Charakterisierung von Fermentationsaromen mittels gaschromatographischer Techniken

In this work, new sources for sustainably produced flavourings were identified. In the first part of the presented thesis, a fermentation medium of Ashbya gossypii, which is a waste product from the industrial biotechnological production of riboflavin (vitamin B2), was investigated for the presence of aroma-active compounds, and more than 1 g/L of volatile aroma-active compounds were detected. For instance, 367 mg of 2- and 3-methylbutanol were determined per L fermentation medium. In addition, 2-phenylethanol was identified with a content of 473 mg/L. These three compounds are typical products of the Ehrlich pathway, which is a metabolic pathway of amino acids in yeasts. In addition, remarkable amounts of various γ-lactones were detected. These included γ-decalactone, γ-dodecalactone as well as some unsaturated derivatives thereof, such as (Z)-γ-dec-7-enlactone or (Z)-γ-dodec-6-enlactone. In nature, these unsaturated lactones occur in low concentrations in peaches or nectarines, for example, and are partly responsible for their typical smell. Sample preparation was carried out by means of Solvent Assisted Flavour Evaporation (SAFE). This method involves a high-vacuum distillation in which an organic extract of the sample is passed to a high-vacuum apparatus in which solvent and volatile compounds evaporate immediately and are cryofocused by liquid nitrogen in another part of the apparatus. The non-volatile components of the organic sample extract, such as triglycerides, collect in a vessel below the sample inlet. Subsequently, the volatile compounds are concentrated, and the obtained flavour extract is analysed by gas chromatography-mass spectrometry-olfactometry (GC-MS- O). During the analysis of the extract, some pleasant-smelling compounds were detected, although the comparison of their mass spectra with those of the database did not provide meaningful results in all cases. Manual interpretation of the mass spectra showed that the unknown compounds were γ-lactones, bearing a double bond in the side chain. The compounds were isolated by high-performance liquid chromatography (HPLC) in order to determine the position of the double bond using Paternò-Büchi reaction. In this reaction, a carbonyl compound, e.g., 3-acetylpyridine, is used to undergo a photo-induced [2+2] cycloaddition on the double bond. Subsequently, fragment ions can be generated by analysing the products by means of MS/MS, whereby the mass of the fragment ions can provide information about the position of the double bond. Based on these results, the substances were subsequently synthesised. The products obtained were converted to Mosher esters and the diastereomers were separated by preparative HPLC, so that after subsequent elimination of the Mosher acids, the enantiopure lactones were available. Thus, the enantiomeric distribution of the lactones in the investigated sample could be determined. In addition, the odour impressions and odour thresholds in air of the racemates and the pure enantiomers were determined by GC-O. In the second part of the thesis, another fermentation medium of an industrial process was investigated. Culture media of the bacterium Basfia succiniciproducens were derived from the biotechnological production of succinic acid. The analysis of the medium by means of Headspace-Solid Phase Microextraction-GC-MS-O (HS-SPME-GC-MS-O) revealed mainly alkylated pyrazines as interesting odour impressions. Alkylated pyrazines are highly potent aroma substances that occur mainly in highly heated foods. For example, alkylated pyrazines are formed during roasting or frying at temperatures above 140 °C in Maillard-type reactions from reducing sugars and amino acids. They are mainly responsible for the smell of foods like coffee or chocolate. Since the preliminarily identified substances were not commercially available as reference compounds, they were chemically synthesised, and quantification could be carried out by means of standard addition. In total, about 10 mg/L of alkylated pyrazines were determined in the fermentation medium. The third project of this thesis addressed the characterisation of aroma substances from the electrochemical oxidation of (R)-limonene. Therefore, (R)-limonene was dissolved in ethanol, and, after the addition of a conductive salt, a current was applied via graphite electrodes, so that various oxidation products of limonene were formed. The resulting mixture of aroma substances was analysed by GC-MS-O to describe the odour impressions of the respective substances. Since the comparison of the mass spectra with the database did not yield any useful suggestions for most of the compounds, attempts were made to isolate the individual oxidation products by column hromatography and subsequently by preparative HPLC. Using various combinations of solvents, 17 substances could be isolated, and the structure of the substances was elucidated by NMR experiments and mass spectrometry. The isolated compounds represented mainly mono- and diethoxyethers of limonene, but also several keto compounds and acetals. In total, the 17 isolated compounds made up more than 95% of the total peak area of the chromatogram of the sample. In addition to these 17 isolates, limonene, carvone and p-cymene were identified. The odour impressions of the isolated compounds were described as fruity, citrus-like, herbaceous, fresh and floral, with nuances in various directions. Most of the isolated compounds have not been described in the literature previously. All of the flavour mixtures investigated in this study were derived from sustainable production processes. In the industrial production of riboflavin, biotechnology has now completely replaced chemical synthesis. As a result, the use of chemicals has been drastically reduced. Since biotechnological production produces large quantities of fermentation media, the extraction of natural flavouring substances from them is an alluring option. Currently, 2- and 3-methylbutanol are chemically synthesised from butenes, but can also be obtained as natural aroma compounds from fusel oils produced by yeasts. 2-Phenylethanol is mostly synthesised chemically, whereby mainly benzene or its derivatives are transformed. However, 2- phenylethanol can also be obtained naturally through fermentation with yeasts. Of the lactones detected in the fermentation medium, only γ-decalactone is produced commercially by biotechnological conversion of ricinoleic acid. The unsaturated lactones are not yet available commercially, so that a sustainable and natural source of these substances is even more desirable. Similarly, the chemical synthesis of succinic acid is performed by oxidation of 1,4-butanediol or through the hydrogenation of other C4 acids, such as maleic or fumaric acid. However, biotechnological production by various microorganisms is also possible. A special characteristic of B. succiniciproducens is that this bacterium also is able to use crude glycerol as a carbon source. Glycerol accrues in large quantities during the production of biodiesel. Thus, a good coupling with the biotechnological production of succinic acid is possible. The alkylated pyrazines formed during fermentation are hardly available as natural flavouring substances. In the industrial production of natural pyrazines, foodstuffs such as potatoes, nuts or coffee are extracted in most cases. Therefore, recovery from the resulting fermentation medium may represent a profitable and more sustainable alternative. Both fermentation processes have currently not been designed nor optimised for the production of flavourings, so that by improving the processes, much higher quantities of natural flavourings could be produced as co-products. The electrochemical oxidation of limonene also represents a sustainable process. Limonene is available in large quantities, as it can be obtained from the fruit juice industry. Furthermore, the chemical oxidation of terpenes often involves the use of heavy metal catalysts and toxic and/or hazardous chemicals. None of these is necessary in the electrochemical oxidation of terpenes. In addition, a wide variety of different aroma substances can be generated quickly and inexpensively, with diverse combination possibilities by varying the parameters (duration, voltage, type of reactants (different alcohols in combination with different terpenes)).     

Interrupted‐Burning Tests of Plasma‐Ignited JA2 and M30 Grains in a Closed Chamber

Enhanced burning of plasma‐ignited propellants has been deduced in recent years from closed bomb pressure traces. The question remains, however, whether the enhanced burning is inherent or possibly the result of propellant grain fracture. To find out, interrupted and non‐interrupted burning tests were conducted with cylindrical perforated grains of JA2 and M30 in a closed bomb with a loading density of approximately 0.2 g/cm³. Both, conventional black powder and plasma igniters were used, and a few tests were carried out with the propellants cooled to −20 °C. The plasma igniter was an ablating capillary, and the electrical energy density was about 0.7 kJ/g of propellant. The diameters of the collected grains yielded the actual burn distance at the time of the interrupted burning. The experimental pressure traces and the conventional burn rate coefficients of the propellants were used to calculate the theoretical depth burned assuming no plasma‐induced burn rate modifications. From overlapping pressure traces at several interrupted pressures, and from comparison to the calculated versus measured burn distances, it was found that there is burn rate enhancement during the plasma pulse–but not much once the pulse has ended. In contrast to the JA2 burn rates, both ambient and cold M30 burn rates, deduced from the non‐interrupted tests using the BRLCB code, were enhanced even after the plasma turn‐off, thus contradicting the interrupted‐tests results. However, vivacity analysis of the non‐interrupted tests indicated that the M30 grains exhibited increased surface area (possible fragmentation) because of the plasma interaction–an effect that would cause erroneous results from the BRLCB. Indeed, simulation of the non‐interrupted M30 tests using the XNOVAKTC code, and assuming partial fragmentation of the propellant charge, yielded vivacities that mimicked the experimental ones.