Browsing by Subject "Short-chain fatty acids"

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    Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
    (2023) Trautmann, Andrej; Seifert, Jana
    The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.
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    Einfluss der mediterranen Ernährung auf das Fettsäuremuster von Erythrozytenmembranen sowie auf Darmmikrobiota- und Darmbarriere-assoziierte Biomarker : Mechanismen und klinische Anwendungen
    (2022) Seethaler, Benjamin; Bischoff, Stephan C.
    Dissertation from Benjamin Seethaler: "Effect of the Mediterranean diet on the fatty acid pattern of erythrocyte membranes and on gut microbiota- and gut barrier-associated biomarkers - mechanisms and clinical applications". In summary, the results of the PhD project offer new insights into the biomedical mechanisms of action and health effects of the Mediterranean diet. Of particular importance is the relationship we have shown between dietary fiber from the Mediterranean diet, its fermentation to short-chain fatty acids, and its beneficial influence on impaired intestinal barrier function. In the future, our studies may provide the basis for personalized nutritional therapy to improve impaired gut barrier function in high-risk breast cancer patients. Furthermore, we were able to establish LBP and zonulin as biomarkers to detect gut barrier function and to determine and assess gut barrier disorders. This method validation simplifies or enables the assessment of intestinal barrier function in clinical practice or clinical trials.
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    Versuche zur Verbesserung der sensorischen Fleischqualität beim Schwein durch nutritive Hemmung der Skatolbildung
    (2006) Lösel, Dorothea; Claus, Rolf
    Skatole is known to occur in pork carcasses. Due to its pronounced faecal odour, it may have an adverse effect on sensorial quality of pork. Skatole has been associated with boar taint, but is also formed in the large intestine of barrows and gilts by bacterial degradation of tryptophan. As apoptotic cells from the gut mucosa are the main source of tryptophan, skatole formation is partly linked to the degree of cell renewal in the gut. A part of microbially formed skatole is absorbed and stored in adipose tissue due to its lipophilic properties. In earlier studies it has been shown that intestinal butyrate formation increased by feeding resistant potato starch. This short-chain fatty acid reduced the apoptotic activity in the gut mucosa and thus decreased skatole formation and tissue accumulation. Therefore, it was the aim of the present study to investigate whether this method is applicable to improve sensorial quality of pork under routine fattening conditions. In the first experiment the amount of potato starch required for sufficient reduction of skatole was determined. Four groups of pigs received diets containing different amounts of potato starch (0 % as a control, 20 %, 30 %, 40 %) for 2-3 weeks prior to slaughter. Skatole concentrations in gut content (proximal and distal colon, rectum) and adipose tissue (flare fat and ventral fat) were determined by High Performace Liquid Chromatography. An amount of 30 % in the diet reduced skatole concentrations in gut content and adipose tissue compared to the control. The reduction was 98 % in the proximal colon, 93 % in the distal colon and 89 % in the rectum. Skatole concentrations in flare fat decreased by 84 %. Feeding a higher amount of potato starch (40 %) did not result in a significant further reduction. Therefore, in the following experiment (consisting of two replicates) diets of treatment groups contained 30 % of potato starch. In each replicate, 80 Baden-Württemberg Hybrids (barrows and gilts) were fed the diets during the whole fattening period. The control groups received a conventional diet based on grain and soya and a diet with a proportion of 30 % gelatinized starch in replicate 1 and 2, respectively. Skatole concentrations were determined in gut content (proximal and distal colon, rectum), blood plasma and adipose tissue (flare fat, ventral fat and back fat). Apoptosis in the colonic mucosa was determined cytochemically. From each replicate cutlets from 12 control pigs and 12 potato starch pigs were used for sensorial evaluation. The panellists evaluated samples for aroma of meat juice, and aroma and taste of meat according to a 5-point scale. The attributes were from 1 (very unpleasant) to 5 (very pleasant). A trend towards reduced apoptosis was demonstrated in the upper crypt compartment in the proximal colon. Intestinal skatole formation decreased from 50-60 µg/g dry matter to values around the lower limit of detection (0.4 µg/g). Thus, less skatole was absorbed and the mean concentrations in blood plasma were reduced by 45-65 %. In adipose tissue, concentrations in the potato starch group decreased by 57-90 % compared to the controls. Therefore, the variation of concentrations was reduced, so that 90 % of the potato starch pigs had values below 20 ng/g. Concentrations in gut content, blood plasma and adipose tissue were significantly correlated. In replicate 1, sensorial evaluation showed that cutlets containing between 0 and 2.78 ng/g skatole in fat had better ratings for aroma of meat juice and meat than samples with mean (up to 28 ng/g) or high (up to 78 ng/g) skatole concentrations. Also, the overall impression was significantly improved. In replicate 2, there was still a tendency to these differences. In conclusion, this study shows that the reduction of intestinal skatole formation by feeding resistant starch can improve the aroma of pork. For routine application, it would be sufficient to feed the potato starch diet for only a few days prior to slaughter.