Browsing by Subject "Fluoreszenz"

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Fluorescence spectroscopy and chemometrics : an innovative approach for characterization of wheat flour and dough preparation
(2016) Ahmad, Muhammad Haseeb; Hitzmann, Bernd
Implementation of process analytical technologies (PAT) in food applications has attained a remarkable motivation due to higher quality and safety standards in this field. PAT applications also include rapid and non-invasive approaches which can be obtained from spectroscopic techniques. Fluorescence spectroscopy together with chemometrics is considered to be an outstanding analytical tool for fast and non-invasive technique for food analysis which can be used in various food applications on industrial scale. It is known for its sensitivity and specificity which can analyze the different foods and its ingredients while chemometrics helps to extract the useful information from the spectral data. The different chemometrics tools used for quantitative and qualitative analysis of spectral data, has increased the importance of this spectroscopic technique in generating the new ideas and hypothesis to develop new analytical methods which lead towards betterment in industrial operations for process and quality monitoring. In this doctoral project, fluorescence spectroscopic together with chemometric has been utilized to develop some new methods for determination of different parameters of wheat which provides the central idea of the thesis. First manuscript presents the potential of fluorescence spectroscopy to predict the analytical, rheological and baking parameters of different wheat flours by just taking the spectral signature without any sample preparation. Twelve different wheat flours milled from wheat cultivars were used to analyze the analytical, rheological and baking parameters using the conventional methods. These measured parameters were predicted from the spectral data taken for different wheat flours using genetic algorithm coupled with partial least square regression. The model obtained for protein, wet gluten and sedimentation value showing high R2 = 0.90, 0.92 and 0.81 respectively. Similarly, the rheological parameters like dough development time and water absorption were also predicted with low root mean square error of cross validation (RMSECV) and high R2 = 0.95 and 0.77 respectively while pasting temperature showed R2 = 0.78. Furthermore, moisture and volume of bread were predicted with high accuracy showing R2 = 0.86 and 0.95 respectively in the baking parameters. Other rheological and baking parameters like dough stability, softening, farinograph quality number, baking loss, crumb hardness and springiness were not predicted well due to poor correlation and high error. In the second paper, characterization of complex farinographic kneading process is performed by using the fluorescence spectroscopy in combination with chemometric tools. The aim of this investigation is to determine the impact of hydration of flour onto the spectral signals, classification of farinographic curve and separation of wheat flours based on their bread making performance. Secondly the middle curve of farinograph was predicted out of the fluorescence spectra using partial least square regression (PLSR) which can help to predict optimal dough development time. The spectra of the flour showed high intensities in protein, NADH and riboflavin regions which reduce to 36 %, 58 % and 61 % respectively after the hydration process depicting its influence due to structural changes in protein and oxidation of NADH. The farinographic curve was divided into four phases and principal component analysis (PCA) has been used to extract the qualitative information regarding the farinographic curve from the fluorescence spectra to categorize all farinographic phases into hydration, dough development, and stability and softening. Similarly, different pre-processing tools like standard normal variate and generalized least square weighting generate good separation of various wheat flours during the farinographic kneading process into different quality groups (E, A, B and C) on the basis of their bread baking performance from the spectral data using PCA. Additionally, PLSR was applied to predict the middle curve of farinograph out of spectral data showing a descent coefficient of determination R2 = 0.75 with RMSECV of 14 Brabender units. However, more research can lead towards the development of a sensor for determination of optimal dough development time. In another study, the nutritional parameters of 26 different types of wheat flour obtained from different vendors from the supermarket were predicted using fluorescence coupled with linear and non-linear chemometric tools. PCA applied on the spectral data for different types of the wheat flours showing a clear separation. On the other hand, PLSR was used to quantify the nutritional parameters of different types of wheat flours showing a good prediction for fat, moisture and carbohydrates using cross-validation, with a R2 of 0.88, 0.86 and 0.89, respectively whereas the protein, sucrose and salt contents presented a little correlation in PLSR. Therefore, locally weighted regression, a non-linear chemometric tool improves the prediction ability of all of the nutritional parameters by decreasing the error with an increasing R2. The energetic value, protein, fat, carbohydrate, moisture, sucrose, salt and saturated fatty acid contents showed R2 of 0.96, 0.93, 0.99, 0.99, 0.98, 0.88, 0.95, and 0.99 respectively, for different wheat flours. The aforementioned results clearly demonstrate the potential of the fluorescence spectroscopy in determination of analytical, rheological, baking and nutritional parameters of the wheat flours. They present that it can be used to characterize and categorize the farinographic kneading process, which is important in the bread-baking industry. More research in this direction can result in developing a sensor for predicting the quality parameters and processing operations in the cereal based industries rapidly and non-invasively which are important for regulatory and screening of the wheat on quality characteristics for marketing and end product evaluations.
Spatial combination of sensor data deriving from mobile platforms for precision farming applications
(2019) Zecha, Christoph Walter; Gerhards, Roland
This thesis combines optical sensors on a ground and on an aerial platform for field measurements in wheat, to identify nitrogen (N) levels, estimating biomass (BM) and predicting yield. The Multiplex Research (MP) fluorescence sensor was used for the first time in wheat. The individual objectives were: (i) Evaluation of different available sensors and sensor platforms used in Precision Farming (PF) to quantify the crop nutrition status, (ii) Acquisition of ground and aerial sensor data with two ground spectrometers, an aerial spectrometer and a ground fluorescence sensor, (iii) Development of effective post-processing methods for correction of the sensor data, (iv) Analysis and evaluation of the sensors with regard to the mapping of biomass, yield and nitrogen content in the plant, and (v) Yield simulation as a function of different sensor signals. This thesis contains three papers, published in international peer-reviewed journals. The first publication is a literature review on sensor platforms used in agricultural research. A subdivision of sensors and their applications was done, based on a detailed categorization model. It evaluates strengths and weaknesses, and discusses research results gathered with aerial and ground platforms with different sensors. Also, autonomous robots and swarm technologies suitable for PF tasks were reviewed. The second publication focuses on spectral and fluorescence sensors for BM, yield and N detection. The ground sensors were mounted on the Hohenheim research sensor platform “Sensicle”. A further spectrometer was installed in a fixed-wing Unmanned Aerial Vehicle (UAV). In this study, the sensors of the Sensicle and the UAV were used to determine plant characteristics and yield of three-year field trials at the research station Ihinger Hof, Renningen (Germany), an institution of the University of Hohenheim, Stuttgart (Germany). Winter wheat (Triticum aestivum L.) was sown on three research fields, with different N levels applied to each field. The measurements in the field were geo-referenced and logged with an absolute GPS accuracy of ±2.5 cm. The GPS data of the UAV was corrected based on the pitch and roll position of the UAV at each measurement. In the first step of the data analysis, raw data obtained from the sensors was post-processed and was converted into indices and ratios relating to plant characteristics. The converted ground sensor data were analysed, and the results of the correlations were interpreted related to the dependent variables (DV) BM weight, wheat yield and available N. The results showed significant positive correlations between the DV’s and the Sensicle sensor data. For the third paper, the UAV sensor data was included into the evaluations. The UAV data analysis revealed low significant results for only one field in the year 2011. A multirotor UAV was considered as a more viable aerial platform, that allows for more precision and higher payload. Thereby, the ground sensors showed their strength at a close measuring distance to the plant and a smaller measurement footprint. The results of the two ground spectrometers showed significant positive correlations between yield and the indices from CropSpec, NDVI (Normalised Difference Vegetation Index) and REIP (Red-Edge Inflection Point). Also, FERARI and SFR (Simple Fluorescence Ratio) of the MP fluorescence sensor were chosen for the yield prediction model analysis. With the available N, CropSpec and REIP correlated significantly. The BM weight correlated with REIP even at a very early growing stage (Z 31), and with SAVI (Soil-Adjusted Vegetation Index) at ripening stage (Z 85). REIP, FERARI and SFR showed high correlations to the available N, especially in June and July. The ratios and signals of the MP sensor were highly significant compared to the BM weight above Z 85. Both ground spectrometers are suitable for data comparison and data combination with the active MP fluorescence sensor. Through a combination of fluorescence ratios and spectrometer indices, linear models for the prediction of wheat yield were generated, correlating significantly over the course of the vegetative period for research field Lammwirt (LW) in 2012. The best model for field LW in 2012 was selected for cross-validation with the measurements of the fields Inneres Täle (IT) and Riech (RI) in 2011 and 2012. However, it was not significant. By exchanging only one spectral index with a fluorescence ratio in a similar linear model, it showed significant correlations. This work successfully proves the combination of different sensor ratios and indices for the detection of plant characteristics, offering better and more robust predictions and quantifications of field parameters without employing destructive methods. The MP sensor proved to be universally applicable, showing significant correlations to the investigated characteristics such as BM weight, wheat yield and available N.
Untersuchung einer Methode zur spezifischen Fluoreszenz – Markierung von Signalproteinen und deren Beobachtung in lebenden Escherichia coli (Einzelmolekültechnik & Perspektiven)
(2016) Ehrhard, Tanja Margret; Herten, Dirk-Peter
Malfunctions in signal transduction often cause diseases such as cancer and metabolic disorders. A thorough understanding of the relevant mechanisms of signal transduction is therefore an important requirement for the development of therapies and pharmaceuticals. In this thesis, a method was developed, which allows the observation of individual signaling proteins and their interactions in living cells. Therefore this method has advantages compared to molecular detection methods which are based on ensemble averages. As a model system for signal transduction, the bacterial chemotaxis with its regulator protein CheY was selected. The experimental studies were carried out with total internal reflection fluorescence microscopy (TIRFM), which requires a fluorescent labeling of the examined molecules. To ensure a specific and background reduced labeling, bright and photostable fluorescent ,tags are needed. In this work, the SNAP-tag system was used, which allows the use of different dyes. An advantage of this system is the possibility of using fluorescence-quenched benzyl guanine (BG)-dyes, which show a strong fluorescence only after binding to SNAP-tag. For development of the labeling method, the dyes Atto 620, Atto 633, Atto 655 and Atto 680 were analyzed in preliminary experiments regarding their fluorescence, photostability and blinking behavior. The thorough knowledge of these properties is essential for the correct interpretation of the experimental results. Dyes which are ideal for the method have a high fluorescent signal over a long observation time, and they are stable and do not interfere with the function of the target molecule. The preliminary investigations have shown that among the dyes tested, Atto 633 had the best photophysical properties for labeling with the SNAP-tag system and also the best cell permeability. This allows, under continuous laser excitation, to observe individual molecules for several seconds. In addition, the labeling efficiency was controlled by the protein expression, the dye concentration, and the incubation time of the dye. For single-molecule detection, a low labeling efficiency is of advantage since too high density of fluorescently labeled molecules makes the identification of individual molecules difficult. Subsequently, a labeling protocol was established which allows a specific, background- reduced fluorescence labeling of individual CheY proteins in living E. coli cells, without impairment of the protein’s functionality. Real-time detection with a time resolution of 30 milliseconds showed that it is possible to observe individual CheY molecules as a fluorescent point during the state of binding to an interaction partner. By means of numerical methods, the state of binding can be extracted from the fluorescence intensity traces as on/ off and their probability distribution can be determined. These quantitative studies gave indications on specific protein interactions, but no detailed information on binding times could be found. Different interactions of the protein, both specific and non-specific nature, could be the reason. Therefore, another important development of this labeling system would be the opportunity of simultaneous staining of two or more proteins with spectroscopically distinguishable fluorescent tags (e.g. CLIP-tag) to perform colocalization with alternating laser excitation. Another cause might be found in the nature of the dye itself. Laser- and temperature-dependent studies could provide further information concerning the behavior of the dyes. Thus, the described fluorescence labeling method provides a new approach for quantitative studies of protein interactions in living cells.
Untersuchungen zur autonomen und YidC-vermittelten Membraninsertion von Pf3 coat-Protein mit Hilfe Fluoreszenz-spektroskopischer Einzelmolekülmessungen
(2011) Schönbauer, Anne-Kathrin; Kuhn, Andreas
Pf3 coat is the capsid protein of the bacteriophage Pf3. The phage leaves the host cell by continuous extrusion without damaging the cell. The protein itself consists of 44 amino acid residues and has a rod-like shape. Because of its simple structure, the protein needs only the help of the insertase YidC to insert into the bacterial inner membrane. 3L-Pf3 coat, a protein mutant with three additional leucine residues in the center of the transmembrane region (TMD), has an increased hydrophobicity. It is independent of YidC and inserts into the membrane autonomously (Serek et al., 2004). In this work, a newly developed physical method was used to find out whether the elongation or the increased hydrophobicity accounts for the autonomous insertion of the protein. For this reason, two new protein mutants were constructed. Each mutant has only one of the changed properties of the 3L-Pf3 coat protein: GAT-Pf3 coat has an elongated TMD with three additional residues (glycine, alanin and threonine). The second mutant, 2M-Pf3 coat, shows an increased hydrophobicity due to the substitution of two alanine residues by two methionine residues at the positions 30 and 31. So it had an increased hydrophobicity like 3L-Pf3 coat. The above mentioned proteins, wt-Pf3 coat and its mutants, were modified with a fluorescent label to follow the proteins with optical methods. The Proteins were first modified with a single cysteine and then labeled by a fluorescent marker, Atto520 maleimid. Proteins with a labeled N-terminal tail were called NC-Pf3 coat, whereas CC-Pf3 coat had a labeled C-terminal tail. In addition, the orientation of the protein in the membrane was identified by quenching the fluorescence of the NC- and CC- labeled proteins. A new method employing single molecules was developed using fluorescence correlation spectroscopy. This method allows real time observations of binding and insertion of the protein into semisynthetical liposomes. By using fluorescent quenching the membrane insertion and binding were distinguished. It became clear that both the elongation of the TMD as well as an increased hydrophobicity play a crucial role in the autonomous insertion of the protein into the membrane. Therefore, the interaction between the hydrophobic region of the protein and the hydrophobic core region of the membrane is important for the binding of the protein and its insertion into the membrane.