzii), yeast microbiota of French sourdoughs was dominated by Saccharomyces cerevisiae. Strains isolated in this study could be a useful base for developing new basic researches on physiology, metabolism, and intraspecific diversity of L. sanfranciscensis, as well as for standardizing the quality of traditional French breads.

The predominance of Lactobacillus sanfranciscensis in French organic sourdoughs and its impact on related bread characteristics.

Fourteen bakeries located in different regions of France were selected. These bakers use natural sourdough and organic ingredients. Consequently, different organic sourdoughs used for the manufacture of French bread were studied by the enumeration of lactic acid bacteria (LAB) and 16S rRNA sequencing of the isolates. In addition, after DNA extraction the bacterial diversity was assessed by pyrosequencing of the 16S rDNA V1-V3 region. Although LAB counts showed significant variations (7.6-9.5log10CFU/g) depending on the sourdough studied, their identification through a polyphasic approach revealed a large predominance of Lactobacillus sanfranciscensis in all samples. In ten sourdoughs, both culture and independent methods identified L. sanfranciscensis as the dominant LAB species identified. In the remaining sourdoughs, culture methods identified 30-80% of the LAB as L. sanfranciscensis whereas more than 95% of the reads obtained by pyrosequencing belonged to L. sanfranciscensis. Other sub-dominant species, such as Lactobacillus curvatus, Lactobacillus hammesii, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus sakei, were also identified. Quantification of L. sanfranciscensis by real-time PCR confirmed the predominance of this species ranging from 8.24 to 10.38log10CFU/g. Regarding the acidification characteristics, sourdough and related bread physico-chemical characteristics varied, questioning the involvement of sub-dominant species or L. sanfranciscensis intra-species diversity and/or the role of the baker's practices.

A polyphasic approach to study the dynamics of microbial population of an organic wheat sourdough during its conversion to gluten-free sourdough.

To develop a method for organic gluten-free (GF) sourdough bread production, a long-term and original wheat sourdough was refreshed with GF flours. The dynamics of the sourdough microbiota during five months of back-slopping were analyzed by classical enumeration and molecular methods, including PCR-temporal temperature gel electrophoresis (PCR-TTGE), multiplex PCR, and pulsed field gel electrophoresis (PFGE). The results showed that the yeast counts remained constant, although Saccharomyces cerevisiae, present in the initial wheat sourdough, was no longer detected in the GF sourdough, while lactic acid bacteria (LAB) counts increased consistently. In the first phase, which was aimed at obtaining a GF sourdough from wheat sourdough, Lactobacillus sanfranciscensis, L. plantarum, and L. spicheri were the main LAB species detected. During the second phase, aimed at maintaining the GF sourdough, the L. plantarum and L. spicheri populations decreased whereas L. sanfranciscensis persisted and L. sakei became the predominant species. Multiplex PCRs also revealed the presence of several L. sakei strains in the GF sourdough. In a search for the origin of the LAB species, PCR-TTGE was performed on the flour samples but only L. sanfranciscensis was detected, suggesting a flour origin for this typical sourdough species. Thus, while replacement of the wheat flour by GF flour influenced the sourdough microbiota, some of the original sourdough LAB and yeast species remained in the GF sourdough.

A polyphasic approach to study the dynamics of microbial population of an organic wheat sourdough during its conversion to gluten-free sourdough

To develop a method for organic gluten-free (GF) sourdough bread production, a long-term and original wheat sourdough was refreshed with GF flours. The dynamics of the sourdough microbiota during five months of back-slopping were analyzed by classical enumeration and molecular methods, including PCR-temporal temperature gel electrophoresis (PCR-TTGE), multiplex PCR, and pulsed field gel electrophoresis (PFGE). The results showed that the yeast counts remained constant, although Saccharomyces cerevisiae, present in the initial wheat sourdough, was no longer detected in the GF sourdough, while lactic acid bacteria (LAB) counts increased consistently. In the first phase, which was aimed at obtaining a GF sourdough from wheat sourdough, Lactobacillus sanfranciscensis, L. plantarum, and L. spicheri were the main LAB species detected. During the second phase, aimed at maintaining the GF sourdough, the L. plantarum and L. spicheri populations decreased whereas L. sanfranciscensis persisted and L. sakei became the predominant species. Multiplex PCRs also revealed the presence of several L. sakei strains in the GF sourdough. In a search for the origin of the LAB species, PCR-TTGE was performed on the flour samples but only L. sanfranciscensis was detected, suggesting a flour origin for this typical sourdough species. Thus, while replacement of the wheat flour by GF flour influenced the sourdough microbiota, some of the original sourdough LAB and yeast species remained in the GF sourdough (AU)

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Characterization of Carnobacterium maltaromaticum LMA 28 for its positive technological role in soft cheese making.

Carnobacterium maltaromaticum is a lactic acid bacterium isolated from soft cheese. The objective of this work was to study its potential positive impact when used in cheese technology. Phenotypic and genotypic characterization of six strains of C. maltaromaticum showed that they belong to different phylogenetic groups. Although these strains lacked the ability to coagulate milk quickly, they were acidotolerant. They did not affect the coagulation capacity of starter lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, used in dairy industry. The impact of C. maltaromaticum LMA 28 on bacterial flora of cheese revealed a significant decrease of Psychrobacter sp. concentration, which might be responsible for cheese aging phenomena. An experimental plan was carried out to unravel the mechanism of inhibition of Psychrobacter sp. and Listeria monocytogenes and possible interaction between various factors (cell concentration, NaCl, pH and incubation time). Cellular concentration of C. maltaromaticum LMA 28 was found to be the main factor involved in the inhibition of Psychrobacter sp. and L. monocytogenes.