Browsing by Subject "Milchsäurebakterien"

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Impact of process parameters on the sourdough microbiota, selection of suitable starter strains, and description of the novel yeast Cryptococcus thermophilus sp. nov.
(2013) Vogelmann, Stephanie Anke; Hertel, Christian
The microbiota of a ripe sourdough consists of lactic acid bacteria (LAB), especially of the genus Lactobacillus, and yeasts. Their composition is influenced by the interplay of species or strains, the kind of substrate as well as the process parameters temperature, dough yield, redox potential, refreshment time, and number of propagation steps (Hammes and Gänzle, 1997). As taste and quality of sourdough breads are mainly influenced by the fermentation microbiota, intense research has been focused on determination of sourdough associated species and search for new starter cultures. In recent years, economic competition pressure and new consumer demands have led to steady research for new cereal products, especially with health benefit or for people suffering from celiac disease. For these reasons, alternative cereals like oat and barley (both toxic for celiac disease patients) as well as the celiac disease compatible cereals rice and maize, sorghum and millets, the pseudocereals amaranth, quinoa and buckwheat as well as cassava got into the focus of interest. However, information about the microbiota of sourdoughs fermented with buckwheat, amaranth, quinoa, oat or barley is not available except for the following recent studies: a study about the microbiota of amaranth sourdoughs by Sterr et al. (2009), a study about barley sourdough by Zannini et al. (2009), a study about oat sourdoughs by Huettner et al. (2010) and a study about buckwheat and teff sourdoughs by Moroni et al. (2011). The microbiota of sourdoughs from the other mentioned cereals as well as cassava was multiply characterised but not systematically. Fermentation conditions were partly not clearly defined, and identification of species was often based on physiological criteria only, known to be insufficient for the exact classification of LAB. Thus, in this thesis, the influence of the process parameters substrate, temperature, refreshment time, amount of backslopping dough as well as the interplay between the different species or strains were examined and potential starter strains were selected. In Chapter III, the effect of the substrate on the sourdough microbiota was examined and suitable starter cultures for fermentation of non-bread cereals and pseudocereals were selected. Eleven different flours from wheat, rye, oat, barley, millet, rice, maize, amaranth, quinoa, buckwheat and cassava were inoculated with a starter mixture containing numerous LAB and yeasts. Sourdoughs were fermented at 30 °C and refreshed every 24 hours until the microbiota was stable. Species were identified by PCR-DGGE as well as bacteriological culture and RAPD-PCR, followed by 16S/26S rRNA sequence analysis. In these fermentations, the dominant yeast was Saccharomyces cerevisiae; Issatchenkia (I.) orientalis was only competitive in the quinoa and the maize sourdough. No yeasts were found in the buckwheat and the oat sourdough. The dominant LAB species were Lactobacillus (L.) paralimentarius in the pseudocereal sourdoughs, L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs, and L. fermentum, L. plantarum and L. spicheri in the cassava sourdough. Competitive LAB and yeasts were inserted as starters for a further fermentation using new flours from rice, maize, millet and the pseudocereals. After ten days of fermentation, most of the starter strains were still dominant, but L. pontis and L. helveticus could not compete with the other species. It is remarkable that from the numerous starter strains which all were adapted to or isolated from sourdoughs, only a few were competitive in these fermentations; but if, then in most cases in a lot of different flours. In Chapter IV, the effects of the exogenous process parameters substrate, refreshment time, temperature, amount of backslopping dough as well as competing species on the two microbial associations L. sanfranciscensis ? Candida (C.) humilis and L. reuteri ? L. johnsonii ? I. orientalis were examined. Both associations had previously been found to be competitive in sourdough (Kline and Sugihara, 1971a; Nout and Creemers-Molenaar, 1987; Gobbetti et al., 1994a; Garofalo et al., 2008; Böcker et al., 1990; Meroth et al., 2003a). 28 sourdough batches were fermented under defined conditions until the microbiota was stable. Dominant LAB and yeasts were characterized by bacteriological culture, RAPD-PCR and 16S/26S rRNA gene sequence analysis. The process parameters for the association L. sanfranciscensis ? C. humilis could be defined as follows: rye bran, rye flour or wheat flour as substrate, temperatures between 20 and 30 °C, refreshment times of 12 to 24 hours and amounts of backslopping dough from 5 to 20 %. In addition, the association was predominating against all competing lactic acid bacteria and yeasts. The association L. reuteri ? L. johnsonii ? I. orientalis was competitive at temperatures of 35 to 40 °C, refreshment times of 12 to 24 hours and the substrates rye bran, wheat flour and rye flour, but only with sufficient oxygen supply. Cell counts of I. orientalis fell rapidly under the detection limit when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 hours. The fermentations depicted in Chapter III and IV give new information about the influence of process parameters on the sourdough microbiota. The studies show that the sourdough microbiota is markedly influenced by the process parameters and kind and quality of substrate. The competitiveness of a single LAB or yeast is strain specific. Interactions between microorganisms also play an important role. However, for the search for suitable starter strains, it would be beneficial to know the reasons, why a single LAB or yeast strain is better adapted to specific process parameters or substrates than others. One of the starter sourdoughs used for fermentation I described in Chapter III was a sourdough made from cassava flour, inoculated with several LAB. No yeast had been inserted, but several yeasts were isolated from the ripe sourdough, which are supposed to originate from the cassava flour. An unknown yeast species constituted 10 % of the isolated yeasts which is described as novel species Cryptococcus thermophilus sp. nov. in Chapter V. This yeast is characterized by budding on small neck-like structures, no fermentative ability, growth at 42 °C and without vitamins, a major ubiquinone of Q-10, as well as the production of green or blue fluorescent substances in the growth medium. It is distinct from related species by the ability to assimilate raffinose and cadaverine, the inability to assimilate soluble starch, xylitol, galactitol, butane-2,3-diol, sodium nitrite and lysine, and the inability to produce starch-like substances. The closest relatives are the yeasts belonging to the Cryptococcus humicola complex.
Molekulare Interaktionen von Milchsäurebakterien mit enterohämorrhagischen Escherichia coli und humanen Darmepithelzellen
(2011) Stöber, Helen; Schmidt, Herbert
The interactions of 19 benign strains of lactic acid bacteria, bifidobacteria and staphylococci with five enterohemorrhagic Escherichia coli (EHEC) strains of different serotypes and virulence gene spectrum were investigated using a HT29 cell culture infection model. As a parameter for the infection the secretion of Interleukin 8 (IL-8) of the infected cells was analyzed by ELISA. None of the used benign strains induced an IL-8 secretion, whereas the infection with the EHEC strains leads ? independent of their virulence profile - to high amounts of IL-8. In coinfection assays with the pathogen EDL933 (O157:H7) and different test strains the secretion of IL-8 of the cultured cells was decreased by a few strains. With 12 of 19 tested strains, a weak reduction < 30 % of IL-8 secretion of HT29 cells after coinfection with EHEC O157:H7 strain EDL933 was observed. Six strains reduced the IL-8 secretion up to 60 % and the strain B. breve DSMZ 20083 decreased the IL-8 production about 73 %. Coinfection assays with different strains of one species (B. adolescentis DSMZ 20083 and DSMZ 20086 as well as L. johnsonii BFE 633 and DSMZ 10533) showed the strain specificity of the observed anti-inflammatory effect, due to different capabilities of IL-8 reduction. In further coinfection assays with different EHEC strains of the serotypes O103:H2, O26:H-, 0157:H- and O113:H21 different abilities of the benign strains to influence the infection with the different pathogen strains were noted. Therefore the protective anti-inflammatory effect is strain specific for the tested benign bacteria and also depends on the application of EHEC strains with different sero- and virulence types. Further investigations indicated the imperative of living bacteria for the observed protective effect; neither culture supernatant nor inactivated bacteria showed an effect on the IL-8 secretion of the EDL933 infected HT29 cells. The analysis of the cell culture supernatants 6 h after infection with different bacteria detected the production of lactic and acetic acid. The application of these acids in infection assays with EDL933 did not lead to an reduced IL-8 secretion of the infected cells. Therefore the production of organic acids did not explain the protective effect. The induction of IL-8 could not be traced back to the influence of a single virulence factor. Four PMK5 strains with deletions in different virulence genes induced similar IL-8 secretions in comparison to cells infected with the wild-type strain. Coinfection assays with the mutants and S. pasteuri LTH 5211 showed also similar IL-8 reductions than coinfection assays with the wild-type strain. It is to suppose that the anti-inflammatory effects of the benign bacteria do not influence a single virulence factor of the tested EHEC strains. As a second parameter the activation of the transcription factor ?Nuclear Factor kappa B? (NF-κB) of coinfected HT29 cells was monitored using a reporter-genassay. In comparison to the single EHEC-infection, the NF-κB activation was reduced by all tested lactic acid bacteria, bifidobacteria and S. pasteuri LTH 5211 in coinfection trials significantly. No strain-specificity and no pathogen-specificity could be observed. Interestingly, stimulation of the HT29 cells with benign bacteria led to inhibition of NF-κB activity, the measured values were less than the values of the negative control PBS. A gene expression analysis of toll-like receptors (TLRs), recognizing bacteria on cell surfaces and initiating the immune response, showed no regulation for TLR2. Infection with EDL933 led to down regulation of TLR4 and to up regulation of TLR9. Stimulation with L. rhamnosus GG, L. johnsonii DSMZ 10533 or L. fermentum DSMZ 20052 led neither to regulation of TLR4 nor TLR9. The benign bacteria did not influence the EHEC-induced TLR4 regulation in coinfection trials; in contrast the regulation of TLR9 was reduced significantly. The model described here is useful for screening basic effects of protective bacteria that are able to counteract EHEC-mediated effects on human cells and to study the molecular interaction between bacteria as well as between bacteria and human cultured cells.
The production of melezitose in honeydew and its impact on honey bees (Apis mellifera L.)
(2021) Seeburger, Victoria; Hasselmann, Martin
Honeydew honey is a honey type which is of high economic importance in Europe. Phloem sap feeding insects of the order Hemiptera (true bugs) excrete honeydew, the key component of honeydew honey. Beekeepers move their hives between forest regions so that their bees can process the honeydew into honey. In case of high osmolality in the phloem sap of the hemipterans’ host trees, they counteract osmotic pressure by osmoregulation and produce oligosaccharides such as melezitose. Melezitose-rich honeydew honey is a major issue for beekeepers; it crystallises and obstructs the combs, leading to an economical loss. Nevertheless, precise analyses of the conditions of the occurrence of melezitose have not been realised. Furthermore, it is not known which impacts the trisaccharide has on honey bee health and the honeydew flow disease documented in beekeepers’ journals can have one explanation in the nutrition on melezitose. In order to determine influence factors for the emergence of melezitose, more than 600 honeydew droplets from defined honeydew producer species were collected under different environmental conditions (hemipteran species (host tree specific), natural area, air temperature, relative humidity, altitude, time of the year and of the day) between 2016 and 2019. The sugar spectra were analysed by high performance anion exchange chromatography with pulsed amperometric detection. To obtain the impact of melezitose on honey bee health, additional feeding experiments with daily evaluation of food uptake, gut-body weight ratio and mortality have been realised between 2017 and 2019. Additionally, comprehensive 16S rRNA Illumina sequencing of the gut microbial community has been performed. Remarkable differences could be found in the amount of melezitose between honeydew samples collected from different honeydew producer species and according to different environmental conditions. Air temperature increases and decreases in relative humidity increased the melezitose production in honeydew by the observed seven hemipteran species. Both, scale insect species on Picea abies and aphid species on Abies alba produced significantly less honeydew containing melezitose than aphid species on Picea abies. Additionally, honeydew with increased melezitose content was significantly more frequent collected in natural areas with limited water reservoir capacities, at higher altitudes and years with low precipitation. All results lead to the conclusion that hemipteran species produce more melezitose when the host trees have less access to water, increasing the osmolality of the phloem sap and indirectly enhancing the osmoregulation with producing melezitose by hemipteran species. Bees fed with melezitose showed increased food uptake and higher gut-body weight ratio than the control groups. Furthermore, melezitose feeding caused disease symptoms such as swollen abdomen, abdomen tipping and impaired movement and a significantly higher mortality than in control groups. Gut microbiota analyses indicated a shift of the bacterial species Lactobacillus Firm-4 and Lactobacillus kunkeei in favour of Lactobacillus Firm-5 in melezitose fed bees. This PhD project provides the important knowledge about the indicators that point out an enhanced melezitose production. This is a valuable contribution to design a warning system for beekeepers that will help to prevent harmful nutrition for honey bees or crystallised honey in the future by timely removal of bee colonies from local regions at risk. Additionally, feeding experiments point out the high effort that is required for the degradation process of the large-molecule melezitose. This effort might lead to a higher uptake of food, heavier guts, shorter lifespan and a higher susceptibility to intestinal diseases. Finally, an evidence was presented that the lactic acid bacteria of the gut microbiota are significantly involved in the digestion of melezitose.