Characterisation of recombinant GH 3 ß-glucosidase from ß-glucan producing Levilactobacillus brevis TMW 1.2112.

Levilactobacillus (L.) brevis TMW 1.2112 is an isolate from wheat beer that produces O2-substituted (1,3)-ß-D-glucan, a capsular exopolysaccharide (EPS) from activated sugar nucleotide precursors by use of a glycosyltransferase. Within the genome sequence of L. brevis TMW 1.2112 enzymes of the glycoside hydrolases families were identified. Glycoside hydrolases (GH) are carbohydrate-active enzymes, able to hydrolyse glycosidic bonds. The enzyme ß-glucosidase BglB (AZI09_02170) was heterologous expressed in Escherichia coli BL21. BglB has a monomeric structure of 83.5 kDa and is a member of the glycoside hydrolase family 3 (GH 3) which strongly favoured substrates with ß-glycosidic bonds. Km was 0.22 mM for pNP ß-D-glucopyranoside demonstrating a high affinity of the recombinant enzyme for the substrate. Enzymes able to degrade the (1,3)-ß-D-glucan of L. brevis TMW 1.2112 have not yet been described. However, BglB showed only a low hydrolytic activity towards the EPS, which was measured by means of the D-glucose releases. Besides, characterised GH 3 ß-glucosidases from various lactic acid bacteria (LAB) were phylogenetically analysed to identify connections in terms of enzymatic activity and ß-glucan formation. This revealed that the family of GH 3 ß-glucosidases of LABs comprises most likely exo-active enzymes which are not directly associated with the ability of these LAB to produce EPS.

Proteomic Analysis Reveals Enzymes for ß-D-Glucan Formation and Degradation in Levilactobacillus brevis TMW 1.2112.

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-ß-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, ß-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the ß-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for ß-glucan biosynthesis involved a ß-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the ß-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples.

Persistence and ß-glucan formation of beer-spoiling lactic acid bacteria in wheat and rye sourdoughs.

Some beverage-spoiling lactic acid bacteria (LAB) produce capsular ß-glucans from UDP-glucose, which is accompanied by cell network formation causing viscosity increases of liquids. This feature of certain LAB is feared in breweries but could be useful for structural and nutritional improvement of baked goods, provided that these LAB are suited for the manufacture of sourdoughs. The aim of this study was to investigate the persistence and ß-glucan formation of the brewery isolates Levilactobacillus (L.) brevis TMW 1.2112 and Pediococcus (P.) claussenii TMW 2.340 in wheat and rye sourdoughs. Both the wild-type strains and the respective ß-glucan-deficient mutants were dominant in wheat and rye sourdoughs and acidified them to characteristic pH ranges. The formation of ß-glucan capsules during sourdough fermentations was stable in L. brevis TMW 1.2112 in contrast to P. claussenii TMW 2.340. Wheat sourdoughs fermented with the ß-glucan producing L. brevis TMW 1.2112 cells were significantly more viscous than doughs fermented by the P. claussenii TMW 2.340 cells and the applied mutant strains. In conclusion, L. brevis TMW 1.2112 and P. claussenii TMW 2.340 were suited and persistent wheat and rye sourdough starters, while the in situ ß-glucan formation in sourdoughs was hardly detectable in case of P. claussenii.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

ß-Glucan Production by Levilactobacillus brevis and Pediococcus claussenii for In Situ Enriched Rye and Wheat Sourdough Breads.

Sourdough fermentation is a common practice spread across the globe due to quality and shelf life improvement of baked goods. Above the widely studied exopolysaccharide (EPS) formation, which is exploited for structural improvements of foods including baked goods, ß-glucan formation, by using lactic acid bacteria (LAB), offers additional values. Through renunciation of sucrose addition for bacterial ß-d-glucan formation, which is required for the production of other homopolysaccharides, residual sweetness of baked goods can be avoided, and predicted prebiotic properties can be exploited. As promising starter cultures Levilactobacillus (L.) brevis TMW (Technische Mikrobiologie Weihenstephan) 1.2112 and Pediococcus (P.) claussenii TMW 2.340 produce O2-substituted (1,3)-ß-d-glucan upon fermenting wheat and rye doughs. In this study, we have evaluated methods for bacterial ß-glucan quantification, identified parameters influencing the ß-glucan yield in fermented sourdoughs, and evaluated the sourdough breads by an untrained sensory panel. An immunological method for the specific detection of ß-glucan proved to be suitable for its quantification, and changes in the fermentation temperature were related to higher ß-glucan yields in sourdoughs. The sensory analysis resulted in an overall acceptance of the wheat and rye sourdough breads fermented by L.brevis and P.claussenii with a preference of the L. brevis fermented wheat sourdough bread and tart-flavored rye sourdough bread.

Complete Genome Sequence and Annotation of the Paracoccus pantotrophus Type Strain DSM 2944.

Paracoccus spp. are metabolically versatile alphaproteobacteria able to perform heterotrophic and chemoautotrophic growth. This study describes the whole-genome sequence of the Paracoccus pantotrophus type strain DSM 2944 (ATCC 35512, LMD 82.5, GB17). The genome sequence revealed the presence of a complete phaZ phaC phaP phaR gene cluster related to polyhydroxyalkanoate metabolism.