LC-MS/MS quantitation of α-amylase/trypsin inhibitor CM3 and glutathione during wheat sourdough breadmaking.

AIMS: This study aimed to quantify α-amylase/trypsin inhibitor (ATI) CM3 and glutathione (GSH) during wheat sourdough breadmaking. METHODS AND RESULTS: Breads were made with two wheat cultivars and fermented with Fructilactobacillus sanfranciscensis, F. sanfranciscensis ΔgshR or Latilactobacillus sakei; chemically acidified and straight doughs served as controls. Samples were analysed after mixing, after proofing and after baking. GSH and CM3 were quantified by multi-reaction-monitoring-based methods on an LC-QTRAP mass spectrometer. Undigested ATI extracts were further examined by SDS-PAGE. CONCLUSIONS: GSH abundance was similar after mixing and after proofing but increased after baking (p < 0.001), regardless of fermentation. In breads baked with cv. Brennan, the samples fermented with lactobacilli had higher GSH abundance (p < 0.001) than in the controls. CM3 relative abundance remained similar after mixing and after proofing but decreased after baking (p < 0.001) across all treatments. This trend was supported by the SDS-PAGE analysis in which ATI band intensities decreased after baking (p < 0.001) in all experimental conditions. The overall effect of baking exerted a greater effect on the abundances of GSH and CM3 than fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to quantify ATI over the course of breadmaking by LC-MS/MS in sourdough and straight dough processes.

Dynamic interactions of lactic acid bacteria in Korean sourdough during back-slopping process.

AIMS: This study aimed to clarify the cause of quality reduction in Korean sourdough after successive back-slopping. METHODS AND RESULTS: We investigated the dynamic changes in lactic acid bacteria during the back-slopping process using genetic fingerprinting techniques. During the initial propagation phases, the dominant lactic acid bacteria were Fructilactobacillus sanfranciscensis (<5 log CFU per g sourdough), Latilactobacillus curvatus (9·5 log CFU per g sourdough) and Levilactobacillus brevis (6·5 log CFU per g sourdough). However, after the 11th propagation, F. sanfranciscensis became more prominent (>9·0 log CFU per g sourdough), whereas L. curvatus and L. brevis rapidly decreased. Monitoring these bacteria in the co-culture system revealed that acid-tolerant F. sanfranciscensis rapidly utilized maltose (1·65 g l-1  h-1 ) and produced large amounts of lactic acid, whereas L. brevis and L. curvatus consumed maltose slowly and L. curvatus was poorly tolerant to lactic acid. CONCLUSION: The results indicate that competition exists between the lactic acid bacteria in sourdough during the back-slopping process, and microbial succession by acid-tolerant species results in quality reduction of sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uncovered the cause of microbial changes during the propagation of Korean sourdough and proposed a strategy to develop starters to produce high-quality bakery products.

Investigating the growth kinetics in sourdough microbial associations.

In this study we investigated the effect of the single strain in stabilization of type I sourdough microbial associations by crossing six different Fructilactobacillus sanfranciscensis with five Kazachstania humilis strains. Furthermore, we compared three predictive models, Zwitwering based on Gompertz's equation, Baranyi and Roberts' function and Schiraldi's function to evaluate which one best fitted the experimental data in determining the behaviour of co-cultivated microorganisms. Specific growth rates (µm) and lag time (λ) values for each mixed population were assessed. Results showed that the different F. sanfranciscensis strains significantly steer the growth kinetics within the pair and affect the ratio bacterial/yeast cells, as data analysis confirmed, whereas K. humilis accommodates to the bacterial strain. To compare the growth models, Root Mean Square (RMS) values were calculated for each predicted curve by implementing an algorithm based on an iterative process to minimize the deviation among observed and calculated data. Schiraldi's function performed better than the others, revealing, on average, the smallest RMS values and providing the best fitting for over 70% of co-cultivation experiments. Models prove to be consistent in predicting growth kinetics of microbial consortia too.

Comparative Genomics Reveals Genetic Diversity and Variation in Metabolic Traits in Fructilactobacillus sanfranciscensis Strains.

Fructilactobacillus sanfranciscensis is a significant and dominant bacterial species of sourdough microbiota from ecological and functional perspectives. Despite the remarkable prevalence of different strains of this species in sourdoughs worldwide, the drivers behind the genetic diversity of this species needed to be clarified. In this research, 14 F. sanfranciscensis strains were isolated from sourdough samples to evaluate the genetic diversity and variation in metabolic traits. These 14 and 31 other strains (obtained from the NCBI database) genomes were compared. The values for genome size and GC content, on average, turned out to 1.31 Mbp and 34.25%, respectively. In 45 F. sanfranciscensis strains, there were 162 core genes and 0 to 51 unique genes present in each strain. The primary functions of core genes were related to nucleotide, lipid transport, and amino acid, as well as carbohydrate metabolism. The size of core genes accounted for 41.18% of the pan-genome size in 14 F. sanfranciscensis strains, i.e., 0.70 Mbp of 1.70 Mbp. There were genetic variations among the 14 strains involved in carbohydrate utilization and antibiotic resistance. Moreover, exopolysaccharides biosynthesis-related genes were annotated, including epsABD, wxz, wzy. The Type IIA & IE CRISPR-Cas systems, pediocin PA-1 and Lacticin_3147_A1 bacteriocins operons were also discovered in F. sanfranciscensis. These findings can help to select desirable F. sanfranciscensis strains to develop standardized starter culture for sourdough fermentation, and expect to provide traditional fermented pasta with a higher quality and nutritional value for the consumers.

Functional Characterization of Different Fructilactobacillus sanfranciscensis Strains Isolated from Chinese Traditional Sourdoughs.

Fructilactobacillus sanfranciscensis, the dominant species of lactic acid bacteria in sourdoughs, impacts the microstructure and flavor of steamed bread through exopolysaccharide production, acidification, proteolysis, and volatile compound generation. The aim of this study is to investigate the phenotypic diversity and technological traits of 28 F. sanfranciscensis strains of different genotypes isolated from Chinese traditional sourdoughs. The results showed that F. sanfranciscensis strains exhibited substantial variation in proteinase and peptidase activities and the amount of acidification and volatiles in fermented sourdoughs. However, we observed no significant differences in exopolysaccharide production among the strains. The strains Sx14 and Ts1 were further chosen for transcriptomics to gain a deep insight into their intraspecies diversity in sourdough fermentation. Significant transcriptome differentiations between these two strains after 12 h fermentation in sourdoughs were revealed. According to the results, the strain Sx14 possessed higher dipeptidase and aminopeptidase activities, galactose utilization, and lactic and acetic acid production abilities, whereas Ts1 showed higher transmembrane transport of substrates and fructose utilization.

The quest for the perfect loaf of sourdough bread continues: Novel developments for selection of sourdough starter cultures.

Sourdough fermentation, one of the oldest unit operations in food production, is currently experiencing a revival in bread production at the household, artisanal, and the industrial level. The expanding use of sourdough fermentation in bread production and the adaptation of fermentation to large scale industrial bread production also necessitate the development of novel starter cultures. Developments in the last years also have expanded the tools that are used to assess the metabolic potential of specific strains, species or genera of the Lactobacillaceae and have identified multiple ecological and metabolic traits as clade-specific. This review aims to provide an overview on the clade-specific metabolic potential of members of the Lactobacillaceae for use in sourdough baking, and the impact of these clade-specific traits on bread quality. Emphasis is placed on carbohydrate metabolism, including the conversion of sucrose and starch to soluble polysaccharides, conversion of amino acids, and the metabolism of organic acids. The current state of knowledge to compose multi-strain starter cultures (synthetic microbial communities) that are suitable for back-slopping will also be discussed. Taken together, the communication outlines the current tools for selection of microbes for use in sourdough baking.

Intraspecies diversity and genome-phenotype-associations in Fructilactobacillus sanfranciscensis.

In this study the intraspecies diversity of Fructilactobacillus (F.) sanfranciscensis (formerly Lactobacillus sanfranciscensis) was characterized by comparative genomics supported by physiological data. Twenty-four strains of F. sanfranciscensis were analyzed and sorted into six different genomic clusters. The core genome comprised only 43,14 % of the pan genome, i.e. 0.87 Mbp of 2.04 Mbp. The main annotated genomic differences reside in maltose, fructose and sucrose as well as nucleotide metabolism, use of electron acceptors, and exopolysacchride formation. Furthermore, all strains are well equipped to cope with oxidative stress via NADH oxidase and a distinct thiol metabolism. Only ten of 24 genomes contain two maltose phosphorylase genes (mapA and mapB). In F. sanfranciscensis TMW 1.897 only mapA was found. All strains except those from genomic cluster 2 contained the mannitol dehydrogenase and should therefore be able to use fructose as external electron acceptor. Moreover, six strains were able to grow on fructose as sole carbon source, as they contained a functional fructokinase gene. No growth was observed on pentoses, i.e. xylose, arabinose or ribose, as sole carbon source. This can be referred to the absence of ribose pyranase rbsD in all genomes, and absence of or mutations in numerous other genes, which are essential for arabinose and xylose metabolism. Seven strains were able to produce exopolysaccharides (EPS) from sucrose. In addition, the strains containing levS were able to grow on sucrose as sole carbon source. Strains of one cluster exhibit auxotrophies for purine nucleotides. The physiological and genomic analyses suggest that the biodiversity of F. sanfranciscensis is larger than anticipated. Consequently, "original" habitats and lifestyles of F. sanfranciscensis may vary but can generally be referred to an adaptation to sugary (maltose/sucrose/fructose-rich) and aerobic environments as found in plants and insects. It can dominate sourdoughs as a result of reductive evolution and cooperation with fructose-delivering, acetate-tolerant yeasts.