Browsing by Subject "Probiotics"

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Application of two-dimensional fluorescence spectroscopy for the on-line monitoring of teff-based substrate fermentation inoculated with certain probiotic bacteria
(2022) Alemneh, Sendeku Takele; Emire, Shimelis Admassu; Jekle, Mario; Paquet-Durand, Olivier; von Wrochem, Almut; Hitzmann, Bernd
There is increasing demand for cereal-based probiotic fermented beverages as an alternative to dairy-based products due to their limitations. However, analyzing and monitoring the fermentation process is usually time consuming, costly, and labor intensive. This research therefore aims to apply two-dimensional (2D)-fluorescence spectroscopy coupled with partial least-squares regression (PLSR) and artificial neural networks (ANN) for the on-line quantitative analysis of cell growth and concentrations of lactic acid and glucose during the fermentation of a teff-based substrate. This substrate was inoculated with mixed strains of Lactiplantibacillus plantarum A6 (LPA6) and Lacticaseibacillus rhamnosus GG (LCGG). The fermentation was performed under two different conditions: condition 1 (7 g/100 mL substrate inoculated with 6 log cfu/mL) and condition 2 (4 g/100 mL substrate inoculated with 6 log cfu/mL). For the prediction of LPA6 and LCGG cell growth, the relative root mean square error of prediction (pRMSEP) was measured between 2.5 and 4.5%. The highest pRMSEP (4.5%) was observed for the prediction of LPA6 cell growth under condition 2 using ANN, but the lowest pRMSEP (2.5%) was observed for the prediction of LCGG cell growth under condition 1 with ANN. A slightly more accurate prediction was found with ANN under condition 1. However, under condition 2, a superior prediction was observed with PLSR as compared to ANN. Moreover, for the prediction of lactic acid concentration, the observed values of pRMSEP were 7.6 and 7.7% using PLSR and ANN, respectively. The highest error rates of 13 and 14% were observed for the prediction of glucose concentration using PLSR and ANN, respectively. Most of the predicted values had a coefficient of determination (R2) of more than 0.85. In conclusion, a 2D-fluorescence spectroscopy combined with PLSR and ANN can be used to accurately monitor LPA6 and LCGG cell counts and lactic acid concentration in the fermentation process of a teff-based substrate. The prediction of glucose concentration, however, showed a rather high error rate.
Probiotic bacteria enhance the antibacterial barrier of enterocytes: insights into their mechanism of action
(2007) Schlee, Miriam; Bode, Christiane
In the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, and immunoregulatory factors may precipitate the onset of ulcerative colitis (UC) and Crohn's disease (CD), the primary manifestations of inflammatory bowel disease (IBD). It is currently believed that IBD results from an aberrant immune response of the intestinal mucosa towards the normal commensal bacterial flora. Alternatively, a primary defect in the mucosal barrier might permit bacterial invasion and trigger inflammation. In our research group the hypothesis was proposed that the defective barrier in Crohn´s disease may be due to a lack of defensins which form a chemical barrier against luminal bacteria. A major gut defensin is the human beta defensin-2 (hBD-2) which is an inducible antimicrobial peptide synthesized and secreted by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. In recent studies, Fellermann et al demonstrated that the defective expression of hBD-2 which was measured in the gut mucosa of patients with Crohn´s disease was due to a reduced copy number of the hBD-2 gene. In patients with ulcerative colitis beta-defensin expression is low in the colon during remission, but readily inducible during inflammation. Probiotic bacteria might act beneficially in the human gut by inducing the expression of defensins and thereby reinforcing the mucosal barrier. Recently, our group has been the first to describe hBD-2 induction by the probiotic strain E. coli Nissle (Mutaflor®) which is an effective treatment for ulcerative colitis during remission. The aim of the present work was to determine the underlying molecular mechanisms. We determined a time- and dose-dependent expression pattern of hBD-2 in Caco-2 cells upon stimulation with IL-1 beta;, E. coli Nissle culture supernatant and diverse other probiotic strains. We further investigated the transcriptional regulation of hBD-2 expression mediated by probiotics. The hBD-2 promoter contains several elements known to be involved in transcriptional upregulation such as the NF-kappa B element, which is believed to be one of the main regulators of the hBD-2 gene expression. However, for certain signals, the expression of the hBD-2 gene has been reported to depend on the activation of a second transcription factor, such as AP-1. Most importantly, E. coli Nissle was shown to shed or secrete factors, contained in the bacterial supernatant, which were sufficient to trigger activation of NF-kappa B and AP-1 and to induce hBD-2. Our results indicated further that the supernatant-induced activation of the MAP kinase pathways ERK½, JNK, and p38 may be directly responsible for the probiotic supernatant-induced activation of the transcription factors AP-1 and NF-kappa B and subsequent synthesis of hBD-2. A further aim of the present study was to identify and isolate the bacterial components which are responsible for E. coli Nissle mediated hBD-2 induction. As E. coli Nissle culture supernatant was found to be a more potent stimulant than the bacterial pellet, we investigated the characteristics of the unknown soluble or shed molecules in the bacterial culture media. The first analysis revealed the factor as a heat resistant and proteinase sensitive molecule. Both, E. coli Nissle specific lipopolysaccharide (LPS) and bacterial DNA, which might contain immunostimulatory DNA motifs, failed to trigger hBD-2 expression. Based on the knowledge of the surface composition several E. coli Nissle deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin, the flagella filament protein, specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Next, we isolated flagellins from different bacteria strains (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle and the uropathogenic E. coli strain CFT073 Delta hly, whose genome structure resembles closely that of E. coli Nissle). In the Western blot anti-H1 flagellin displayed immunoreactivity against the different types of flagellins, due to the highly conserved central region of the flagellin filament structure. Incubation of Caco-2 cells with isolated E. coli Nissle flagellin (molecular size 60.81 kDa) induced hBD-2 promoter activation in a dose-dependent manner. The induction of hBD-2 expression by flagellin was confirmed with a positive control (Salmonella flagellin). Interestingly, the serotype-identical CFT073 Delta hly flagellin expressed only moderate hBD-2 inducing ability compared to E. coli Nissle flagellin. Thus, differences in extracellular matrix e.g. the glycosilation degree might underlie the differentially modulated hBD-2 response of Caco-2 cells by the two flagellins. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle. In conclusion, flagellin of E. coli Nissle provides reinforcement of mucosal antimicrobial function, apparently without inducing inflammation. This might explain the beneficial effects of E. coli Nissle on remission maintenance in ulcerative colitis. In patients with Crohn´s disease there is evidence against a therapeutic effect of probiotics and this may be explained by a defective defensin system. Future investigations about strain-specific beneficial functions might contribute to the therapeutic application of science-based probiotic products.