Browsing by Subject "NMR"

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Enrichment and structural assignment of geometric isomers of unsaturated furan fatty acids
(2023) Müller, Franziska; Conrad, Jürgen; Hammerschick, Tim; Vetter, Walter
Furan fatty acids (FuFAs) are valuable minor fatty acids, which are known for their excellent radical scavenging properties. Typically, the furan moiety is embedded in an otherwise saturated carboxyalkyl chain. Occasionally, these classic FuFAs are accompanied by low amounts of unsaturated furan fatty acids (uFuFAs), which additionally feature one double bond in conjugation with the furan moiety. A recent study produced evidence for the occurrence of two pairs of E -/ Z -uFuFA isomers structurally related to saturated uFuFAs. Here, we present a strategy that allowed such trace compounds to be enriched to a level suited for structure determination by NMR. Given the low amounts and the varied abundance ratio of the four uFuFA isomers, the isolation of individual compounds was not pursued. Instead, the entire isomer mixture was enriched to an amount and purity suitable for structure investigation with contemporary NMR methods. Specifically, lipid extracted from 150 g latex, the richest known source of FuFAs, was subsequently fractionated by countercurrent chromatography (CCC), silver ion, and silica gel column chromatography. Analysis of the resulting mixture of four uFuFAs isomers (2.4 mg in an abundance ratio of 56:23:11:9) by different NMR techniques including PSYCHE verified that the structures of the two most abundant isomers were E -9-(3-methyl-5-pentylfuran-2-yl)non-8-enoic acid and E -9-(3-methyl-5-pent-1-enylfuran-2-yl)nonanoic acid. Additionally, we introduced a computer-based method to generate an averaged chromatogram from freely selectable GC/MS runs of CCC fractions without the necessity of pooling aliquots. This method was found to be suitable to simplify subsequent enrichment steps.
Factors determining the surface oil concentration of encapsulated lipid particles: impact of the emulsion oil droplet size
(2020) Linke, Annika; Weiss, Jochen; Kohlus, Reinhard
Microencapsulation of oxidation sensitive oils aims to separate lipids from the environmental oxygen by embedding oil droplets in a solid matrix, which builds a physical barrier. Some oil droplets are not fully incorporated and are in contact with the powder surface generating surface oil. It is proposed that the probability of oil droplets being in contact with the particle surface increases with the oil droplet size. The aim of the study is to investigate the impact of the oil droplet size on the encapsulation efficiency (EE). Two sets of feed emulsions differing in the applied homogenization pressure and in the protein to oil ratio were spray dried using a pilot plant spray dryer. The oil droplet size of the emulsion was determined by static light scattering (SLS). In addition, nuclear magnetic resonance (NMR) was used to measure the d3,2 of oil droplets in the emulsion and in the powder before and after surface oil removal. Encapsulates were analyzed regarding aw, moisture content, particle size, oil load and EE. The oil droplet size in the emulsion decreased with increasing protein to oil ratio as well as with the homogenization pressure. Large oil droplets and in particular droplet clusters resulted in more non-encapsulated oil. The experimentally determined EE was in accordance with the theoretical one, calculated based on the droplet and particle diameter. For emulsions with a diameter > 1 µm, the d3,2 decreased in the powder and further by removing the surface oil, which was related to the deformation of oil droplets contributing to the non-encapsulated oil.
Metaomic studies of the dietary impact on the structural and functional diversity of the rumen microbiome
(2018) Deusch, Simon; Seifert, Jana
Ruminant production efficiency and related emission of greenhouse gases are mainly determined by the rumen microbiome. The structure and activity of the microbial communities in turn are mostly influenced by the animal’s feed intake. The most widely used forage sources for ruminant production in Europe are corn silage, grass silage and grass hay. Progress in animal production requires optimized feeding strategies which presuppose an improved understanding of the dietary impact on the complex bionetwork residing in the rumen. A broad range of different methods are applicable to investigate archaea and bacteria which represent the most active members of the rumen microbiome. Most rumen studies available are restricted to nucleic acid-based approaches with limited functional insights. To improve knowledge about the prokaryotic communities and their adaptation responses to different animal feeds, it is essential to focus on the actual functions out of numerous possibilities that are encoded by the genomes of the rumen microbiome. Therefore proteins are best suited since representing the actual function of investigated cells combined with phylogenetic information. The major aim of this project was the feasible, first-time establishment of a metaproteomics-based characterization of the ruminal prokaryotic communities to further investigate the dietary impact on the prokaryotic rumen metaproteome. The first part was providing an overview about research that used state of the art technologies to investigate the microbiome of the gastrointestinal tract of farm animals. Yet, Omics-technologies and their combination are rarely employed in livestock science. The considered studies relied mainly on stand-alone, DNA-based molecular methods which clearly emphasized the importance of introducing contemporary methods such as shotgun metaproteomics to study the rumen microbiome and to gain deeper, more complete insights into the actual functions carried out by the specific members of the prokaryotic communities. The second part of the current project focused on a suitable, mass spectrometry-based analysis of the prokaryotic communities in the rumen ecosystem. Metaproteomic studies are challenged by the heterogeneity of the rumen sample matter that contains, besides archaeal and bacterial cells, also eukaryotic cells of rumen fungi and protozoa as well as enormous amounts of plant cells from ingested feed and epithelial cells of the animals. Shotgun metaproteomic studies require the extraction of proteins preferably of the desired target organisms to increase the coverage of the respective metaproteome and the reliability of subsequent protein identifications. This entails the avoidance of undesired proteins present in the rumen samples. In contrast to nucleic acids, proteins cannot be enriched or amplified by PCR thus, optimized sample preparation protocols are necessary in order to retrieve enhanced amounts of prokaryotic instead of plant-derived or other eukaryotic cells before protein extraction and subsequent LC-MS/MS analysis. The final step and the major aim of this project was the in depth analysis of the metaproteome of archaea and bacteria and their adaptive response to the most common forages, corn silage, grass silage and grass hay accessing as well host-related influences and variations between different ecological niches within the rumen. Improved mass-spectrometric measurements and the construction of a customized, sample-specific in-house database for enhanced bioinformatic quantification of proteins yielded comprehensive datasets comprising 8,163 bacterial and 358 archaeal proteins that were identified across 27 samples from three different rumen fractions of three Jersey cows, fed rotationally with three different diets. The functional and structural data of the metaproteomic analysis was further flanked by 16S rRNA gene-based analyses of the archaeal and bacterial community structures and the metabolomes of the rumen fluid fractions were quantified by nuclear magnetic resonance. So far, to the best of our knowledge, there are no studies investigating the metaproteome expressed by the entirety of archaeal and bacterial communities in the different phases of the rumen ecosystem under varying dietary influence. Dietary treatments revealed significant variations in the metaproteome composition and community structures of ruminal bacteria. Host-related effects were not significant. In conclusion, within this project the application of shotgun metaproteomics to characterize the prokaryotic rumen metaproteome was successfully implemented and the obtained results clearly emphasized the benefits of using complementary, state of the art methods to study the microbiome of complex ecosystems like the rumen. Considering the specific functional niches of the rumen microbiome have been shown to be of great importance.