Fakultät Naturwissenschaften

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Biologie, Ernährungs-wissenschaften und Lebensmittelwissenschaften sind die Schwerpunkte der Fakultät. Die Forschung befasst sich mit Schlüsselthemen der Life Sciences.

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Analysis of the structure of tomato mosaic virus movement protein based on virus host interactions
(2011) Tanwir, Fariha; Pfitzner, Artur J. P.
Viruses are obligatory plant pathogens causing sever diseases, and ultimately great losses in crop yield. Plant viruses, once entered in the cell, make use of host machinery for its own replication and moves from one cell to the other. Natural resistance against virus attack is achieved by the presence of resistance genes (R genes). R genes recognize viral avirulence (Avr) factors in elicitor-receptor manner to initiate resistance cascade. In tomato, the resistance genes Tm-I, Tm-2 and Tm-22 are used to protect the plants against infection by tomato mosaic virus.Tm-2 and Tm-22 require recognition of the viral 30kDa movement protein (MP) for triggering resistance response. Sequence analysis of Tm-2 and Tm-22 resistance breaking viruses have shown an amino-acid exchange at position 133 (E>K) is found in all Tm-2 resistance breaking virus strains, whereas, amino-acid exchange at position 130 (K>E) is associated with Tm-22 resistance breaking phenotype (Calder and Palukaitis, 1992). This suggests a physical interaction between resistance genes and 30kDa MP. In the present study, a unique Split GFP approach is used to analyse the structure and localization of different domains of 30kDa MP in S. cerviceae and N. benthamiana. Different deletion mutants were fused between two non-overlapping halves of GFP and expressed. Results showed that both N and C terminus as well as the middle part of 30kDa MP (aa 80-150) is present in the cytoplasm with two integral membrane loops. These findings are in contrast with previous in-vitro results, which suggest that middle part of 30kDa MP is present in ER lumen, whereas N and C terminus in cytoplasm (Brill et al., 2000). Fluorescence microscopy revealed that GFP fused 30kDa MP deletion mutants were localized on the cytoplasmic side of plasmamembrane and near plasmodesmata. Membrane association of fusion protein confirmed the proper folding and functionality of deletion mutants. Therefore, the structural model of ToMV 30kDa MP has to be revised. Secondly, to identify the host factors involved in resistance mechanism, initiated by Tm-2 and Tm-22 resistance genes, tomato mosaic virus based vectors were constructed. Two different types of in-vivo transcription vectors were constructed, one containing both right and left border of the T-DNA (pBinSLN) and one without the right border (pBinSLN-RB). Self replication of these vectors were analysed in N. benthamiana, N. tabacum and S. lycopersicum. It was found that the deletion of RB does not affect virus replication, when agro-infiltrated in N. benthamiana. pBinSLN-RB was used further for the isolation of a stable and vigorous Tm-2 and Tm-22 resistance breaking ToMV strain through a novel selection scheme. ToMV2-22 contains two amino-acid exchanges at position 54(N>D) and 133(E>K). ToMV2-22 is the first mutant strain of ToMV, which can escape both Tm-2 and Tm-22 resistance simultaneously.
Antimikrobielle Aktivität der Histone bei chronisch entzündlichen Darmerkrankungen
(2017) Kunkel, Yasmin; Stange, Eduard F.
The human intestine harbours a multitude of microorganisms. In addition to its func-tion as a protective layer against pathogens, it has to prevent an excessive immune answer against commensales simultaneously. Antimicrobial Peptides (AMPs) with their cationic character are playing an essential role in protection, because they are able to form voltage dependent channels on the surface of microorganism, which kill pathogens. In addition to the classical AMPs more antimicrobial active polypeptides, such as members of the histone family, were isolated. Histones are alkaline proteins, which are components of the chromatine. They are foremost responsible for packaging the DNA and for posttranslational modifications. Five different families can be differentiated: the core histones H2A, H2B, H3 and H4, as well as the linker histone H1. While extracellular histones show strong antimicrobial activity against a broad spectrum of microorganisms, the mechanism of their toxicity has not yet been sufficiently determined. If the antimicrobial protection layer of the intestine is weakened, due to a diminished expression of AMPs for instance, microorganisms can penetrate the mucosa and trigger inflammations. These findings have been confirmed in different tissues of patients with inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC). The aim of this work is to determine whether histones play a role in IBD. In the first part a systematic analysis of the transcriptome (Q-PCR) and the transla-tome (Western Blotting) of the core histones of colonic tissue was performed. In tis-sues of patients with CD gene expression data showed generally an increase of his-tones. In the cases of H2A and H2B the increase was significant. The quantification on the protein level offered an extreme variance of the expression of all core histones, irrespective of the analysed group. Significant differences were not detected. However, in trend H2B was lower in inflammation. After the systematic analysis, histones were then isolated of human colonic tissue. Before the extracted histones were fractionated via RP-HPLC and screened via MALDI-TOF-MS, different methods for the isolation of histones had been compared. The antimicrobial activity of the isolated histones of different intestinal tissues and mucus showed no differences between healthy controls and patients with IBD in flow cytometric tests. A significant increase of the histone activity in inflamed tissues of UC was only detected against S. aureus. The impact of the extracted histones seems to be strain-specific and higher against gram-positive species. As expected, extracel-lular histones could be detected in the mucus by immunehistological staining. Through ELISAs, protein concentrations of H2A and H2B were determined in the mucus and thus a slight increase of the histone proteins in UC was observed. In the last part of this work, the interactions of recombinant histones among them-selves and with other AMPs were analysed by flow cytometric viability assays. A strain-specific increase of the antimicrobial activity of histones among themselves and with AMPs was found. Thereby synergistic effects occurred frequently. The in-teractions of histones against several bacteria were visualised by electron microscope images and furthermore an agglutination of the microorganisms as well as a massive loss of cell integrity were detected. Variantions of the histones’ transcriptome and the translatome, as well as variations of the antimicrobial activity of histones in CED would have been evaluated as patho-logic defects. However, in this work such effects could not been confirmed. Because of their enormous antimicrobial activity histones still play an important role in the protection against microorganisms in the colon. Further studies have to show, if his-tones possess a therapeutic potential, and if they can be used as new antibiotics. This work was able to verify the strong potential of histones against different pathogens, which is absolutely comparable with the potential of classic AMPs, and could pro-mote inspiration for subsequent studies.
Application of nature-inspired optimization algorithms to improve the production efficiency of small and medium-sized bakeries
(2023) Babor, Md Majharul Islam; Hitzmann, Bernd
Increasing production efficiency through schedule optimization is one of the most influential topics in operations research that contributes to decision-making process. It is the concept of allocating tasks among available resources within the constraints of any manufacturing facility in order to minimize costs. It is carried out by a model that resembles real-world task distribution with variables and relevant constraints in order to complete a planned production. In addition to a model, an optimizer is required to assist in evaluating and improving the task allocation procedure in order to maximize overall production efficiency. The entire procedure is usually carried out on a computer, where these two distinct segments combine to form a solution framework for production planning and support decision-making in various manufacturing industries. Small and medium-sized bakeries lack access to cutting-edge tools, and most of their production schedules are based on personal experience. This makes a significant difference in production costs when compared to the large bakeries, as evidenced by their market dominance. In this study, a hybrid no-wait flow shop model is proposed to produce a production schedule based on actual data, featuring the constraints of the production environment in small and medium-sized bakeries. Several single-objective and multi-objective nature-inspired optimization algorithms were implemented to find efficient production schedules. While makespan is the most widely used quality criterion of production efficiency because it dominates production costs, high oven idle time in bakeries also wastes energy. Combining these quality criteria allows for additional cost reduction due to energy savings as well as shorter production time. Therefore, to obtain the efficient production plan, makespan and oven idle time were included in the objectives of optimization. To find the optimal production planning for an existing production line, particle swarm optimization, simulated annealing, and the Nawaz-Enscore-Ham algorithms were used. The weighting factor method was used to combine two objectives into a single objective. The classical optimization algorithms were found to be good enough at finding optimal schedules in a reasonable amount of time, reducing makespan by 29 % and oven idle time by 8 % of one of the analyzed production datasets. Nonetheless, the algorithms convergence was found to be poor, with a lower probability of obtaining the best or nearly the best result. In contrast, a modified particle swarm optimization (MPSO) proposed in this study demonstrated significant improvement in convergence with a higher probability of obtaining better results. To obtain trade-offs between two objectives, state-of-the-art multi-objective optimization algorithms, non-dominated sorting genetic algorithm (NSGA-II), strength Pareto evolutionary algorithm, generalized differential evolution, improved multi-objective particle swarm optimization (OMOPSO) and speed-constrained multi-objective particle swarm optimization (SMPSO) were implemented. Optimization algorithms provided efficient production planning with up to a 12 % reduction in makespan and a 26 % reduction in oven idle time based on data from different production days. The performance comparison revealed a significant difference between these multi-objective optimization algorithms, with NSGA-II performing best and OMOPSO and SMPSO performing worst. Proofing is a key processing stage that contributes to the quality of the final product by developing flavor and fluffiness texture in bread. However, the duration of proofing is uncertain due to the complex interaction of multiple parameters: yeast condition, temperature in the proofing chamber, and chemical composition of flour. Due to the uncertainty of proofing time, a production plan optimized with the shortest makespan can be significantly inefficient. The computational results show that the schedules with the shortest and nearly shortest makespan have a significant (up to 18 %) increase in makespan due to proofing time deviation from expected duration. In this thesis, a method for developing resilient production planning that takes into account uncertain proofing time is proposed, so that even if the deviation in proofing time is extreme, the fluctuation in makespan is minimal. The experimental results with a production dataset revealed a proactive production plan, with only 5 minutes longer than the shortest makespan, but only 21 min fluctuating in makespan due to varying the proofing time from -10 % to +10 % of actual proofing time. This study proposed a common framework for small and medium-sized bakeries to improve their production efficiency in three steps: collecting production data, simulating production planning with the hybrid no-wait flow shop model, and running the optimization algorithm. The study suggests to use MPSO for solving single objective optimization problem and NSGA-II for multi-objective optimization problem. Based on real bakery production data, the results revealed that existing plans were significantly inefficient and could be optimized in a reasonable computational time using a robust optimization algorithm. Implementing such a framework in small and medium-sized bakery manufacturing operations could help to achieve an efficient and resilient production system.
Beschreibung und Optimierung der Vorgänge der dynamischen Gefriertrocknung
(2018) Pliske, Roland; Kohlus, Reinhard
Freeze-drying is a gentle but also time-consuming drying method. One reason for the longer drying times is the formation of a dry layer during drying, which is a heat and mass transfer resistance. One approach for reducing the drying time is removing these resistances. The detail of an approach to remove the dry layer within a special powder mixer has been investigated. The process of freeze-drying while agitating has been termed ‘dynamic freeze-drying’. The used mixer was a plow-share type, in which the dry layer is actively rubbed-off permanently during the drying process. In this process the drying always takes place on the moister particle surface. This corresponds to the characteristics of a constant drying rate period, which can be considered confirmed by independent dynamic freeze-drying experiments. Freeze-drying process typically do not show a constant drying rate period. The drying front retreats immediately at the start of drying into inside of the particle. Therefore, drying rate of dynamic-freeze drying could be increased. The drying rate can be furthermore increased applying higher heating temperature in the case of dynamic freeze-drying compared to static freeze-drying. The danger of a collapse is prevented by abrasion of the dry layer during dynamic freeze drying. It has also been shown that under identical drying conditions, dynamic freeze-drying has an up to tenfold faster drying rate compared to conventional, static freeze-drying. One reason for this is a higher conductive heat flux into the bed. Another reason is the conversion of the kinetic energy into heat energy during the mixing of the bed, which is additionally used for the sublimation. Since the dry layer is removed during dynamic freeze-drying, the advantage should lie by larger initial diameters, because there are greater heat and mass transfer resistances compared to smaller initial particle diameters. This effect is overcompensated by the number of particles that are present if the same initial mass will be used for creation smaller particles than bigger particles. The contact number of particles to mixer wall determines the heat transfer by conduction and particle to particle determines the heat transfer by friction. For this reason, the drying time of the dynamic freeze-drying of smaller diameter beds is always lower. All results indicate that the number of contact points of particles to the mixer wall and other particles is relevant for the energy transfer to the bed during dynamic freeze-drying. As the particles become smaller during the drying process, however their number remains constant, and so is the effective heat transfer coefficient. A positive effect on drying rate was determined for the dried powder, which is within the mixer during the drying process. While drying with low rotational frequency less dried powder was discharged from the mixer and the experimental drying times always were lower than the modeled ones. The powder is heated at the mixer wall and is then afterwards reintroduced into the bed. At high rotational frequencies the powder is fluidized up more intensively and discharged with the water vapor from the mixer. During the drying process the water vapor leaves the mixer and partially the dried final product, too, and the load decreases and the energy input as well. Freeze-drying covers a large part of microorganism conservation so called starter culture conservation. First trials in using dynamic freeze-drying for this application have been conducted. Dynamic freeze-drying has been used in the drying of microorganisms in order to compare the viable count and the activity of the dried microorganisms with those from static freeze-drying. The presented results show that the viable count of the dynamic freeze-dried microorganisms is reduced. The activity however is partly higher than that of static freeze-dried microorganisms, which indicates a stress activation. These results were found using starter cultures that were frozen without adding "protective medium". Whether trials using protective medium will show similar results is currently unclear. The phenomenon of stress activation has to be confirmed using a large variety of lactic acid bacteria.
Bioprospecting for novel lipopeptide-producing strains for potential application in food and agriculture
(2024) Akintayo, Stephen Olusanmi; Hausmann, Rudolf
The need for sustainable alternatives to chemical products has been a huge topic in recent years and has put a demand on researchers and biotechnological companies to come up with bio-based alternatives to several chemical products. In line with this, interest in biosurfactants as alternatives to chemical surfactants is on the rise. Biosurfactants produced by microorganisms have great potential for application in detergents, personal care products, and pharmaceuticals, as well as in environmental, food processing, and agricultural applications. There are a few types of biosurfactants, including lipopeptides, which are primarily produced by Bacillus species and exhibit antimicrobial properties in addition to the well-known surface activity, surface tension reduction, and emulsifying ability of biosurfactants. Like other biosurfactants, lipopeptides have found more use in environmental applications such as bioremediation and microbial enhanced oil recovery (MEOR), while their use in agriculture and food industries remains limited due to concerns that may be related to acceptability, compatibility, and low yield by wild-type strains. To overcome these challenges, this thesis sought to find novel wild-type lipopeptide-producing strains from food-related sources that could be presumably safe for use in agriculture and food applications. To achieve this goal, a screening approach that combined several methods was adopted to identify potential high-yield wild-type, and possibly novel lipopeptide-producing strains. The ability of selected strains as promising biocontrol agents in agriculture was also evaluated. In Publication 1, potential lipopeptide-producing strains were isolated from food-related sources and screened for lipopeptide production. The screening approach combined microbiological and molecular identification of strains, with screening methods based on biosurfactant properties, as well as chemical analysis of surfactin production. Strains with promising lipopeptide-production potential belonging to three genera of Bacillus, Lysinibacillus and Priestia were identified. These strains included several exotic species that were either previously unknown or minimally studied with respect to LP production. Multiple strains that produced more than 150 mg L-1 surfactin, including a B. subtilis strain with a yield of about 1.5 g L−1 were discovered. In Publication 2, two promising LP-producing B. velezensis strains ES1-02 and EFSO2-04 were evaluated for their biocontrol potential and compared with commercial biocontrol strains B. velezensis QST713 and FZB42. The isolated strains demonstrated biocontrol ability comparable to QST713 against Diaporthe spp., which are notorious fungal pathogens of soybeans and other economically important crops. Co-incubation of strain ES1-02 with the phytopathogen D. longicolla induced a 10-fold increase in surfactin production. The broader molecular response of B. velezensis to plant pathogens investigated through an associated global proteome analysis showed the adaptation and response mechanisms of B. velezensis to plant pathogens. In general, B. velezensis seemed to adopt LP- modulation, physiological adaptation, and increased abundance of antimicrobial compounds as antagonistic and adaptation strategies for interaction with the phytopathogen D. longicolla. In Publication 3, genomic techniques were used in the discovery and description of a novel lipopeptides-producing species of the genus Lysinibacillus for which the name Lysinibacillus irui sp. nov. was proposed. This Gram-positive, motile, aerobic, rod-shaped, endospore-forming strain designated IRB4-01T was isolated from fermented African locust beans (Iru) and as such was named after Iru. A comprehensive chemotaxonomic analysis of the strain showed that the cell wall peptidoglycan type is A4α (Lys–Asp), and MK-7 is the major respiratory quinone. Detailed information about the polar lipids and major cellular fatty acids was also obtained. The G+C content of the genomic DNA was 37.4 mol%. Surfactin production by this novel strain was described in Publication 1 of this work.
Characterization of the aroma properties in fragrant rapeseed oil and aroma variation during critical roasting phase
(2023) Zhang, Youfeng; Zhang, Yanyan
Rapeseed oil is one of the third most-produced vegetable oil in the world, which is appreciated for its characteristic flavor and high nutritional value. Fragrant rapeseed oil (FRO) produced by a typical roasting process is popular for its characteristic aroma, which has an annual consumption exceeding 1.5 million tons. However, the changes in aroma blueprint of FRO during the typical roasting processing are still unclear, which challenges rapeseed oil quality and consumer acceptance. Accordingly, the aim of this work was to investigate the aroma characteristics and their precursors pyrolysis behavior of FRO to provide a basis and guidance for the control of FRO aroma quality during production processing. First, a systematic review on summarizing, comparing, and critiquing the literature regarding the flavor of rapeseed oil, especially about employed analysis techniques (i.e., extraction, qualitative, quantitative, sensorial, and chemometric methods), identified representative/off-flavor compounds, and effects of different treatments during the processes (dehulling, roasting, microwave, flavoring with herbs, refining, oil heating, and storage) was performed. One hundred and thirty-seven odorants found in rapeseed oil from literature are listed, including aldehydes, ketones, acids, esters, alcohols, phenols, pyrazines, furans, pyrrolines, indoles, pyridines, thiazoles, thiophenes, further S-containing compounds, nitriles, and alkenes, and possible formation pathways of some key aroma-active compounds are also proposed. Nevertheless, some of these compounds require further validation (e.g., nitriles) due to lack of recombination experiments in the previous work. To wrap up, advanced flavor analysis techniques should be evolved toward time-saving, portability, real-time monitoring, and visualization, which aims to obtain a “complete” flavor profile of rapeseed oil. Aparting from that, studies to elucidate the influence of key roasting processing on the formation of aroma-active compounds are needed to deepen understanding of factors resulting in flavor variations of rapeseed oil. Following, a systematic comparison among five flavor trapping techniques including solid-phase microextraction (SPME), SPME-Arrow, headspace stir bar sorptive extraction (HSSE), direct thermal desorption (DTD), and solvent-assisted flavor evaporation (SAFE) for hot-pressed rapeseed oil was conducted. Besides, methodological validation of these five approaches for 31 aroma standards found in rapeseed oil was conducted to compare their stability, reliability, and robustness. For the qualification of the odorants in hot-pressed rapeseed oil, SAFE gave the best performance, mainly due to the high sample volumes, but it performed worse than other methods regarding linearity, recovery, and repeatability. SPME-Arrow gave good performances in not only odorant extraction but also quantification, which is considered most suitable for quantifying odorants in hot-pressed rapeseed oil. Taking cost/performance ratio into account, SPME is still an efficient flavor extraction method. Multi-method combination of flavor capturing techniques might also be an option of aroma analysis for oil matrix. Afterwards, by application of the Sensomics approach the key odorants in representative commercial FRO samples were decoded. On the basis of the aroma blueprint, changes of overall aroma profiles of oils and their key odorants were studied and compared in different roasting conditions. To better simulate industrial conditions, high temperatures (150-200 ºC) were used in our roasting study, which was rarely studied before. Identification and quantitation of the key odorants in FRO were well performed by means of the Sensomics concept. Glucosinolate degradation products were a special kind of key odorants existing in rapeseed oil. Most of the odorants showed first rising and then decline trends as the roasting process progressed. Aroma profile results showed that high-temperature-short time and low-temperature-long time conditions could have similar effects on the aroma profiles of roasted rapeseed oils, which could provide a reference for the time cost savings in industrial production. To gain the fundamental knowledge of the aroma formation in FRO, the thermal degradation behavior of progoitrin (the main glucosinolate of rapeseed) and the corresponding generated volatile products were investigated in liquid (phosphate buffer at a pH value of 5.0, 7.0, or 9.0) and solid phase systems (sea sand and rapeseed powder). The highest thermal degradation rate of progoitrin at high temperatures (150-200 ºC) was observed at a pH value of 9.0, followed by sea sand and then rapeseed powder. It could be inferred that bimolecular nucleophilic substitution reaction (SN2) was mainly taken place under basic conditions. The highest degradation rate under basic conditions might result from the high nucleophilicity of present hydroxide ions. Under the applied conditions in this study, 2,4-pentadienenitrile was the major nitrile formed from progoitrin during thermal degradation at high temperature compared to l-cyano-2-hydroxy-3-butene, which might be less stable. The possible formation pathways of major S-containing (thiophenes) and N-containing (nitriles) volatile (flavor) compounds were proposed. Hydrogen sulfide, as a degradation product of glucosinolates, could act as a sulfur source to react further with glucose to generate thiophenes. Overall, the present work comprehensively documented the effects of thermal conditions and matrices on the aroma characteristics, aroma profiles, and key odorants of hot-pressed rapeseed oil, which could provide data and theoretical basis for the flavor control of FRO under thermal treatment at actual production temperatures (150-200 °C).
Characterization of the rehydration behavior of food powders
(2019) Wangler, Julia; Kohlus, Reinhard
The rehydration behavior of food powders is of high importance in terms of powder processing and product quality. Rehydration of powders mainly depends on the physical powder characteristics particle size, porosity and wettability, the latter being expressed by the contact angle between solid and rehydrating liquid. With focus on food powders, it could be shown that the rehydration behavior is strongly influenced by dynamic changes of these physical characteristics. This includes the initiation of dissolution and swelling directly after powder-liquid contact. Especially in case of biopolymers, which were investigated in detail by the example of xanthan gum, guar gum and alginate, these processes are important to describe their rehydration behavior. Due to the special characteristics of these biopolymers dissolution and swelling result in an increase of viscosity as well as in a decrease of bulk porosity. The kinetics and interactions of these processes significantly affect the individual steps of rehydration and have to be considered in describing the process of food powder rehydration. For inert powder-liquid systems capillary liquid uptake into a powder bulk can be described by the Washburn equation which equates the capillary pressure and the hydrodynamic flow resistance. This approach was used as basic equation to describe capillary liquid uptake of food powders. The validity of the original approach is restricted to the case of constant powder and liquid properties. With regard to food powders, changes within the powder-liquid system were considered by a stepwise adaption of the variables of the Washburn equation. Thus, the first part of this thesis focused on establishing and defining methods to characterize the dynamics of the physical properties particle size, bulk porosity, viscosity and contact angle. This enabled a more detailed characterization of the interactions between food powder and liquid during rehydration. Wettability of food powders in contact with dist. water was assessed by contact angle measurements. Contact angles were 52° for alginate, 58.1° for xanthan gum and 70° for guar gum which confirmed their hydrophilic character. To describe the change of the bulk porosity a rheological measurement set-up was constructed to quantify the swelling behavior. Influence of viscosity on rehydration was determined by measuring the concentration dependent viscosity increase and the rate of viscosity increase over time. The change of viscosity as a consequence of dissolution allowed conclusions about the dissolution rate of biopolymers in highly concentrated situations. These results indicated that rehydration of guar gum is mainly influenced by viscosity effects whereas swelling has the highest impact on the rehydration behavior of xanthan gum and alginate. Further methods such as Nuclear Magnetic Resonance analysis enabled a more detailed characterization concerning the dynamics of powder-liquid interactions and the strength of water binding within these biopolymer gels. The strength of water binding was found to correlate with the stability of highly concentrated biopolymer aggregates. The aggregate stability was determined by rheological analyses and is of importance, particularly with regard to powder dispersability. To predict food powder rehydration, a model was established using a VoF approach. To simulate capillary liquid rise based on physical characteristics, dynamic changes were resolved both spatial and temporally. To describe particle and liquid properties more precisely, a model system consisting of biopolymer coated glass beads was developed by fluid bed technology. By the variation of the coating layer thickness and the coating material, dynamic changes within the system could be controlled which enabled a more differentiated description. A parameter variation study was conducted to simulate the influence and interaction of dynamic processes on capillary liquid uptake into such powder systems. Capillary liquid uptake into the coated glass beads was investigated experimentally. It could be shown that even with coating layers of 0.5 µm dynamic effects are sufficiently strong to cause a stop of capillary liquid uptake. It has been shown that viscosity development dominates guar gum rehydration whereas swelling is the prevalent mechanism in xanthan gum and alginate rehydration. Simulation of capillary liquid rise demonstrated that the influence of the coating layer thickness is not significant. This result could be explained by the slow dissolution rates of the biopolymer samples. Calculations indicated that even a coating layer of 0.5 µm could only be dissolved partially after a dissolution time of 250 s. This explains the little impact of coating layer thickness on viscosity development and thus on capillary liquid uptake. Further explanations focus on biopolymer swelling. Simulation showed that coating layers of 0.5 µm are sufficient to cause swelling-induced pore-blocking conditions.
Climate dynamics : the performance of seasonal ensemble forecast for improving food security in Ethiopia
(2023) Ware, Markos Budusa; Wulfmeyer, Volker
Part one of this thesis aims to define homogenous climatic regions using objective clustering methods and characterize seasonal cycles, trends, and anomalies in precipitation and temperature. Climate-based on amplifies inherent spatiotemporal climate variability in the Horn of Africa due to global, regional, coastal, and local processes. The homogeneous climatic regions and synoptic circulation types were defined using Principal Component Analysis (PCA) PCA–K-means and PCA–Ward’s. Using the decision criteria of respective algorithms, four homogenous climatic regions were determined for Ethiopia. These climatic regions were distinctive in their seasonal cycles, trends, and anomalies in annual and seasonal precipitation and temperature. These results highlight that the trends in precipitation and temperature vary not only between climatic regions but also by rainy seasons. The short rains (received between November and December) increased by 50 mm/decade in the southwestern region where the evergreen forest meets with the long rainy season. The mean annual and seasonal temperature increased between 0.3 and 0.6 °C/decade virtually in all climatic regions. Regionalization methods were sensitive to spatial domain size but not to the length of the time series. Climatology of sea-level air pressure showed decreasing northward trend over the study domain, as did the temperature, wind velocity, and relative humidity at 500 hPa. However, geopotential height at 500 hPa and temperature at 850 hPa decreased toward the south over the domain. Circulation types were defined by applying PCA on a composite matrix of the six variables. From the first five Principal Components (PCs), ten circulation types (CTs) were defined over East Africa and then associated with environmental events. CTs clearly distinguished rainy seasons comprising different atmospheric states responsible for varying weathers. The summer season was described by a combination of strong positive anomalies in temperature at 850 hPa, northeasterly winds, and Somali jet at 500 hPa, and weak negative anomalies in temperature at 500 hPa. Trends in the number of days categorized in different CTs showed a significant variation among the groups. The drought events, defined using the consecutive dry days (CDD), correspond with positive anomalies in temperature at 850 hPa, northwesterly and Somali Jet, and negative anomalies in relative humidity at 500 hPa. Flooding, defined using a proxy of 80 mm/day per grid cell, was associated with strong westerly winds at 500 hPa, strong positive anomalies in temperature at the lower troposphere, strong easterlies and southwesterly, and positive anomalies in relative humidity at 500 hPa. Part two of the thesis aims to assess the performance of the seasonal ensemble forecast over the Horn of Africa for improving food security. A seasonal forecast with a horizon of up to seven months offers a great opportunity for agricultural optimization, which results in an improved economy and food security. For this purpose, the Weather Research and Forecasting (WRF) model was applied for dynamical downscaling of the latest seasonal forecasting system version 5 (SEAS5) for summer 2018 with different microphysics parameterizations, and initial and boundary conditions. Downscaling was performed by a horizontal resolution of 3 km over the topographically complex domain of East Africa. The seasonal ensemble forecast was evaluated using probabilistic metrics like the Brier skill score, probability ranking score, continuous probability ranking score, discrimination score, and ignorance score. The results of the WRF showed that the model has a strong warm bias in the 2m temperature and a wet bias in precipitation. The relative operating characteristics (ROC) curve showed a higher predicting probability of 2m temperature in below-normal and above-normal terciles over northern Ethiopia and the Indian Ocean, where the model performed better, highlighting the advantage of high-resolution simulations compared to ERA5. The median and distribution of WRF, SEAS5, and ERA5 showed remarkable variation between the homogenous climatic regions. Especially the summer of 2018 was wetter relative to climatology, and WRF overestimated this condition in the region.
Convective-scale data assimilation of thermodynamic lidar data into the weather research and forecasting model
(2022) Thundathil, Rohith Muraleedharan; Wulfmeyer, Volker
This thesis studies the impact of assimilating temperature and humidity profiles from ground-based lidar systems and demonstrates its value for future short-range forecast. Thermodynamic profile obtained from the temperature Raman lidar and the water-vapour differential absorption lidar of the University of Hohenheim during the High Definition of Clouds and Precipitation for advancing Climate Prediction (HD(CP)2) project Observation Prototype Experiment (HOPE) are assimilated into the Weather Research and Forecasting model Data Assimilation (WRFDA) system by means of a new forward operator. The impact study assimilating the high-resolution thermodynamic lidar data was conducted using variational and ensemble-based data assimilation methods. The first part of the thesis describes the development of the thermodynamic lidar operator and its implementation through a deterministic DA impact study. The operator facilitates the direct assimilation of water vapour mixing ratio (WVMR), a prognostic variable in the WRF model, without conversion to relative humidity. Undesirable cross sensitivities to temperature are avoided here so that the complete information content of the observation with respect to the water vapour is provided. The assimilation experiments were performed with the three-dimensional variational (3DVAR) DA system with a rapid update cycle (RUC) with hourly frequency over ten hours. The DA experiments with the new operator outperformed the previously used relative humidity operator, and the overall humidity and temperature analyses improved. The simultaneous assimilation of temperature and WVMR resulted in a degradation of the temperature analysis compared to the improvement observed in the sole temperature assimilation experiment. The static background error covariance matrix (B) in the 3DVAR was identified as the reason behind this behaviour. The correlation between the temperature and WVMR variables in the background error covariance matrix of the 3DVAR, which is static and not flow-dependent, limited the improvement in temperature. The second part of the thesis provides a solution for overcoming the static B matrix issue. A hybrid, ensemble-based approach was applied using the Ensemble Transform Kalman Filter (ETKF) and the 3DVAR to add flow dependency to the B matrix. The hybrid experiment resulted in a 50% lower temperature and water vapour root mean square error (RMSE) than the 3DVAR experiment. Comparisons against independent radiosonde observations showed a reduction of RMSE by 26% for water vapour and 38% for temperature. The planetary boundary layer (PBL) height of the analyses also showed an improvement compared to the available ceilometer. The impact of assimilating a single lidar vertical profile spreads over a 100 km radius, which is promising for future assimilation of water vapour and temperature data from operational lidar networks for short-range weather forecasting. A forecast improvement was observed for 7 hours lead time compared with the ceilometer derived planetary boundary layer height observations and 4 hours with Global Navigation Satellite System (GNSS) derived integrated water vapour observations. With the help of sophisticated DA systems and a robust network of lidar systems, the thesis throws light on the future of short-range operational forecasting.
Development of rapid analytical methods for coffee quality assessment: Spectroscopy and chemometrics approach
(2024) Munyendo, Leah Masakhwe; Hitzmann, Bernd; Zhang, Yanyan
The assessment of coffee quality is based on the physical characteristics (bean quality), chemical constituents, and cup quality. Different factors, including altitude, genetics, management conditions, presence of adulterants, roasting, geographical origin, processing methods, and storage, affect the coffee quality. To meet the consumers' expectations regarding quality, the development of fast, new, and advanced analytical techniques for assessing the factors affecting coffee quality is a central aspect. Therefore, this research aimed to develop spectroscopic techniques complemented with chemometrics for evaluating the factors affecting coffee quality. The first specific objective was to investigate the ability of a deep autoencoder neural network to detect adulterants in roasted Arabica coffee and to determine a coffee’s geographical origin using near‐infrared (NIR) spectroscopy. Arabica coffee was adulterated with Robusta coffee or chicory at adulteration levels ranging from 2.5 % to 30 % in increments of 2.5 % at light, medium, and dark roast levels. Based on the results, all the samples adulterated with chicory were detectable by the autoencoder at all roast levels. For Robusta-adulterated samples, the detection was possible at adulteration levels above 7.5 % at medium and dark roasts. One can attribute the observations to potential differences in the chemical composition among the samples. Additionally, it was possible to differentiate coffee samples from different geographical origins. As a continuation of the first objective, the potential of NIR spectroscopy to quantify Robusta coffee or chicory in roasted Arabica coffee using different regression models constructed from the linear discriminant analysis (LDA) or principal component analysis (PCA) features was investigated. In addition, two classification methods (k-nearest neighbor regression (KNR) and LDA) were used. The regression models derived from LDA-extracted features exhibited better accuracies than those derived from PCA-extracted features. The two feature extraction methods exhibit differences in their working principle. PCA focuses on identifying the direction of maximum variance regardless of the adulteration levels. In contrast, LDA identifies the feature subspace that optimizes the separability of the classes (adulteration levels) and minimizes the variance within the class. Therefore, LDA extracted the features better than PCA, explaining the better performance of the regression models constructed from its features. The models provided satisfactory results with the coefficient of determination (R2) values above 0.92 for both the adulterants, indicating their efficiency in quantifying Robusta coffee or chicory in roasted Arabica coffee. For the classification methods, the LDA model performed better than KNR. Another focus of this doctoral research was to develop analytical tools based on Raman and NIR spectroscopy for real-time monitoring of the coffee roasting process by predicting chemical changes in coffee beans during roasting. Green coffee beans of Robusta and Arabica species were roasted at 240 °C for 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 minutes. Four process runs were performed for each coffee species. The spectra of the ground samples were taken using the two spectrometers and modeled by the KNR, partial least squares regression (PLSR), and multiple linear regression (MLR). All the models based on the NIR spectra provided satisfactory results for the prediction of chlorogenic acid, trigonelline, and DPPH radical scavenging activity with low relative root mean square error of prediction (pRMSEP < 9.469 %) and high R2 (> 0.916) values. Similarly, all the models based on the Raman spectra provided acceptable prediction accuracies for monitoring the dynamics of chlorogenic acid, trigonelline, and DPPH radical scavenging activity (pRMSEP < 7.849 % and R2> 0.944). In conclusion, this research proposes different approaches that would allow valuable decisions regarding coffee quality to be made quickly and efficiently. The study suggests using NIR spectroscopy to determine a coffee’s geographical origin and detect and quantify adulterants in roasted coffee. The findings reveal that the method could be a promising tool for routine coffee quality control applications in the coffee industry and other legal sectors. The study also proposes using different spectroscopic methods (NIR and Raman) to monitor a coffee roasting process. One can consider the presented approaches as essential steps toward optimizing the roasting process at an industrial scale as they permit instantaneously taking significant process decisions.
Entwicklung eines computergestützten Assessment-Tools zur Erfassung des Ernährungszustandes von Senioren
(2011) Ott-Renzer, Cornelia; Biesalski, Hans-Konrad
Introduction: Worldwide a demographic change of population is to be observed, thus also in Germany. Special expertise in geriatric diagnostics and therapy will gain in importance. Thereby, a special relevance comes up to determination of nutritional status by corresponding screening and assessment. Conventional assessment mostly equates to questionnaire methods. Purpose/Question: Focus was on developing a software (geroMAT-Malnutrition Assessment and Therapy for gerontologic patients) for identification Senior´s nutritional state, a kind of "management tool" for diagnostics and intervention. Methods: Investigation was open, cooperative and multi-centric, as well as clinical-experimentally invested. In three partial studies (I: Suitability of the MNA® as a reference method; II: Anthropo-metry, biochemistry, body composition; III: Food patterns and intake, not-nutritive factors) indicators of malnutrition were initially selected. In a final analysis Model I (prognosis of malnutritional risk) and accordingly Model II (prognosis of the MNA®) have been developed for their use in geroMAT. Results: Prevalence of malnutrition was (in this random sample) 5%, 44% were at risk and 51% were well nourished. Due to inhomogeneity in class range by assessment with the MNA®, modelling of a dichotomic risk variable ("RiskMal", homogeneous) occurred. All up 25 features and 12 (optional) additional items from the partial studies I-III could be generated and attached to further analysis. Model I prognosticated "RiskMal" reliably (auROC=.739). Although firstly Model II predicted MNA® well (r=0,5167), model quality could be improved even further by the well-chosen parametres of a feature subset selection for Models I and II (Í: r=0,822;II: r=0,6634). Discussion: The Models I/II reached the requirements made on developing geroMAT. According to the features, geroMAT would be multi-centric usable, simple to learn and operable, documentable and reproduceable, interprofessionally and without high expense, as well as modern. Advantages of geroMAT, compared with the MNA® lay in its detailedness and its choice of further options, its capture of documented information off the normal anamnesis process and the initiation or monitoring of individual interventions. Conclusions: Mean aim of the study, the identification of indicators and model development, was reached. Other model validation studies should follow before the final clinical practice of geroMAT.
Entwicklung und Charakterisierung verschiedener humaner 3D Fettgewebemodelle mit und ohne Hydrogelmatrix als in vivo nahe Alternative
(2023) Albrecht, Franziska Brigitte; Kluger, Petra
Humanes Fettgewebe sekretiert hunderte regulatorisch aktive Hormone, die bei der Entstehung und Manifestierung schwerwiegender Krankheiten wie Diabetes oder kardiovaskulären Erkrankungen beteiligt sind. Um die Vorhersagekraft von in vitro Modellen zu verbessern und die Komplexität des nativen Fettgewebes besser nachzubilden, werden dringend dreidimensionale (3D) in vivo nahe Fettgewebemodelle benötigt. Um solch ein flexibel anwendbares möglichst physiologisches Modell aufzubauen wurde in dieser Arbeit der Aufbau und die Evaluierung verschiedener 3D Fettgewebemodelle teils mit artifizieller Matrix angestrebt und im Anschluss mit dem nativen Zustand vergleichen. Beginnend wurden aus primärem humanem Fett Lobuli in verschiedenen Größenbereichen isoliert, kultiviert und der Zelltod sowie Zeichen der Lipolyse bestimmt. Es hat sich gezeigt, dass Lobuli mit einem Gewicht von 27 – 70 mg über 15 Tagen die stabilsten Ergebnisse geliefert haben und wurden somit tiefergehend analysiert. Bei der Analyse der Viabilität und Zellfunktionalität in Form der Lipidakkumulation und der Expression von Perilipin A konnte bewiesen werden, dass Lobuli als Fettgewebemodell genutzt werden können. Als weiteres Modell erfolgte die Etablierung von Sphäroiden aus humanen primären Stammzellen aus dem Fettgewebe (adipose-derived stem cells, ASC). Die Sphäroide wurden für 14 Tage adipogen differenziert und währenddessen die Veränderung des Volumens und der Rundheit sowie die Expression von Zelltodmarkern und das adipogene Differenzierungspotenzial betrachtet. Dabei haben Sphäroide mit mehr als 100.000 Zellen ihr Volumen über die Zeit verkleinert und Apoptose- und Nekrosemarker an den Tagen 0 und 14 gezeigt. Sphäroide bis 100.000 Zellen haben ihr Volumen während der Differenzierung leicht vergrößert und haben die meisten Lipide akkumuliert, Perilipin A und Kollagen Ⅵ exprimiert. Somit konnten ASC-basierte Sphäroide aus 10.000 Zellen, mit signifikanter Lipidzunahme, als weiteres Fettgewebemodell mit hohem Potenzial identifiziert werden. Im Anschluss erfolgte die Etablierung von methacrylierter Gelatine (GelMA) als Biomaterial für die Verkapselung von primären ASCs und reifen Adipozyten (adipocyte, AC) mit anschließendem extrusions-basierten 3D Druck. Es konnte gezeigt werden, dass die homogene Verteilung der lipidgefüllten ACs in eine wässrige Biotintenlösung nur durch schnelles Herunterkühlen auf Eis möglich war. Der anschließende 3D Druck hatte, weder auf die Viabilität noch Funktionalität bzw. adipogene Differenzierung der Zellen signifikante Einflüsse. Die additiv aufgebauten Modelle wiesen an Tag 1 und 8 bzw. 15 in der Lebend-Tot-Färbung keinen vermehrten Zelltod im Vergleich zu den manuellen Modellen auf. Die Färbung der intrazellulären Lipide und Perilipin A sowie die Glycerolfreisetzung als Funktionalitätsmarker, haben dies bestätigt. Im Vergleich zu nativem Gewebe haben differenzierte ASCs (diffASC) nach der Differenzierung signifikant weniger lipidpositive Zellen und freigesetztes Glycerol gezeigt. Auch morphologisch betrachtet, haben ACs in GelMA mehr Ähnlichkeiten zum nativen Gewebe als diffASCs. Aufgrund der begrenzten Langzeitstabilität von GelMA, des Ursprungs und der Vernetzung wurden Biomaterialien ohne tierischen Ursprung evaluiert. Dabei hat sich das niedrig acetylierte Polysaccharid Gellan Gum (GG) als vielversprechendes Material erwiesen. Es konnten stabile und transparente Hydrogele durch die Vernetzung mit divalenten Ionen im Zellkulturmedium aufgebaut werden. Die 1 %-igen Hydrogele waren weder bei indirekter noch direkter Testung zytotoxisch oder haben Monozyten aktiviert. Bestätigt wurde es durch die Laktatdehydrogenase (LDH)-Freisetzung, den Resazurinumsatz und eine Lebend-Tot-Färbung. Azelluläre Gele wurden über 98 Tagen ohne signifikante Veränderungen erhalten und zeigten für Fettgewebe geeignete Materialeigenschaften. Aufgrund dieser Langzeitstabilität konnten diffASCs für 98 Tage in GG kultiviert werden und zu univakuolären Fettzellen reifen. Dies wurde durch die Färbung von intrazellulären Lipiden und Perilipin A sowie der Leptinsekretion und Glycerolfreisetzung bewiesen. Verglichen mit nativem Gewebe wiesen die diffASCs eine vergleichbare, wenn auch kleinere Morphologie, ähnliche Anteile an lipidpositiven und univakuolären Zellen auf. Auf Basis von GG konnten ACs erfolgreich verkapselt und aufgebaut werden. Hierbei erwies sich eine GG Konzentration von 0,5 % als geeignet, da homogen gemischte Hydrogele mit hohem Resazurinumsatz und geringer Glycerolfreisetzung kultiviert werden konnten. Für den additive Aufbau der GG-basierten Modelle, fand eine Optimierung zur Biotinte statt, was durch die initiale Zugabe divalenter Ionen zur Erhöhung der Viskosität und damit Druckbarkeit ermöglicht wurde. Die additiv gefertigten Modelle zeigten keine signifikanten Unterschiede in Bezug auf Viabilität oder Funktionalität. Nach 32 Tagen in Kultur konnten univakuoläre und funktionale Zellen gezeigt werden. Durch die Erhöhung der GG Konzentration auf 1,5 % wurde ein erfolgreiches 6D Bioprintingverfahren etabliert. Auch hier zeigten die in GG verkapselten ASCs keine Viabilitäts-, Morphologie-, oder Differenzierungsunterschiede nach dem Druckprozess. Abschließend erfolgte ein Vergleich der etablierten Modelle wobei die Adipogenese der ASC-basierten Modelle und zum anderen der adipozytenspezifische Phänotyp evaluiert wurde. Hinsichtlich der Viabilität konnten keine Unterschiede festgestellt werden. Das adipogene Differenzierungspotenzial zeigte sich am stärksten im diffASC Hydrogel. Sowohl hier als auch in den Sphäroiden konnten eingelagerte Lipide ohne hormonelle Induktion mit Medium nachgewiesen werden. Die Betrachtung der adipozytenspezifischen Morphologie der ausgereiften Modelle erlaubte den Rückschluss, dass die Zellen in den Monolayern den unausgereiftesten Zustand in Form von multivakuolären, elongierten diffASCs mit Aktin-Stressfasern aufweisen. Die Zellen der anderen Modelle zeigen deutlich größere Lipidvakuolen mit einem abgerundeten Zytoskelett. Die quantitative Bestimmung der Lipid-, Leptin- und Glycerolmenge legt nahe, dass sich die Zellen, außer die im Monolayer, in einem Steady-State befinden, was die relative Genexpression adipozytenspezifischer Gene untermauert. Zusammengefasst wurden in dieser Arbeit sechs unterschiedliche Fettgewebemodelle entwickelt und mit nativem Gewebe verglichen. Jedes Modell weist individuelle Stärken auf, wodurch sich der Anwendungsbereich und die Forschungsfragen unterscheiden. Um ein möglichst in vivo-nahes Modell zu erreichen, sind diffASCs in GG Hydrogelen ein vielversprechendes, langzeitstabiles, ausgereiftes Fettgewebemodell und können daher als flexible Plattform für in vitro Testungen genutzt werden.
Entwicklung von datengetriebenen Auswerteverfahren zur Analyse und Schätzungder Reaktorleistung von Biogasanlagen
(2020) Beltramo, Tanja; Hitzmann, Bernd
The production of biogas is very complex process, which runs in some stages involving different microorganisms. Microbiological diversity of the process depends mainly on the composition of substrate and ambient conditions, such as process temperature. The fact is, the development and composition of the microbiological communities of the process are difficult to predict. Thus, the control and evaluation of such complex biological processes are very time consuming and expensive. In Germany the evaluation of the biogas plants can be performed according to the VDI-Norm 4630, which describes the methods for the evaluation of fermentation of organic materials including characterization of the substrate, sampling, collection of material data and fermentation tests. For that specially equipment and skilled personnel are required. Moreover, the evaluation procedure is very time consuming. That is why a new state-of-the-art alternative for the evaluation purposes is necessary to simplify and to speed up the assessment of the biogas production processes. The aim of this doctoral thesis is the development of a fast and reliable method for the evaluation of the biogas production processes. Therefore the mathematical modelling should identify significant process variables able to evaluate the whole process. For the optimization of mathematical models metaheuristic tools were used. In this doctoral thesis two different data sets were used – experimental data and simulated data. The experimental data were collected in projects “Biogas-Biocoenosis” (FKZ 22010711, Dr. Michael Klocke, Leibnitz-Institute für Agrartechnik und Bioökonomie e.V., Potsdam) and “Biogas-Enzyme” (FKZ 22027707, Dr. Monika Heiermann, Leibnitz-Institute für Agrartechnik und Bioökonomie e.V., Potsdam). The simulated data set was generated using the Anaerobic Digestion Model No.1 (ADM1). The chemical process variables were used as the independent process variable set, while the biogas production output represented the dependent process variable. Prediction of the biogas production was done using linear and nonlinear mathematic models. Here, Partial-Least-Square-Regression (PLSR), Locally-Weighted-Regression (LWR) and Artificial Neural Networks (ANN) were implemented. In order to identify the most significant undependable process variables optimization algorithms were used, Ant Colony Optimization (ACO) and Genetic Algorithm (GA). Prediction capacity was evaluated using two model evaluation variables, Root Mean Square Error (RMSE) and Coefficient of Determination (R2). Figure 1 in Supplementary represents the flow chart of the developed methodology applied for ADM1 generated data set. In Figure 2 (Supplementary) there is a flow chart of the developed methodology applied for the experimentally collected data. The developed approaches could be successfully used for the prediction of the desired process variable, biogas production rate. The variable selection done with the help of metaheuristic optimization algorithms improved the prediction results and reduced number of the independent process variables. Hydraulic retention time, dry matter, neutral detergent fibre, acid detergent fibre and n-butyric acid were identified as the most significant ones. The best prediction was obtained using ANN models. Here, the error of prediction was low and the coefficient of determination high. The successful implementation of the developed methodology proved mathematical models to be an effective alternative method capable to evaluate and to optimize complicated biological processes. Furthermore, it would be mandatory further experimental evaluation of the developed strategy, using the model-based process information.
Impact of oxygen on quality of white wine
(2013) Morozova, Ksenia; Schwack, Wolfgang
Today?s wine market is becoming more and more saturated. At given conditions, the understanding of the consumer needs and preferences determines the success of a wine producer. The value of white and rosé wines appreciated by the consumers lies in their fruity and fresh character. Wine oxidation is one of the major wine failures. Recently, it has been reported that up to 48% of the wines rated as faulty by judges in wine competitions exhibited off-flavours that can be linked to the erroneous management of oxygen. Wine exposure to oxygen is possible throughout the whole winemaking process and depends on process conditions and applied equipment. Oxygen can be dissolved in wine up to 8.8 mg/L at normal conditions. After dissolution, oxygen concentration in liquid phase is normally decreasing to undetectable content due to chemical reactions of molecular oxygen with other wine constituents. Wine oxidation is a complex process. The effects may vary significantly influenced by numerous factors, including a type of wine, operation, oxygen amount chemical composition of wine, pH, storage temperature, light exposure, metal content and redox state of wine. Oxygen has a potential to support positively, and subsequently to ruin aroma and colour of wine. Most white wines are negatively affected by small amounts of dissolved oxygen provoking rapid aroma loss and oxidative browning, thus decreasing attractiveness for consumers, whereas in red wines microoxygenation may help to stabilise wine colour and remove reductive off-flavours. Up to now, most of the research on wine oxidation was focused on experiments in model solutions. Although these studies deliver valuable information about oxidation mechanisms, there was, however, limited data published on real wine experiments linking analytical data and sensory analysis. Consequently, the background of the present investigations was comprehensive evaluation of the effectiveness of the various factors influencing wine oxidation in real wine medium, and finally the development of innovative strategy for quality improvement and shelf-life extension of white wines. Comparison of different methods for O2 and CO2 measurement in wine The initial part of the present work was to determine a reliable quantification method for dissolved oxygen quantification in wine medium. Since dissolved carbon dioxide present in wine may influence the oxygen concentration, its amount should also be taken into account. Thus, development of dissolved carbon dioxide determination was of major importance for the further experiments described in this thesis. The main aim of the first study was to give a review of the oxygen and carbon dioxide measurement principles and to compare several instruments for carbon dioxide and oxygen measurement in wine available in the market. For carbon dioxide determination, CarboQC, Orbisphere 3658 and agitation cylinder were used. Oxygen measurements were conducted with PreSens, OxyQC and Orbisphere 3650. Sample bottles were prepared with concentrations in the range from 0 to 2200 mg/L for carbon dioxide and from 0 to 12 mg/L for oxygen, respectively, dissolved in white wine of Müller Thurgau variety. O2 and CO2 measurements with six instruments were sequentially conducted at 20°C after 10 minutes shaking of trial bottles. Subsequently calculated concentrations of oxygen and carbon dioxide were used for correlation regression tests. From the data obtained for CO2, three investigated instruments showed good repeatable measurement results (R2=98%) in the range from 0 to 1500 mg/L. However, in the wine samples oversaturated with CO2 (>1500 mg/L at 20°C) CarboQC and Orbisphere 3658 showed significantly higher precision (R2=99%), compared to the agitation cylinder, due to carbon dioxide losses during filling of the cylinder. As for oxygen data, results obtained with each of the measurement devices (PreSens, OxyQC and Orbisphere 3650) demonstrated good correlation with the initial oxygen amount in wine samples (R2=98%) in the whole monitored range of dissolved oxygen concentrations. However, due to the flexibility of PreSens mini-sensor application inside the bottle and noninvasive measurement, this technique was found to be more advantageous, compared to the other two. Additionally, it allowed not only determination of dissolved oxygen, but also of the gaseous oxygen concentration in a bottle headspace. For these reasons, the PreSens device was chosen for further experiments. Effect of Headspace Volume and Iron and Copper Addition on Oxidation Processes in Model Solution and Riesling Wine: Chemical and Sensory Changes Since previous studies in model solutions published by other authors clearly showed the key role of iron and copper in oxidation reactions in wine, the main aim of this part of the thesis was to compare the effects of oxygen and iron and copper additions on oxygen consumption rate, sulphur dioxide and colour of bottled model solution and actual wine with similar properties. Model solution was prepared of deionised water, glycerol, (+)-tartaric acid, ethanol, gallic acid and potassium metabisulfite to reach a free sulphur dioxide concentration of 50 mg/L. 200 L Riesling wine (vintage 2010) was prepared using standard winemaking techniques and equipment, and was subsequently treated with potassium ferrocyanide to eliminate iron and copper. Model solution and Riesling wine were bottled in 500 mL bottles with and without small additions of iron (0.1 mg/L) and copper (0.05 mg/L). Oxygen concentration in bottles was adjusted using various headspace volumes (0, 50 and 100 mL for model solution, and 0, 20 and 40 mL for Riesling, respectively) full of ambient air. Iron and copper concentration, total consumed oxygen, sulphur dioxide, browning rate (E420) were monitored. Additionally, sensory analysis of bottled Riesling wines (triangle tests and descriptive analysis) was conducted after 90 and 240 days of storage. The results revealed major differences between model solution and real wine. In model solution the headspace volume and the metal addition contributed to significant changes in total consumed oxygen, colour, and free sulphur dioxide. The metal addition increased the rate of the molecular oxygen consumption and resulted in elevated consumption of free SO2. Enhanced colour was observed in all wines, where iron was added. The experiment with Riesling wine with similar parameters showed strong influence of the headspace volume. The rate of oxidative browning and oxygen consumption rate strongly correlated with the headspace volume at the bottling. In contrast to model solution experiment, addition of small concentrations of iron and copper did not contribute to the colour and oxygen consumption rate of Riesling wine. Sensory analysis showed that the wines bottled with 0, 20 mL and 40 mL HS volume became significantly different already after three months of storage at 15°C, which was proved by the triangle tests. Descriptive analysis after six and nine months of storage confirmed negative influence of headspace volume, thus proving the significance of oxygen ingress at bottling on wine quality. Moreover, traces of metals in Riesling wines even in the wines with no iron and copper addition were sufficient to initialise oxidation processes. However, since small iron and copper additions had significant impact on model solution, further studies with Riesling wine needed to be conducted. The Impact of Headspace Oxygen and Copper and Iron Addition on Oxygen Consumption Rate, Sulphur Dioxide Loss, Colour and Sensory Properties of Riesling Wine For further investigation of the impact of iron and copper on white wine oxidation, Riesling wine was bottled with the addition of 1 mg/L of iron and 0.5 mg/L of copper, which correspond to average iron and copper concentrations in wines of Baden-Württemberg. Oxygen concentrations were determined, as previously reported, by the headspace volume in the bottle (0 mL, 10 mL and 20 mL) full with ambient air. In contrast to the previous experiment, addition of 1 mg/L of iron and 0.5 mg/L of copper had significant influence on the oxygen consumption rate, on the loss of free SO2 during storage, and on the sensory changes in wine. Addition of iron and copper significantly catalysed the oxygen consumption. Free sulphur dioxide loss was found to be proportional to the total consumed oxygen after bottling. Moreover, in all wines with iron and copper addition free sulphur dioxide decay was significantly elevated compared to wines with no iron and copper addition. Although colour changes were not observed in wines after 90 days of storage, significant sensory changes were detected. Both oxygen and iron and copper addition made an impact on sensory evaluation of wines. At low oxygen concentration (0 mL headspace) metal addition had positive effect resulting in elimination reduced aromas. In contrast, for 10 mL and 20 mL headspace, wines with iron and copper addition showed lower scores in fruity, citrus, tropical aromas, and elevated scores in untypical aging and had pronounced oxidised character. The results of the study indicate that, in the case of Riesling wine, excessive oxygen exposure due to oxygen present in the headspace of the bottle should be avoided. Moreover, iron and copper concentration also seems to make significant impact on oxygen and SO2 consumption rates and on sensory perception of wines. These findings suggest that iron and copper concentrations should also be taken into account, when oxygen management strategy is defined. Effect of Headspace Volume, Ascorbic Acid and Sulphur Dioxide on Composition and Sensory Profile of Riesling Wine In the last part of the present work the effects of different oxygen and free SO2 levels, and ascorbic acid addition on the development of white wine were investigated. Riesling wine was bottled in 500 mL bottles with four different headspace volumes (0 mL, 10 mL, 20 mL, 30 mL), two levels of free SO2 (50 mg/L and 70 mg/L), and with and without ascorbic acid (250 mg/L) addition. Dissolved oxygen and the oxygen in headspace were measured in the resulting 17 wines. Free and total SO2 concentrations, ascorbic acid concentration, colour, redox potential, and antioxidative capacity were measured regularly in wine samples. After six months of storage, the wines were evaluated using sensory descriptive analysis. It was again proved that wine exposure to oxygen at bottling plays a key role in white wine development during storage. High oxygen ingress may greatly influence the redox state of wine and affect the important quality parameters including colour, free and total SO2, and the overall sensory quality of wine. Free and total sulphur dioxide loss and the decline in ascorbic acid could be linked to the total consumed oxygen content. It was observed that in the presence of ascorbic acid less sulphite was consumed. Ascorbic acid addition also contributed to the fruity and fresh character of the bottled wines, which indicates its strong antioxidant. However, when combined with high oxygen concentration, ascorbic acid addition promoted enhanced white wine browning. This was scavenged in the wines with higher free SO2 concentrations. Based on the data shown, careful control of the oxygen ingress during bottling is crucial for white wine quality. Ascorbic acid addition seems to have positive sensory effects on the development of wines during the post-bottling period. However, the possible wine browning, associated with ascorbic acid, should be taken into account. On the contrary, sulphur dioxide, in case of the Riesling wine studied, seems to be less effective to prevent negative sensory effects in wines due to excessive exposure to oxygen, but may simultaneously decrease oxidative browning in wines with ascorbic acid addition. In summary, low oxygen ingress at bottling combined with low iron and copper concentrations, moderate additions of ascorbic acid and sulphur dioxide seem to be a good oxygen management strategy and offer a good potential to improve quality and extent the shelf-life of white wines.
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.
Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.
Isolierung universell einsetzbarer und mikrobiologisch stabiler Sauerteigstarterkulturen durch spontane Fermentationen mit Amaranth.
(2009) Sterr, Yasemin Arzu; Schmidt, Herbert
Spontaneous fermented sourdoughs prepared from five amaranth flours were screened for the presence of lactic acid bacteria (LAB) that predominate the autochthonous microbiota and thus may be suitable as starter cultures. The doughs were fermented with daily backslopping on the laboratory scale for 10 days with a dough yield of 200 at 30°C. Every 24 hours, the pH-values and total titratable acidity degrees were determined and samples were analyzed for the presence of LAB and yeasts by cultural methods. The identity of the isolates was traced during the fermentation with RAPD-PCR and two different primers, and the strains were identified by sequence analysis of the 16S rDNA genes. The strains Lactobacillus plantarum RTa12, L. sakei RTa14, and Pediococcus pentosaceus RTa11 were selected and applied as starters in further laboratory fermentations. All strains were predominant in repeated experiments, both, as single strains and in combination. During the first 24 h, L. plantarum RTa12 and P. pentosaceus RTa11 grew quite similar. The pH-value dropped earlier with P. pentosaceus RTa11, while both strains gave the same pH-values after 10 h of fermentation. In the challenge test with the autochthonous mikrobiota both strains overgrew the other LAB of the spontaneous fermented dough within the first eight hours, and were therefore considered dominant over the resident microbiota. Influences of the incubation temperature on the fermentation characteristics were mainly assessed in the viable cell counts, the pH-values and the titratable acidity degrees at 25°C. The pH-values for both strains were at high incubation temperatures (30 and 35°C) during the fermentation lower than at 25°C, respectively. However, after 24 h of fermentation both strains reached a pH-value of approximately 4.0 after 24 h. Further sugar, organic acid, mannitol and ethanol profiles of fermented doughs were determined with HPLC. Mainly analyzed metabolites in the doughs were glucose, sucrose, lactate, and acetate. To compare the potential starter cultures with commercial available startercultures, fermentations with two industrial startercultures were performed for 24 h at 30°C and a dough yield of 200. Both strains were able to compete with the commercial available starter cultures concerning viable cell counts, total titratable acidity and pH-values. Because of the dominance of both strains in sourdough fermentations with amaranth, the ability for acidification in a short time, the capacity to compete with the autochthonous mikrobiota, the robustness against lesser effects of the environment, e. g. variation of the temperature, and at least because of the ability to compete with commercial available startercultures, thus, the characterized strains L. plantarum RTa12 and P. pentosaceus RTa11 are interesting candidates as starter cultures for amaranth sourdoughs.
Nutrition and tuberculosis in Ethiopia : the role of vitamin D2 derived from sun exposed oyster mushroom on the treatment outcomes of tuberculosis
(2019) Keflie, Tibebeselassie Seyoum; Biesalski, Hans-Konrad
Tuberculosis (TB) is an old infectious disease which causes ill-health among millions of people each year. Effective anti-TB drugs are available since 1950’s, but still the global burden of TB remains enormous. The disease is very complex and there is a need to look for supportive treatment to the standard anti-TB drugs. Cognizant of this, the present doctoral study was undertaken by giving emphasis on nutrition and TB in Ethiopia. The aim of this doctoral dissertation thesis was to deal with the nutritional situation of people with and without TB and come-up with solutions that could support the effort of combating TB. In this thesis, five papers (four published and one submitted) were included. The first paper encompassed the study of dietary and nutritional assessment. In this study, dietary inadequacy, poor nutritional quality and high risk of micro nutrient deficiencies were identified. The main dietary pattern included cereals, vegetables and legumes. About one-third of the population consumed animal source food (ASF). Malnutrition was the common problem in people with and without TB. This suggested that malnutrition may pave the way for TB. The case-control study in the second paper revealed that more than one-half of TB patients had vitamin A and zinc deficiencies. More than three-fourth of TB patients had below half of the energy fulfillment. The protein intake was above the average fulfillment, but most TB patients relied on cereal-based diets. Patients with TB used a larger proportion of proteins from oral feeding for oxidation and hence for energy production. About half of the patients were undernourished. Thus, vitamin A and zinc deficiencies along with protein-energy malnutrition need to be addressed in the management program of TB. The third paper included systematic review which explored the existence of vitamin D deficiency (VDD). Sunshine, which is very important for the synthesis of vitamin D under the skin is widely available in Africa throughout the year. Surprisingly, more than three-fourth of TB patients in Africa had VDD and vitamin D insufficiency (VDI). Statistically significant variables such as use of sun protection (lack of sun-exposure), inadequate dietary intake, low body mass index (BMI), high skin pigmentation, use of drugs (anti-retro viral and /or anti-TB), low socioeconomic status, rainy season, covering body skin with clothes, old age and co-morbidity were identified as the main predictor variables that hampered the status of vitamin D. Vitamin D can be obtained from dietary intakes apart from endogenous synthesis after sun exposure. Mushroom as such, is a potential non-animal source of vitamin D. The experimental study in the fourth paper revealed that sun-exposure significantly increased the content of vitamin D2 in oyster mushroom. Increasing the surface area for sun-exposure enhanced the production of vitamin D2. Other factors such as duration of sun-exposure and moisture content determined the production of vitamin D2. Exposing slices of oyster mushroom to direct sun for brief period provided enough vitamin D2 that could satisfy the current recommended dietary allowance (RDA) of vitamin D without any visible changes in color and texture. The study in the fifth paper was a randomized controlled trial and demonstrated for the first time the role of mushroom-derived vitamin D2 on the treatment outcomes of TB. Intervention with vitamin D2 derived from sun-exposed oyster mushrooms brought significant improvement in vitamin D status, clinical outcomes and immunological responses, but not in sputum smear and culture conversion. The intervention corrected VDD in more than one-third of TB patients. About one-third of the variability in TB score in the intervention group was accounted for by the change in the serum 25 hydroxy (OH) vitamin D level. There were also significant improvements in the serum IFN-gamma and cathelicidin LL-37 peptide levels after intervention. The balance of cytokines was skewed to TH1 responses due to high level of IFN-gamma. Thus, mushroom-derived vitamin D2 could serve as potential, safe, easily available and cost-effective adjunctive therapy for TB. Taken collectively, foods enriched with vitamin D need to be included in the national TB control program to support the first line anti-TB drugs, increase the cure rate and reduce the infectiousness of TB.
Promoting dietary diversification in the ASEAN region : exposing food taboos, and exploring the nutrient profiles of underutilized, indigenous food resources
(2020) Köhler, Realm; Biesalski, Hans-Konrad
The Association of Southeast Asian Nations (ASEAN) is composed of Brunei Darussalam, Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam. The ASEAN region is ailing from moderate to serious incidence of malnutrition. Among the member countries, Brunei Darussalam has the highest prevalence of child obesity (17.8%), while Malaysia has the highest prevalence of adult obesity (15.6%). Indonesia has the highest percentage of young children suffering from wasting at 13.5%. Lao PDR has the highest percentage of the undernourished in the population (16.5%), at the same time, having the highest percentage of stunted children under five years of age (43.85%). It also has the highest HHI score with 38.7, which corresponds to having a severe case of micronutrient deficiencies, and the highest death rates for both children under five years of age (63 deaths per 1,000 live births) and mothers (197 deaths per 100,000 live births). To fight malnutrition, nutrition-specific interventions address the immediate determinants of nutrition of specific vulnerable groups – young children, pregnant and lactating women, and others. Dietary diversification is an example of a nutrition-specific intervention. This dissertation was conducted to turn the spotlight towards the ASEAN region, its triple burden of malnutrition, and to dietary diversification as a sustainable way to lighten the load. It tackled one of the stumbling blocks to the acceptance of dietary diversification – food taboos, and one of the stepping stones towards its successful implementation – nutrient profiling of underutilized, indigenous resources in the region. This dissertation postulated that plant- and animal-based food taboos adhered to by pregnant, post-partum, and lactating Southeast Asian women can hinder dietary diversification in the most vulnerable and crucial moment of the first 1,000 days of life. The two review papers generated were the first to consolidate and showcase researches on food taboos covering the region. They highlighted the need for culture-sensitive health interventions to address maternal and child health problems that could lead to the attainment of the sustainable development goals of reducing the maternal and under-five mortality ratios and empowering women in Southeast Asia, as well as the priority health goals of the ASEAN. The underutilized, indigenous resources in the ASEAN region have the potential to be valuable components of a diversified diet. To prove this statement and to further promote dietary diversification, the dissertation tackled the nutrient profiling of the edible insects – Bombay locust (Patanga succincta), scarab beetle (Holotrichia sp.), house cricket (Acheta domesticus), and mulberry silkworm (Bombyx mori) from Thailand, and the sago grub (Rhynchophorus bilineatus) from Indonesia. For the pigmented rice varieties, the Camoros (red), Tinta (purple) and Malinao black rice from the Philippines were analyzed, while a review of pigmented rice varieties from Thailand was also conducted. The results of the analysis showed, and based on the Codex Alimentarius on food labelling, that the edible insects are “high in” protein and can be “sources of” or “high in” minerals. Also, data showed that the pigmented rice varieties from the Philippines and the pigmented Thai rice varieties have higher mineral and vitamin contents in comparison with white Jasmine rice. The findings in this dissertation have shown that edible insects and pigmented rice varieties can be added to diversify and improve the nutritional quality of people’s diets and to fight malnutrition from the household level. The novel research into indigenous food resources contributes to the advancement of knowledge in the field of entomology and biodiversity conservation, and of course, in food science and nutrition. Most importantly, the dissertation’s contribution to the promotion of dietary diversification in the hope of attaining improved human health and nutrition will benefit the whole ASEAN region.
Recombinant production and characterization of metalloproteins from bacterial pathogens and the innate immune response
(2024) Göbel, Katharina; Fritz, Günter
The challenges and potential solutions of drug development are highlighted by discussing the identification, production and characterization of potential new drug targets in this study. The successful development of new and specific pharmaceuticals requires that the target for the respective new drug is available as a pure and homogenous molecule in its native state. Typically, the target molecule is a protein. E.g. antibacterial drugs target proteins from a bacterial pathogen or in human diseases pharmaceuticals predominantly target proteins of signaling pathways or receptors. These proteins are usually not available directly from the organism itself and have to be produced in an expression host and purified to homogeneity. Despite the advances in the field of recombinant protein expression and purification many proteins are very difficult to produce and thus represent the major bottleneck in the development of new pharmaceuticals. In particular demanding is the expression of metalloproteins, which make up to 30% of all proteins coded in the human genome and represent a major challenge in recombinant protein production. Metalloproteins are a diverse class of proteins that is crucial for various biological processes. They play an important role in the regulation, catalysis, and maintenance of biomolecular structure. Alone, 10% of all human proteins contain zinc ions and 2% contain iron, and both metal ions are often inserted by specific but so far unknown chaperones impeding the recombinant production of correctly folded and active proteins. The challenges in studying these metalloproteins arise from their complex structures and the difficulty of their expression and isolation. To overcome these problems new approaches and solutions are highlighted and exemplified by the production and characterization of potential new drug targets in this study. The focus lies particularly on metalloproteins that play a role in infectious diseases. Global health challenges include the persistent threat of infectious diseases despite advances in healthcare, hygiene and therapeutics. The COVID-19 pandemic and rising antibiotic resistance are prime examples of the ongoing risks. This research focuses on three different proteins: (1) the maturation factor NqrM from the bacterial pathogen Vibrio cholerae, (2) the human regulator of the interferon response ubiquitin-specific protease 18 (USP18) and its interaction partners, as well as (3) the viral Papain-like protease (PLpro) from the pathogenic virus SARS-CoV-2. All three proteins belong to the class of metalloproteins and bind either iron as in the case of NqrM or zinc as for USP18 and PLpro. New methods and strategies were developed to produce, isolate and investigate these metalloproteins and since all three proteins represent potential drug targets the results presented here provide the basis for future drug development. The production of proteins requires the selection of appropriate expression host systems such as bacteria, yeast, mammalian cells, etc., depending on the desired application. The study emphasizes the versatility of expression host E. coli due to its well-studied genetics, rapid growth kinetics and ease of handling. However, challenges such as the lack of post-translational modifications can lead to the production of non-functional proteins. Optimization of expression strategies is crucial, and the study describes various factors affecting protein production, including protein engineering, growth conditions, media composition and induction parameters expanding and enhancing the well-established E.coli expression system also for very challenging target proteins. The successful isolation of the proteins formed the fundamental basis for a detailed functional and structural characterization of the proteins. The research presented here takes a forward approach and encompasses the new strategies in cloning, recombinant expression and purification of proteins from bacteria, viruses and humans, emphasizing the advantages and disadvantages of homo- and heterologous recombinant expression. The results obtained highlight also the need for extensive experimental testing to establish optimal conditions, particularly for challenging proteins such as the metalloproteins studied here.

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