Functional Characterization of the Proteolytic System ofLactobacillus sanfranciscensisDSM 20451^Tduring Growth in Sourdough

Abstract

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.Wheat sourdough fermentation has a positive influence on the overall bread quality because it improves flavor (14,29,32) and texture (18) and prolongs shelf life due to the formation of antifungal compounds (20) and delayed staling (6). The fate of the protein fraction of the flour during sourdough fermentation is of crucial importance for bread quality. The protein network in wheat doughs determines dough rheology, gas retention, and thus bread volume and texture. Proteolytic events during fermentation provide the substrates for microbial growth and conversion of amino acids to flavor precursor compounds and antifungal metabolites (20,32). Furthermore, some peptides from wheat proteins are involved in human cereal intolerance and their levels in bread hydrolysates are reduced by selected sourdough lactic acid bacteria (9).The most abundant proteins in wheat flour are gluten proteins. After dough mixing, the proteins are linked to each other by disulfide and hydrogen bonds, resulting in an insoluble network with a molecular weight of up to several million (35). Proteolytic enzymes in the flour are associated with wheat gluten. The pH-optimum of activity of these aspartic proteinases is below 4.0 (2, 3). Accordingly, the proteolytic activity in wheat doughs is strongly increased in acidified doughs independent of the presence of lactic acid bacteria (32, 33) and prote...

Metatranscriptome Analysis for Insight into Whole-Ecosystem Gene Expression during Spontaneous Wheat and Spelt Sourdough Fermentations

“…In the present study, no expression of the only proteinase represented on the microarray was found, namely, of the cell wall-associated serine proteinase gene. This confirmed that proteinase activity might not be of utmost importance for the microbiota present, as a sufficient amount of peptides is already generated by the action of flour proteinases (65,76). However, the LAB functional gene microarray did not contain oligonucleotides targeting this gene in L. plantarum or L. fermentum, and so no clear conclusion can be drawn regarding the importance of extracellular proteolytic activity for competitiveness, although strains of these species elaborating proteinase activities have been isolated from sourdough (14,21).…”

“…In general, peptides present in a food matrix (e.g., milk) are taken up by LAB and are degraded intracellularly (38). The resulting amino acids may be used for biosynthesis or other metabolic activities (12,17,65). In the case of sourdough LAB, in particular, it has been shown that accumulated amino acids that are not further metabolized, such as phenylalanine, are transported out of the cell (66).…”

Population Dynamics and Metabolite Target Analysis of Lactic Acid Bacteria during Laboratory Fermentations of Wheat and Spelt Sourdoughs

“…This is reflected by the considerable levels of maltose retrieved after each fermentation step and an increase of total amino acids upon fermentation (27,39,40,69). The present study showed that the competitiveness of sourdough LAB is enhanced by both the use of the arginine deiminase pathway (in an early stage) and the use of alternative electron acceptors.…”

“…Lactic acid bacteria (LAB) and yeasts play a key role in sourdough fermentation processes (9,21,26,28,29,55). Sourdough LAB have been intensively studied with respect to their carbohydrate metabolism (16,24,60), proteolysis and amino acid metabolism (16,23,58,69), lipid metabolism (16), and production of volatile compounds (7,31,32). Besides these general metabolic traits, specific metabolic properties have been recognized in sourdough LAB, such as the use of alternative electron acceptors (59, 61), the production of antifungal compounds (38,45,57), the biosynthesis of exopolysaccharides (36, 63, 64), and arginine catabolism (8, 53).…”

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology

“…4) (69). Extracellular protease activity is required for growth in milk but not for growth in other substrates (68,70,71). Accordingly, the gene coding for the extracellular protease PrtH was frequently found in dairy isolates of the L. delbrueckii, L. casei, L. salivarius, and L. buchneri groups but was rarely present in other lactobacilli.…”