Clarissa Schwab

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen

Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.

Phylotype-level 16S rRNA analysis reveals new bacterial indicators of health state in acute murine colitis.

Human inflammatory bowel disease and experimental colitis models in mice are associated with shifts in intestinal microbiota composition, but it is unclear at what taxonomic/phylogenetic level such microbiota dynamics can be indicative for health or disease. Here, we report that dextran sodium sulfate (DSS)-induced colitis is accompanied by major shifts in the composition and function of the intestinal microbiota of STAT1−/− and wild-type mice, as determined by 454 pyrosequencing of bacterial 16S rRNA (gene) amplicons, metatranscriptomics and quantitative fluorescence in situ hybridization of selected phylotypes. The bacterial families Ruminococcaceae, Bacteroidaceae, Enterobacteriaceae, Deferribacteraceae and Verrucomicrobiaceae increased in relative abundance in DSS-treated mice. Comparative 16S rRNA sequence analysis at maximum possible phylogenetic resolution identified several indicator phylotypes for DSS treatment, including the putative mucin degraders Akkermansia and Mucispirillum. The analysis additionally revealed strongly contrasting abundance changes among phylotypes of the same family, particularly within the Lachnospiraceae. These extensive phylotype-level dynamics were hidden when reads were grouped at higher taxonomic levels. Metatranscriptomic analysis provided insights into functional shifts in the murine intestinal microbiota, with increased transcription of genes associated with regulation and cell signaling, carbohydrate metabolism and respiration and decreased transcription of flagellin genes during inflammation. These findings (i) establish the first in-depth inventory of the mouse gut microbiota and its metatranscriptome in the DSS colitis model, (ii) reveal that family-level microbial community analyses are insufficient to reveal important colitis-associated microbiota shifts and (iii) support a scenario of shifting intra-family structure and function in the phylotype-rich and phylogenetically diverse Lachnospiraceae in DSS-treated mice.

Exopolysaccharide-Forming Weissella Strains as Starter Cultures for Sorghum and Wheat Sourdoughs

The addition of sourdough fermented with lactic acid bacteria synthesizing organic acids and oligo- and exopolysaccharides (EPS) from sucrose enhances texture, nutritional value, shelf life, and machinability of wheat, rye, and gluten-free bread. This study compared acetate, mannitol, and oligosaccharide formation of EPS-producing strains of Weissella and Leuconostoc spp. to the traditional sourdough starter Lactobacillus sanfranciscensis. In broth, Leuconostoc strains generally formed acetate and mannitol, whereas Weissella produced only small amounts of acetate and no mannitol in the presence of sucrose. In the presence of sucrose and maltose, Weissella and Leuconostoc strains synthesized glucooligosaccharides and EPS. Strains of Weissella were employed as starter cultures for wheat and sorghum sourdough and formed 0.8-8 g kg(-1) EPS and gluco-oligosaccharides but only low amounts of acetate and mannitol. In contrast, the formation of EPS from sucrose led to the production of high amounts of acetate and mannitol by L. sanfranciscensis LTH 2950 in wheat sourdough. This study indicates that Weissella strains are suitable starter cultures for wheat and sorghum sourdoughs and efficiently produce gluco-oligosaccharides and EPS.

Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract.

Members of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii #21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.

Influence of in-situ synthesized exopolysaccharides on the quality of gluten-free sorghum sourdough bread.

The majority of gluten-free breads on the market are of poor sensory and textural quality. Exopolysaccharides (EPS) formed from sucrose during sourdough fermentation can improve the technological properties of gluten-free breads and potentially replace hydrocolloids. In this study, the influence of in situ formed EPS on dough rheology and quality of gluten-free sorghum bread was investigated. Dextran forming Weissella cibaria MG1 was compared to reuteran producing Lactobacillus reuteri VIP and fructan forming L. reuteri Y2. EPS containing bread batters were prepared by adding 10% and 20% of sourdough. As control served batters and bread containing sourdoughs fermented without sucrose and batters and bread without sourdough addition. The amount of EPS formed in situ ranged from 0.6 to 8.0 g/kg sourdough. EPS formed during sourdough fermentation were responsible for the significant decrease in dough strength and elasticity, with in situ formed dextran exhibiting the strongest impact. Increased release of glucose and fructose from sucrose during fermentation enhanced CO₂ production of yeast. Organic acids in control sourdough breads induced hardening of the bread crumb. EPS formed during sourdough fermentation masked the effect of the organic acids and led to a softer crumb in the fresh and stored sorghum bread. Among EPS, dextran showed the best shelf life improvements. In addition to EPS, all three strains produced oligosaccharides during sorghum sourdough fermentation contributing to the nutritional benefits of gluten-free sorghum bread. Results of this study demonstrated that EPS formed during sourdough fermentation can be successfully applied in gluten-free sorghum flours to improve their bread-making potentials.

Formation of Oligosaccharides and Polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10M in Sorghum Sourdoughs

ABSTRACT Gluten-free breads, which are composed of gluten-free flours, starch, and hydrocolloids, differ from wheat and rye breads in relation to texture, volume, and crumb structure. Moreover, the dietary fiber content is lower compared with wheat or rye breads. Cereal isolates of lactic acid bacteria frequently produce oligo- and homopolysaccharides from sucrose, which can improve the nutritional and technological properties of gluten-free breads as prebiotic carbohydrates and hydrocolloids, respectively. Sorghum sourdough was fermented with Lactobacillus reuteri LTH5448 or Weissella cibaria 10M, which synthesize fructooligosaccharides (FOS) and levan, and isomaltooligosaccharides and dextran, respectively. The gluten-free bread was produced with 14% sourdough addition. L. reuteri LTH5448 formed FOS and 1.5 g of levan/kg DM in quinoa sourdoughs. FOS were digested by the bakers yeast during proofing, and the levan could be qualitatively detected in the bread. W. cibaria 10M produced >60 g of isomaltooli...

Longitudinal study of murine microbiota activity and interactions with the host during acute inflammation and recovery

Although alterations in gut microbiota composition during acute colitis have been repeatedly observed, associated functional changes and the recovery from dysbiosis received little attention. In this study, we investigated structure and function of the gut microbiota during acute inflammation and recovery in a dextran sodium sulfate (DSS)-colitis mouse model using metatranscriptomics, bacterial 16S rRNA gene amplicon sequencing and monitoring of selected host markers. Parallel to an increase of host markers of inflammation during acute colitis, we observed relative abundance shifts and alterations in phylotype composition of the dominant bacterial orders Clostridiales and Bacteroidales, and an increase of the low abundant Enterobacteriales, Deferribacterales, Verrucomicrobiales and Erysipelotrichales. During recovery, the microbiota began to resume, but did not reach its original composition until the end of the experiment. Microbial gene expression was more resilient to disturbance, with pre-perturbation-type transcript profiles appearing quickly after acute colitis. The decrease of Clostridiales during inflammation correlated with a reduction of transcripts related to butyrate formation, suggesting a disturbance in host-microbe signalling and mucosal nutrient provision. The impact of acute inflammation on the Clostridiales was also characterized by a significant downregulation of their flagellin-encoding genes. In contrast, the abundance of members of the Bacteroidales increased along with an increase in transcripts related to mucin degradation. We propose that acute inflammation triggered a selective reaction of the immune system against flagella of commensals and temporarily altered murine microbiota composition and functions relevant for the host. Despite changes in specific interactions, the host–microbiota homeostasis revealed a remarkable ability for recovery.

Lactic acid bacteria fermentation of human milk oligosaccharide components, human milk oligosaccharides and galactooligosaccharides

Human milk contains about 7% lactose and 1% human milk oligosaccharides (HMOs) consisting of lactose with linked fucose, N-acetylglucosamine and sialic acid. In infant formula, galactooligosaccharides (GOSs) are added to replace HMOs. This study investigated the ability of six strains of lactic acid bacteria (LAB), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus and Leuconostoc mesenteroides subsp. cremoris, to digest HMO components, defined HMOs, and GOSs. All strains grew on lactose and glucose. N-acetylglucosamine utilization varied between strains and was maximal in L. plantarum; fucose utilization was low or absent in all strains. Both hetero- and homofermentative LAB utilized N-acetylglucosamine via the Embden-Meyerhof pathway. Lactobacillus acidophilus and L. plantarum were the most versatile in hydrolysing pNP analogues and the only strains releasing mono- and disaccharides from defined HMOs. Whole cells of all six LAB hydrolysed oNP-galactoside and pNP-galactoside indicating β-galactosidase activity. High β-galactosidase activity of L. reuteri, L. fermentum, S. thermophilus and L. mesenteroides subsp. cremoris whole cells correlated to lactose and GOS hydrolysis. Hydrolysis of lactose and GOSs by heterologously expressed β-galactosidases confirmed that LAB β-galactosidases are involved in GOS digestion. In summary, the strains of LAB used were not capable of utilizing complex HMOs but metabolized HMO components and GOSs.

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth. In conclusion, cofermentation with L. buchneri and L. diolivorans represents a process to increase antifungal capacities of bread.

Structural and rheological characterisation of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sourdough.

Hydrocolloids improve the volume, texture, and shelf life of bread. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough fermentation can replace hydrocolloids. It was the aim of this study to determine whether heteropolysaccharides (HePS) synthesized intracellularly from sugar nucleotides by glycosyltransferases are produced in wheat and gluten-free sorghum sourdough at effective levels. The HePS-producing strains Lactobacillus casei FUA3185, L. casei FUA3186, and Lactobacillus buchneri FUA3154 were used; Weissella cibaria 10M producing no EPS in the absence of sucrose served as control strain. Cell suspensions of L. buchneri in MRS showed the highest viscosity at low shear rate. Glycosyltransferase genes responsible of HePS formation in LAB were expressed in sorghum and wheat sourdough. However, only HePS produced by L. buchneri influenced the rheological properties of sorghum sourdoughs but not of wheat sourdoughs. Sorghum sourdough fermented with L. buchneri exhibited a low |G*| compared to the control, indicating a decrease in resistance to deformation. An increase in tan δ indicated decreased elasticity. The use of LAB producing HePS expands the diversity of EPS and increases the variety of cultures for use in baking.