Institut für Lebensmittelwissenschaft und Biotechnologie

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Analysis of aging-related changes and influencing factors on the metabolome of beef
(2023) Bischof, Greta; Gibis, Monika
Aging of beef is necessary to improve its flavor and tenderness. There are two most common aging types, dry-aging and wet-aging. Dry-aged beef is often associated with a higher eating quality than wet-aged beef. The term “dry-aged beef” is not legally defined, so authentication methods are needed to protect the consumers from food fraud. During beef aging, the metabolome of beef changes due to the postmortem metabolism. This dissertation focuses on the aging method as a postmortem process and the resulting changes in the metabolome. As a hypothesis of this study, it was postulated that the detection of these metabolic changes due to aging of beef is feasible by 1H NMR spectroscopy and based on these measurements the evaluation of an authentication model for the aging method of beef is possible. In order to test this hypothesis, a sample preparation and measurement method was developed and based on this, potential influencing factors such as sampling position in muscle, breed and sex were investigated on the metabolome of fresh and aged beef. In the first part of this thesis, the sample preparation and the 1H NMR measurement method were developed. In the sample preparation, the polar fraction of the metabolome was extracted from 200 mg of beef, allowing 24 samples to be prepared in parallel. The sample preparation and the measurement method were validated, and the first aged beef samples were analyzed to check if the aging-related changes in the metabolome could be detected by this method. In the second part of this thesis, the sampling position in the muscle were analyzed for changes or differences in the metabolome due to its location in the muscle. The results showed that the metabolome changes along the length of the M. longissimus thoracis et lumborum, but the influence of the aging type and aging time was more pronounced in the metabolome of beef. The comparison of the surface and the inner part of wet-aged and dry-aged beef showed that the metabolome of dry-aged beef differed greatly between the surface and the inner part, despite the exclusion of the moisture content by freeze-drying and the low microbial load. There were only slight differences between the surface and the inner part for wet-aged beef, which could be due to the influence of microbiota and their metabolites. Therefore, the sampling location in the M. longissimus thoracis et lumborum was determined as precisely as possible for the further studies. The muscles were cut into ten pieces from cranial to caudal and dry-aged or wet-aged for 0, 7, 14, 21, and 28 days, in duplicates. The third part of this thesis focuses on the potential influencing factors such as breed and sex of the animals. Fresh and aged beef samples from three cattle types (heifer, cow, and young bull) and two different breeds (‘Fleckvieh’ and ‘Schwarzbunt’) were analyzed by targeted and non-targeted 1H NMR spectroscopy. Both factors were shown to influence the metabolome of fresh and aged beef. Therefore, these factors had to be included in the authentication model based on both targeted and non-targeted model. The calculation of the authentication model was the main part of this thesis and showed a good prediction of cattle type, breed, aging time and aging type of beef. The authentication model was based on the combination of multiple models of PLS-R and PLS-DA. The model for predicting the cattle type showed an accuracy of 99 %, and the models for predicting the breed depending on the cattle type showed an accuracy of 100 %. Aging time could be predicted with an error of 2.28 days. The statistical models for aging type were separated by aging time based on the determination of aging time. The model for predicting the aging type of 28-day aged samples had an accuracy of 99 %. The other statistical models for predicting aging type were additionally separated by cattle type and breed, and their accuracy ranged from 90 % to 100 %. In conclusion, an authentication model to determine the cattle type, breed, aging time and aging type of beef was developed in this dissertation. Therefore, it is possible to authenticate beef samples using a single 1H NMR spectrum. In future studies, it would be useful to extend this authentication model to other samples of other breeds and influencing factors.
Applied molecular bioprocess control using RNA thermometers : exploiting temperature responsive elements for rhamnolipid production
(2022) Noll, Philipp; Hausmann, Rudolf
The highest titer reported for heterologous Rhamnolipid (RL) production is 14.9 g/L. However, biomass generation, as a large carbon sink, was a significant drawback in this process with roughly 50 more biomass than product produced. This problem is addressed in this thesis leveraging temperature as control variable and a molecular temperature sensor, an RNA thermometer (RNAT). RNAT generally refers to secondary loop structures, in the 5’ untranslated region of the mRNA, that form at certain temperatures and therefore regulate translation in dependence of temperature. The ROSE (repression of heat shock gene expression) RNAT evaluated in the first original research article in the heterologous system P. putida KT2440 pSynpro8oT_rhlAB originates from P. aeruginosa. The ROSE element regulates, in dependence of ambient temperature, the translation of rhlA and via a polar effect also the translation of rhlB therefore indirectly RL synthesis. It was found that in the ROSE RNAT-controlled system, the RL production rate was 60% higher at cultivations of 37°C than at 30°C. However, besides the regulatory effect of the RNAT, as revealed by control experiments, multiple unspecific metabolic effects may be equally responsible for the increase in production rate. After screening for even more efficient regulatory structures, a fourU RNAT was identified. Natively, this fourU RNAT regulates the expression of the heat shock gene agsA of Salmonella enterica and its regulatory capability can easily be modified by site-directed mutagenesis. The experimental data collected in the second original research article confirms the functionality of the fourU RNAT in the heterologous RL production system. The data suggested improved regulatory capabilities of the fourU RNAT compared to the ROSE element and a major effect of temperature on RL production rates and yields. The average RL production rate increased by a factor of 11 between 25°C and 38°C. Control experiments confirmed that a major part of this increase originates from the regulatory effect of the fourU RNAT rather than from an unspecific metabolic effect. With this system YP/X values well above 1 (about 1.4 gRL/gBM) could be achieved mitigating the problem of high biomass formation compared to product synthesis. Also, YP/S values of about 0.2 gRL/gGlc at elevated temperatures of 37-38°C were reached in shake flasks. The system was subsequently tested in a proof-of-concept bioreactor process involving a temperature switch. With this simple batch experiment and a temperature switch from 25°C to 38°C not only a partial decoupling of biomass formation from product synthesis was achieved but also an around 25% higher average specific rhamnolipid production rate reached compared to the so far best performing heterologous RL production process reported in literature (average specific production rate: 24 mg/(g h) vs. 32 mg/(g h)). However, to achieve higher titers while reducing side product formation a suitable feeding strategy and more complex temperature profiles may be required. Temperature variations in turn cause several metabolic changes, many of which are complex and interdependent. Models that describe biological processes as a function of temperature are thus essential for improved process understanding. The goal of the peer reviewed review article “Modeling and Exploiting Microbial Temperature Response”, shown in this thesis, was to present an overview of various temperature models, aid comprehension of model intent and to facilitate selection and application. Since not all metabolic interdependencies and mechanisms during temperature variation are known for the reasonable connection of input-output relationships, a suitable modeling approach seemed to be neural networks. Neural networks as black box models do not require mechanistic a priori knowledge but representative historic datasets. To collect training data, different temperature profiles or constant temperatures for a bioreactor process with P. putida KT2440 pSynpro8oT_rhlAB were applied and concentration curves for biomass, glucose and RL recorded. Subsequently, the data was fed into the neural network to compute RL titer as output. An exponential temperature profile yielded at the highest RL value of approx. 9 g (around 13 g/L) less biomass (around 12 g/L) than product. These values were reached after only 30 h consuming just 45 g of glucose. Hence, at this timepoint 36 weight-% of the consumed glucose could be assigned to mono-RL (YP/S = 0.19 gRL/gGlc) and biomass (YX/S = 0.17 gBM/gGlc. The so far best performing heterologous RL production process, yielded 23.2 g (14.9 g/L) mono-RL from >250 g of consumed glucose (YP/S = 0.10 gRL/gGlc) in >70 h using the same strain and medium but a constant temperature of 30°C.
Backmittel mit fermentativ angereicherten Hydrokolloiden
(2021) Seitter, Michael Friedrich Hermann; Hertel, Christian
Lactic acid bacteria (LAB) are involved in fermentation of sourdoughs and able to produce exopolysaccharides (EPS). Screening of 190 LAB of different species and genera showed that 82% are able to produce EPS. Whereby, 28% a strong or very strong production exhibited. It becomes evident that strains of species L. reuteri, L. sanfranciscensis, L. frumenti and L. pontis, could be identified as effective EPS producer. The molecular weight of the synthetized EPS was larger than 5*106 Dalton. Glucan was formed almost of L. reuteri strains. To identify the effect of commercial hydrocolloids on bread staling, baking trials were performed. The parameter crumb hardness using Texture-Profile-Analysis and retrogradation of starch using Differential Scanning Calorimetry were chosen. Staling of wheat breads was dependent on the flour quality. Breads produced using weak flours and straight dough method showed faster staling. Addition of isolated EPS produced by L. sanfranciscensis LTH 1729 (Glu/Fru ratio: 1:6) and LTH 2590 (Glu/Fru ratio: 1:45) was more effective in retarding the rate of staling compared to hydrocolloids guar gum and xanthan. Baking trials with chemical acidified sponges showed that swelling and endogenous enzyme activities exerts no positive effect on the rate of staling. In contrast to sponges with fermentative enriched EPS, which exhibits a delayed rate of staling. This effect could be verified in mixed wheat breads (rye : wheat, 50:50). Frozen storage of doughs revealed no influence on the rate of staling. Production of an EPS enriched dried sourdough (baking improver) using optimized fermentation conditions was performed using L. sanfranciscensis LTH 1729. 3% dosage of the baking improver showed similar staling rate compared to control, however with 2% higher water absorption. Thus, addition of hydrocolloids and EPS, respectively, leads to an increase in dough yield of 1 1.5%. The width-height ratio was comparable in all doughs, except the xanthan supplemented. After adjusting the doughs to 500 FE, all doughs showed similar results in measurements with Bohlin-Rheometer. Doughs with added hydrocolloids as well as EPS were less sticky. Fermented sponge doughs with enriched EPS showed higher stickiness compared to not enriched. This could be traced back to residual not metabolized amounts of sucrose. EPS addition affects extensibility of doughs less compared to gum guar and xanthan. Negative influence on dough structure using acidic sponges was compensated with EPS enriched ones. Addition of guar gum and xanthan effect in a viscosity increase during gelatinization. Whereas, EPS and EPS containing sponges showed no effect on viscosity. Frozen storage of 10 days reveals lower dough stability and gas retention. Doughs were less elastic and stickier. Dough resistance decreased and elasticity increased. By addition of EPS these effects could be compensated. The gas retention capability of EPS supplemented frozen doughs was identical not frozen ones. Addition of 3% baking improver produced by spray dried EPS enriched sourdough to doughs increased the water absorption by 2%, whereas almost no change on dough rheological parameters resulted. Dough stability and gas retention was considerably improved. Dough stickiness and resistance decreased. No effect in viscosity during gelatinization. Summarized, the results of the present work show the optimization and manufacturing of a “clean label” baking improver, produced thru EPS enriched fermentation of sourdoughs. As well as the application of the improver and the impact of on dough processing and fresh keeping of frozen dough and baked goods.
Bioethanol production from lignocellulosic biomass
(2023) Hoppert, Luis; Kölling, Ralf
The aim of this thesis was to develop a high gravity second-generation bioethanol process and investigate the effects of a high solid loading. The insights gained from the initial experiments helped to understand the underlying mechanism behind the limitations of a high solid loading. Based on these findings, strategies were developed to overcome these limitations.
Biotechnological conversion of lignocellulose hydrolyzates : model microorganisms for a bio-based economy
(2020) Horlamus, Felix; Hausmann, Rudolf
Lignocellulose has substantial potential as a carbon source in a bio-based economy. It is the most abundant renewable raw material on earth and is available in large quantities as waste from the agriculture, food and wood industry. It is composed mainly of the polymers lignin, cellulose and hemicellulose. In contrast to glucose derived from cellulose, hemicellulose sugars often remain unused although 60 billion tons of hemicelluloses are produced annually. Hemicelluloses are a group of heterogeneous polysaccharides consisting of different monomers such as D xylose, D arabinose, D mannose and D galactose. Lignocellulose is mostly depolymerized in order to obtain fermentable sugars. During the depolymerization process, inhibitors such as organic acids or furan aldehydes can be formed or released, which could be problematical for biotechnological processes. The aim of this thesis was to develop and evaluate bacterial-based biotechnological processes capable of using hemicellulose sugars as a source of carbon. First, Pseudomonas putida KT2440 was chosen. Pseudomonades are claimed as a promising chassis in biotechnology due to their versatile and robust metabolism. Unlike other Pseudomonades, the strain KT2440 is classified as biosafety level 1 in the American Type Culture Collection (ATCC). However, these bacteria can metabolize glucose as the only lignocellulose monosaccharide. Cellvibrio japonicus was the second selected bacterium. This strain is not yet established as a microbial host in biotechnology, but can degrade a huge portfolio of plant cell wall polysaccharides and is also classified as biosafety level 1 in ATCC. The topic of the first publication was to engineer P. putida KT2440 strains for metabolizing the hemicellulose monosaccharides xylose and arabinose and characterize their growth behavior. Initially, an arabinose metabolizing strain with the araBAD operon and a xylose metabolizing strain with xylAB operon was constructed. Later on, these strains were cultivated in minimal salt medium with glucose, xylose and arabinose as carbon sources in Erlenmeyer flasks. The recombinant P. putida KT2440 strains metabolized xylose and arabinose with high growth rates comparable to glucose. It turned out that both engineered strains were able to grow on both pentoses as well as on mixtures of glucose xylose and arabinose. The intent of the second publication was to evaluate P. putida KT2440 as a platform model organism for bioconversion of lignocellulose hydrolyzates. Strains were cultivated in minimal salt medium with several hydrolyzates as carbon source in Erlenmeyer flask and bioreactor. In addition, the growth-inhibiting effect of major toxic substances contained in lignocellulose hydrolyzates on P. putida KT2440 was analyzed via cultivation experiments. Several suitable hydrolyzates were figured out for this strain. Formic acid and acetic acid proved to be relatively unproblematic under pH neutral conditions, whereas furfural and hydroxymethylfurfural (HMF) had a negative effect on the bacterial growth. A diauxic-like growth behavior was revealed via fed batch bioreactor cultivations, since pentoses were almost not consumed with sufficient glucose supply. Consequently, feed-medium was added step-by-step in the next experiment. The applied feed profile did lead to an almost complete metabolization of xylose. The purpose of the third publication was to evaluate C. japonicus as a potential host strain for the one‐step bioconversion of xylans into rhamnolipids. Cultivation experiments were performed in Erlenmeyer flasks filled with minimal salt medium and containing different carbon sources. Furthermore, the strain was transformed with the plasmid pSynPro8oT carrying rhlA (encodes acetyltransferase) and rhlB (encodes rhamnosyltransferase I) to complete the rhamnolipid metabolism. The strain grew on all main lignocellulose monosaccharides as well as, on different xylans. Mono rhamnolipids were produced with the engineered strain using xylans as carbon source. This is particularly interesting as most industrially relevant bacteria are not able to depolymerize wood polymers. As the product yields were quite low, there are still many challenges in order to achieve an economically efficient process. Nevertheless, to the best of our knowledge, it is the first published one step bioconversion of hemicellulose polymers into rhamnolipids. In total, P. putida KT2440 turned out as a flexible and powerful model organism and two xylose and arabinose metabolizing strains were constructed. Moreover, bioreactor cultivations with lignocellulose hydrolyzates were performed and a feeding strategy to overcome diauxic-like growth behavior was presented. A proof of concept for a one-step bioconversion of xylans into rhamnolipids with a recombinant C. japonicus strain was successfully demonstrated.
Broad time‐dependent transcriptional activity of metabolic genes of E. coli O104:H4 strain C227/11Φcu in a soil microenvironment at low temperature
(2023) Detert, Katharina; Währer, Jonathan; Nieselt, Kay; Schmidt, Herbert
In the current study, metabolic genes and networks that influence the persistence of pathogenic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soil microenvironments at low temperature were investigated. The strain was incubated in alluvial loam (AL) and total RNA was prepared from samples at time point 0, and after 1 and 4 weeks. Differential transcriptomic analysis was performed by RNA sequencing analysis and values obtained at weeks 1 and 4 were compared to those of time point 0. We found differential expression of more than 1500 genes for either time point comparison. The two lists of differentially expressed genes were then subjected to gene set enrichment of Gene Ontology terms. In total, 17 GO gene sets and 3 Pfam domains were found to be enriched after 1 week. After 4 weeks, 17 GO gene sets and 7 Pfam domains were statistically enriched. Especially stress response genes and genes of the primary metabolism were particularly affected at both time points. Genes and gene sets for uptake of carbohydrates, amino acids were strongly upregulated, indicating adjustment to a low nutrient environment. The results of this transcriptome analysis show that persistence of C227/11Φcu in soils is associated with a complex interplay of metabolic networks.
Characterization and modulation of technofunctional properties of pea proteins
(2023) Moll, Pascal Bernd; Weiss, Jochen
Plant-derived ingredients for food formulation have gained increasing interest in recent years as animal products pose a higher burden on the environment. Among plant proteins, those from pea (Pisum sativum L.) are of particular interest because of their low allergenicity, low cost, high availability, and good reputation among consumers. However, the technofunctionality of pea proteins is often inferior to animal-derived proteins limiting a more widespread use in food products. These technofunctional properties include - among others - foaming, gelling, and binding of other ingredients and it depends on the food product, which functionality food scientists must utilize and optimize. Cost effective approaches to improve the technofunctionality of pea proteins are therefore desirable and would allow the industry to further implement the use of sustainable ingredients in foods. In line with these overall goals, the aim of the first section of this thesis was to characterize a commercial pea protein isolate and to modulate the physicochemical and technofunctional properties through homogenization for foaming application. The main goal of the second section was to mix pea proteins with pectin to obtain a suitable binder with desired properties for the application in meat alternatives. The mixing approach was based on previous research data that had shown that interacting protein-polysaccharide systems display a synergistic behaviour in terms of their functional properties. First section: Foams are two phase systems consisting of gas bubbles that are stabilized by surface-active ingredients such as proteins in the discontinuous, aqueous phase. The physico-chemical properties of proteins such as their solubility determines foaming performance. In Chapter I, a commercial pea protein isolate was fractionated into a water-soluble and a water-insoluble fraction for characterization. Although the two fractions were similar in protein composition, they showed distinct differences in physicochemical properties. For instance, the particle size of soluble pea proteins was around 40-50 µm at acidic pH (3-5), while no measurable particles were detected at neutral The insoluble pea proteins were large at pH 3 and 7 (> 80 µm) and ca. 40-50 µm close to their isoelectric point at pH 5. The results suggest that commercial pea protein isolates consisted of several fractions with differences in their physico-chemical properties. The yield of the water-insoluble fraction was higher and therefore used in Chapter II, where experimental results illustrated that dispersions of insoluble pea protein aggregates (5% w/w, pH 7) could be disrupted from 180 ± 40 µm (control) to 0.2 ± 0.0 µmm upon homogenization at pressures ≥ 125 MPa. This was attributed to a cleavage of intermolecular interactions such as disulphide bonds, hydrogen bonds, and hydrophobic interactions. The decrease in insoluble pea protein aggregate size was accompanied by an increase in solubility from 23 ± 1% to ≥ 80% that may be beneficial for its technofunctionality. Consequently, homogenization was applied to the same material at pH 3 and 5 with the aim of investigating its foaming performance in Chapter III. In general, unhomogenized dispersions of pea protein aggregates (5% w/w, pH 3 or 5) did not foam at both tested pH values due to large pea protein aggregates with low solubility and surface activity. At pH 3, the dissociation of pea protein aggregates into smaller, more soluble, and more surface-active proteins was responsible for a high foam capacity (FC = 360-520%) with medium foam stability as measured by drainage (FS = 19-30 min). Only a limited particle size reduction upon homogenization was observed at pH 5, which was close to the isoelectric point of the pea proteins. Nevertheless, the still large aggregates consisted of re-aggregated smaller protein particles that were able to form a smaller amount of rather stable foams with thick interfacial films (FC = 213-246%, FS = 32-42 min). Overall, homogenization of insoluble pea protein aggregates was shown to change its physicochemical properties thereby benefitting technofunctional properties such as foaming. Second section: Another technofunctionality of interest is binding of different structural elements in e.g., meat alternatives. For this, the binder must be i.) sticky to glue heterogeneous components together and ii.) able to readily solidify upon further processing thereby ensuring a coherent bulk matrix. In Chapter IV, the influence of pH (3.50, 4.75, 6.00) and biopolymer concentration (17.5-50.0% w/w) on the stickiness of a pea protein isolate – apple pectin mixture (mixing ratio r = 6:1) was investigated. It was found that biopolymer concentrations of 17.5-20.0% w/w led to low stickiness due to a lack of cohesive forces (WoA = 0.29-0.51 mJ). At high biopolymer concentrations of 40-50% w/w, the biopolymer mixtures were also not sticky because of adhesion being limited (WoA = 0.02-0.05 mJ). There was a good balance of adhesion and cohesion that facilitated a high stickiness (WoA = 0.48-0.65 mJ) at intermediate concentrations of 25-30% w/w, which was also indicated by a viscoelastic behavior (G’ ≈ G’’). At those concentrations, the mixtures at pH 6 were stickier due to increased swelling of the pea proteins. The importance of viscoelasticity for stickiness of biopolymer mixtures was confirmed in Chapter V, where pea protein isolate and apple pectin (25% w/w, pH 6) were mixed in different ratios r. Mixtures of pea protein and apple pectin and particularly the sample with r = 2:1 possessed high stickiness due to the development of a multiphase morphology that allowed for a good balance of adhesion and cohesion with distinct frequency dependency. Pea protein alone (r = 1:0, c = 25% w/w) had an elastic but soft texture with low stickiness due to limited viscous properties, whereas a sample solely consisting of apple pectin (r = 0:1, c = 25% w/w) was also not sticky because of its high cohesion and stiffness. The results of Chapter VI revealed that pea protein homogenization prior to mixing with apple pectin led to smaller protein particles in the blend that contributed to a higher cohesive strength. Interestingly, vacuum-dried pea proteins resulted in a higher network strength as this drying method prevented reaggregation of small protein particles to a higher extent as compared to freeze-drying. Overall, the mixture with homogenized and vacuum-dried pea proteins was nearly twice as sticky as the mixture with untreated pea proteins. In Chapter VII, sticky mixtures of different pea protein preparations (soluble, homogenized and unhomogenized pea proteins) and pectin (25% w/w, pH 6, r = 2:1) were tested for their ability to solidify upon different treatments, namely heating as well as the addition of transglutaminase, laccase, calcium, and combinations thereof. Calcium was found to facilitate crosslinking of pectin chains and thus induced solidification of the mixtures. For instance, the consistency coefficient K’ increased from 2800 ± 1000 Pasn for pea protein isolate – apple pectin mixtures to around 19000 Pasn when calcium was added. Heat treatment and transglutaminase did not lead to solidification indicating that pectin made up the continuous phase. Furthermore, laccase led to the highest degree of solidification when sugar beet pectin was used (K’ > 30000 Pasn) due to ferulic acid and pea protein tyrosine crosslinking. Consequently, the sticky mixture of pea protein and sugar beet pectin (25% w/w, pH 6, r = 2:1) with the addition of laccase for solidification was identified as the most suitable binder for a bacon type meat analogue, which was the object of the study carried out in Chapter VIII. This binder had the highest binding strength (W = 2.0-4.3 mJ) between textured protein, fat mimic, and both layers at 25 °C due to the introduction of covalent bonds by laccase within the binder and between the binder and the adherends. A control sample without laccase addition had lower binding properties (W = 0.7-1.0 mJ) and the binding strength of a methylcellulose hydrogel (6% w/w) serving as benchmark was only higher between two fat mimics at 70 °C (W = 1.8 ± 1.1 mJ) due to increased hydrophobic forces. Finally, the pea protein – sugar beet pectin binder (22.5% w/w, pH 6, r = 2:1) was tested in burger patty type meat analogues to glue textured vegetable protein and fat particles together (Chapter IX). The binder system did not influence the hardness of the burger patties suggesting that this property was governed by the structural elements and not the binder. However, the cohesiveness as determined by sensory analysis was found to be superior when the pea protein – sugar beet pectin binder was used (-0.7 ± 0.2) as compared to the methylcellulose benchmark (-2.9 ± 0.3). This was attributed to the sticky character of the biopolymer mixture that enabled improved binding of the different structural elements. Overall, this novel binder based on plant-derived ingredients was shown to be applicable in different meat alternatives. Last, Chapter X reviewed the functionality and binding mechanism of currently used binders in foods and showed that stickiness, hardening/solidification, and water holding capacity are of great importance. In many food products, the binder transitions from a sticky food glue to a solid matrix triggered by different process operations that depend on the characteristics of the applied binder. From the presented results, it can be concluded that pea proteins are useful functional ingredients in various application scenarios. The desired technofunctionality can be improved through different process operations such as fractionation, homogenization, or mixing with other plant-derived ingredients. For this, knowledge regarding structure-function relationship and other influential factors is needed. In some cases – such as in binders – process operations must be well orchestrated to induce structural transitions and therefore changes in functionality at the desired time during manufacturing. Overall, the results of this thesis contributed to a better understanding for a more widespread use of pea proteins to promote a more sustainable food system. The appended graphical abstract summarizes the key steps undertaken in this thesis to come to this conclusion.
Characterization of function and regulation of the subtilase cytotoxin and Shiga toxin of pathogenic Escherichia coli
(2021) Heinisch, Laura; Schmidt, Herbert
Food-borne diseases caused by enterohemorrhagic Escherichia coli (EHEC) constitute a great threat to human health worldwide. Pathogenicity of EHEC strongly depends on the ability to produce virulence factors such as amongst others bacterial toxins. One of these toxins are the so-called Shiga toxins (Stx), which is why EHEC are assigned to the group of Shiga toxin-producing Escherichia coli (STEC). Stx belong to the family of AB5 protein toxins consisting of two subunits. One of them, the StxA-subunit causes depurination of the 28S rRNA in eukaryotic ribosomes by exhibiting N-glycosidase activity subsequently leading to inhibition of the protein biosynthesis followed by apoptosis of the host cell. The second one is the homopentameric B-subunit, which mediates binding to the host cell surface via the receptor glycolipid globotriaosylceramide (Gb3). Besides Stx, the subtilase cytotoxin (SubAB) has been described in STEC in recent years. SubAB, also assigned to the family of AB5 toxins, generates its cytotoxic activity via cleavage of the endoplasmic chaperone binding immunoglobulin protein (BiP) by its A-subunit. This cleavage leads to an unfolded protein response, resulting in apoptosis of the host cell. The B-subunit forms a ring-like homopentameric structure which is responsible for the binding to the receptor N-glycolylneuraminic acid (Neu5Gc) and other O-glycans. Although the mode of cytotoxicity of AB5 toxins have been studied extensively, some mechanisms remain unsolved. The scope of this thesis was to analyze further the mode of action of AB5 toxins and the gene regulation of stx and subAB. Both publications included in this thesis combine the characterization of the cytotoxic activity of AB5 toxins, the regulation of their genes, their subunits, and the combination of subunits of Stx and SubAB. In the first publication the regulation of gene expression of AB5 toxins was investigated in more detail. In this study, the gene expression of subAB1 was analyzed with a luciferase reporter gene assay and by quantitative real-time polymerase chain reaction. To unravel the regulatory mechanisms, both the laboratory E. coli strain DH5α and the STEC O113:H21 strain TS18/08 were used. Expression of subAB1 and promoter activity was studied using standard cultivation methods. Moreover, this work shed light on the impact of the global regulatory proteins host factor of bacteriophage Qβ (Hfq) and histone-like nucleoid structuring protein (H-NS) on subAB1 gene expression. Therefore, isogenic deletion mutants of hfq and hns gene were generated in the respective strains. Afterwards, plasmid-based complementation was conducted to verify that the observed effects were due to the deletion. Analysis of subAB1 promoter activity revealed impact of both Hfq and H-NS during different growth phases in both strains. In addition, the influence of both regulatory proteins on the expression toxin genes in STEC strain TS18/08 was investigated. This study did not only focus on the expression of stx2a and subAB1, but also the gene expression of the gene of the cytolethal distending toxin V (cdtV) was analyzed. Interestingly, all three toxin genes studied were upregulated in the deletion mutants of Δhfq and Δhns. Those results demonstrate the impact of global regulatory proteins on AB5 toxin gene expression and show that all three toxin genes investigated are integrated into the same regulatory network. In the second publication, the mode of action of AB5 toxins on the example of Stx2a was analyzed in more detail. The paradigm of AB5 toxin was known as the receptor binding B-subunit which mediates uptake of the enzymatic A-subunit and the subsequent cytotoxic activity. Previous studies have questioned this paradigm by showing cytotoxic effects of the SubA-subunit in absence of its corresponding B-subunit. This work analyzed whether this cytotoxic effect of the A-subunit is not only true for SubAB, but also for Stx. Thus, seperate recombinant expression of StxA2a subunits and subsequent His tag-based purification was performed. Both StxA2a-His and StxB2a-His were analyzed on cytotoxicity separately or in combination with the other subunit. Strikingly, cytotoxic effects of the StxA2a-His was observed in the absence of its corresponding B-subunit cell-type independently on HeLa, Vero B4, and HCT-116 cells. Studies on the B-subunit revealed no cytotoxicity on all cell lines. Additionally, combinations of different A- and B-subunits of Stx2a and SubAB1 proteins were analyzed. The hybrid combination showed that the cytotoxic effect of StxA2a-His on HeLa and HCT-116 cells could be reduced in the presence of the SubB1-His. Contrary, the cytotoxic effects of SubA1- His were unaltered in combination with StxB2a-His. Those results give the assumption that the Stx2aA-subunit binds to a target cell receptor blocked by SubB1-His. Additional experiments on the binding capacity of the Stx2a-subunits to Gb3 revealed that while StxB2a-His was able to bind to the receptor, no binding of the recombinant A-subunit was observed. The results indicate a cytotoxic effect of StxA2a on different cell types in absence of its corresponding B-subunit, which is designated as “single-A” effect in this work. The role of this effect in STEC pathogenicity, the uptake mechanism and subsequent transport inside the host cells of StxA-subunit need to be further analyzed in the future.
Characterization of the effects of chia gels on wheat doughand bread rheology as well as the optimization of breadroll production with the Nelder-Mead simplex method
(2016) Zettel, Viktoria; Hitzmann, Bernd
Chia (Salvia hispanica L.) is becoming increasingly popular as ingredient for baked goods. The aim of the first part of this thesis was to investigate the influence of gel from ground chia on the rheology of different wheat dough systems and the resulting baked goods. The evaluated products were wheat bread and sweet pan bread. The effects of chia incorporated as gel in wheat bread dough as hydrocolloid were characterized using empirical and fundamental rheological methods and differential scanning calorimetry. To avoid competition of starch and ground chia with respect to the water uptake, chia was incorporated as gel. The gel was prepared of ground chia with 5 g/g and 10 g/g water, respectively. The doughs were prepared with 1-3 % chia related to the amount of wheat flour. The effects of gel from ground chia were studied also as fat replacer in sweet pan breads. The main focus of the work was to study the effects of the fat substitution on the dough rheology. The dough rheology was characterized using a rotational rheometer and a Rheofermentometer. The end products were evaluated with a texture analyser and two samples were additionally evaluated with respect to their fatty acid profile. The substitution was secondly addressed to reduce the total amount of fat in the product and to improve the nutritional value of the products regarding the fatty acid composition. The fat was replaced in four steps, and the ratio among the ingredients was held constant to ensure a better comparability. Within this thesis it was shown that addition of gel from ground chia can affect wheat doughs and the resulting baked products in a positive way. The approach of using ground chia as gel seems to be fruitful to avoid competition between starch and chia with respect to the water uptake while the crumb formation during the baking process takes place. The evaluation of the pasting profiles of wheat flour suspensions with chia gel addition reinforced this assumption. The gel from ground chia affected the pasting properties in a way that the viscosities decreased with increasing amount of chia. The rheological properties of the doughs were affected in negative ways with respect to further processing by the addition of too high amounts of chia gel. The dough stability was reduced and the resulting baked products were less and irregular porous and therefore compact. All doughs showed weakening regarding the rheometer measurements, however the linear viscoelastic region was not affected. The frequency sweep measurements showed for all doughs a decrease with increasing content of gel from ground chia. The creep-recovery tests of the sweet pan bread doughs revealed that the zero viscosity η0 decreased and the creep compliance J0 increased with increasing chia gel content. The weakening of the doughs may not absolutely be caused by the incorporated chia, but by the additional water. There seems to be a kind of interaction between ground chia particles, wheat flour constituents and water, because nearly the same results were achieved for 2 % and 1 % of ground chia with 5 g/g and 10 g/g water, respectively. These experiments lead also to the best results for incorporating gel from ground chia to wheat breads. The best results for sweet pan breads were obtained with 25 % fat replacement through gel from ground chia. This gel was prepared of 2.3 g ground chia with 5 g/g water. Summarizing the incorporation of defined amounts of gel from ground chia has a positive effect on the rheology and the resulting baked products. The retrogradation of the baked products was decreased over storage and the dietary fibre content was increased. Thus chia acts like a hydrocolloid. The nutritional values of the evaluated baked products, wheat bread and sweet pan bread, were increased. For the sweet pan breads an increase of omega-3 fatty acids was determined. The resulting best sweet pan bread exhibited an amount of 5 % linolenic acid. Gel from ground chia can therefore be incorporated into bakery products as hydrocolloid and for improving the nutritional values regarding the dietary fibre and omega-3 fatty acid contents. Another part of the work was the optimization of the production parameters, proofing time and baking temperature, for bread rolls. The optimization was performed with the Nelder-Mead simplex method. The optimization was necessary for a new oven type, where the oven walls were coated with a ceramic, that increased the infrared radiation during the baking process. The quality criterion for the optimization were the specific volume, the baking loss, the colour saturation, crumb firmness as well as the elasticity of the bread rolls. Within 11 experiments the optimal baking result defined by the results of a conventional oven was obtained. The optimal processing parameters for the bread rolls were a proofing time at 117 minutes and a baking temperature of 215 °C for 16 minutes.
Dekontamination von pharmazeutischen Isolatoren mit verdampftem Wasserstoffperoxid : Charakterisierung von Einflussparametern und Optimierung des Maschinendesigns
(2010) Unger-Bimczok, Beatriz; Kottke, Volker
In the pharmaceutical industry sterile drugs, which can not be terminally sterilized, have to be prepared and handled under aseptic conditions. The application of isolator technology with physical separation of the process from the environment and the operator is commonly used. Prior to the aseptic processing, the inner isolator surfaces have to be treated with a sterilant to reduce the microbial contamination to a defined acceptable level. Today vaporized hydrogen peroxide (VPHP) is most commonly used for this purpose. Different parameters like hydrogen peroxide concentration, humidity, and condensation have an influence onto the microbicidal activity of the decontamination cycle. Also isolator design factors (e.g. material of construction, geometrical structure) can impact the inactivation results. The objective of the presented thesis was to investigate the mode of action of the VPHP and the relationship between different influencing cycle parameters in order to develop a recommendation for optimum decontamination conditions. An additional goal was to analyze the impact of different construction materials, surface finish and geometrical structures onto the inactivation efficiency of the sterilant to improve the design of aseptic processing machines regarding VPHP decontamination. For the studies an pharmaceutical isolator connected to a VPHP generator was used. Standard decontamination cycles with varying combinations of hydrogen peroxide and water concentration, cycle time and condensation levels were developed. Biological indicators (BIs) with defined initial spore population of Geobacillus stearothermophilus were exposed to the different VPHP cycles. By determination of inactivation kinetics for the microbial test challenge, the sporicidal activity for each set of cycle conditions was evaluated. The applied microbial methods were Most Probable Number (MPN) technique as well as the determination of decimal reduction times (D-values). BIs were not only tested when openly exposed to the sterilizing atmosphere, but also inside of defined gaps to challenge the penetration capability of the VPHP into small lumens under diffusive conditions. Different construction materials were inoculated with defined spore populations to investigate the resistance behaviour of the spores on varying surfaces. Supplementary the physico-chemical characteristics of the respective materials were analyzed in detail to draw conclusions regarding correlation of surface quality and inactivation properties. The results demonstrate that the decisive factor for a successful decontamination is the overall microscopic interaction with the bioburden on the surface. It is shown, that the microcondensation in the sub-visible range is effective for good inactivation performance and that further condensation in the visible range does not enhance the microbicidal activity. The data illustrate that the microbial inactivation is accelerated by increasing hydrogen peroxide concentration. An H2O2 level of 800 ppm ensures a sufficient deposition of sterilant onto the surface and results in excellent and reproducible kill. For sterilant levels > 800 ppm no further improvement in inactivation is detectable. It is shown that for openly exposed BIs a lower H2O2 level (400 ppm) can be compensated by higher humidity. The elevated water content in the decontamination atmosphere promotes the sterilant deposition. The higher the hydrogen peroxide level is, the more independent from humidity becomes the inactivation effect. For H2O2 levels of 800 ppm, the microbicidal activity of the VPHP is found to be independent from the water concentration. In contrast to the openly exposed BIs, for the inactivation of spores exposed under diffusive conditions inside of gaps, a lower hydrogen peroxide level can not be compensated by higher humidity. Solely the hydrogen peroxide concentration and the overall cycle duration are able to influence the decontamination success inside of the trenches. It is demonstrated that in principle complex structures can be decontaminated by the means of VPHP but the penetration capability is limited. The inactivation is impeded with decreasing gap cross section and with increasing gap depth. It is shown that different construction materials and surface textures have an impact onto the resistance behaviour of spores towards VPHP.
Design and evaluation of a 3D‐printed, lab‐scale perfusion bioreactor for novel biotechnological applications
(2023) Merkel, Manuel; Noll, Philipp; Lilge, Lars; Hausmann, Rudolf; Henkel, Marius
3D‐printing increased in significance for biotechnological research as new applications like lab‐on‐a‐chip systems, cell culture devices or 3D‐printed foods were uncovered. Besides mammalian cell culture, only few of those applications focus on the cultivation of microorganisms and none of these make use of the advantages of perfusion systems. One example for applying 3D‐printing for bioreactor development is the microbial utilization of alternative substrates derived from lignocellulose, where dilute carbon concentrations and harmful substances present a major challenge. Furthermore, quickly manufactured and affordable 3D‐printed bioreactors can accelerate early development phases through parallelization. In this work, a novel perfusion bioreactor system consisting of parts manufactured by fused filament fabrication (FFF) is presented and evaluated. Hydrophilic membranes are used for cell retention to allow the application of dilute substrates. Oxygen supply is provided by membrane diffusion via hydrophobic polytetrafluoroethylene membranes. An exemplary cultivation of Corynebacterium glutamicum ATCC 13032 supports the theoretical design by achieving competitive biomass concentrations of 18.4 g L−1 after 52 h. As a proof‐of‐concept for cultivation of microorganisms in perfusion mode, the described bioreactor system has application potential for bioconversion of multi‐component substrate‐streams in a lignocellulose‐based bioeconomy, for in‐situ product removal or design considerations of future applications for tissue cultures. Furthermore, this work provides a template‐based toolbox with instructions for creating reference systems in different application scenarios or tailor‐made bioreactor systems.
Detektion von Schadhefen in Wein mittels mit Flusszytometrie analysierter in situ Hybridisierung (Flow-FISH)
(2021) Willberger, Ilka Nadine; Scharfenberger-Schmeer, Maren
In oenological practice, mostly unpasteurised grape musts are used. This leads to an increased introduction of non-saccharomyces, which can have a lasting effect on the fermentation process. Disturbances in the fermentation process are usually only detected in practice on the basis of abnormalities in selected parameters such as sugar content or temperature or the occurrence of off-flavours. The fermenting yeast population may already be so affected at this point that intervention in the fermentation process can no longer prevent the occurrence of off-flavours in the end product or incomplete fermentation. With the help of flow cytometry, an efficient method using FISH (Fluorescence In Situ Hybridisation) was developed to detect and quantify the common representatives of the fermentation population such as Sacchoromyces cerevisiae and the harmful yeast population such as Hanseniaspora uvarum, Dekkera bruxellensis and Pichia anomala in the course of fermentation. Rapid detection enables countermeasures to be taken in good time before a harmful yeast population can have too great of an influence on the course of fermentation and the metabolites formed. Flow-FISH was established with pure cultures from strain collections in defined medium (YPD) and pasteurised white grape must. Samples are extracted and fixated directly from fermentation mixtures. For hybridisation, 18S- and 26S-rRNA probes with FITC-labelling are used. For the evaluation of the flowcytometric data, the Overton-subtraction method is used in this work. This allows a more accurate assessment of the hybridised cell population than the usual setting of a marker. For this purpose, an effective negative control with complementary sequence to the universal eukaryote probe (Euk516) is introduced. Subsequently, the method already known from the literature was optimised with regard to hybridisation conditions and cell fixation and thus adapted to the requirements of a quantitative flow cytometric analysis. With fixation in formaldehyde or in ethanol, fixation methods were developed that fulfil the requirements of both rapid and reliable fixation in the laboratory and rapid fixation in the cellar, if transport to the laboratory is not possible in a timely manner.Helper probes were designed to increase the fluorescence intensity. They are unlabelled and bind in the direct proximity of the specific probe. In all yeast species investigated, S. cerevisiae, H. uvarum, D. bruxellensis and P. anomala, the fluorescence intensity can be considerably increased by using the helper probes. In the case of D. bruxellensis and P. anomala, detection is only possible with the use of the helper probes. The helper probes allow the Flow-FISH assay to be used in a broader growth range of the yeast culture. Without helper probes, quantitative detection is limited to the middle logarithmic growth phase. With helper probes, hybridised cells can be reliably detected starting in the early logarithmic growth phase up until the stationary phase. This covers the critical phase of fermentative activity so that increasing contamination can be detected in the fermentation.The specificity of the probes is given. In part, there are slightly increased fluorescence intensities compared to the negative control, especially with the D. bruxellensis probe combination and non-specific yeasts, which can probably be attributed to increased binding due to the composition of this probe combination.The Flow-FISH assay is also reliable in mixtures of different yeast species and up to a cell count of 10³ cells / ml in the initial fermentation. This detection limit is also achieved by other methods in molecular biology for yeast detection. In contrast to most of these methods, Flow-FISH can also quantify the number of yeasts present. Additionally the use of the flow cytometer offers a simple variant to determine the total cell count of all yeasts in the fermentation. The detection limit of Flow-FISH allows detection before the damage threshold values of the yeasts examined are reached. The Flow-FISH method presented in this dissertation can also be applied to other yeast strains, some of which also originate from wild isolates. A transferability to native fermentations from oenological practice is given. It was possible to examine both spontaneous fermentations and inoculated fermentations in practice fermentations in steel tanks for their yeast population composition and to follow their development in the course of fermentation. Due to the use of flow cytometry and the helper probes and negative control used in this dissertation, the optimised Flow-FISH assay offers a stable basis for the continued development of a test system for use in oenological practice.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Development of an on-line process monitoring for yeast cultivations via 2D-fluorescence spectroscopy
(2019) Assawajaruwan, Supasuda; Hitzmann, Bernd
An optimum process is required in the field of food, pharmaceutical and biotechnological industry with the ultimate goal of achieving high productivity and high-quality products. In order to achieve this goal, there are many different parameters to be realized and controlled, e.g., physical, chemical and biological aspects of microbial bioprocesses. Microbial cultivations are a very complex process, therefore, reliable and efficient tools are required to receive as much real-time information for an on-line monitoring as possible, so that the processes can be controlled in time. The primary objective of this research was to apply a two-dimensional (2D) fluorescence spectroscopy to monitor glucose, ethanol and biomass concentrations of yeast cultivations. The measurement of one spectrum has 120 fluorescence intensity variables of excitation and emission wavelength combinations (WLCs) without consideration of the scattered light. To investigate which WLCs carry important and relevant information regarding the analyte concentrations, the three wavelength selection methods were implemented: a method based on loadings, variable importance in projection (VIP) and ant colony optimization. The five selected WLCs from each method for a particular analyte were evaluated by multiple linear regression (MLR) models. The selected WLCs, which showed the best predictive performance of the MLR models, were relevant to the analyte concentrations. Regarding the results of the MLR models, the most significant WLCs contained seven different excitation and emission wavelengths. They can be combined to have 38 WLCs for one spectrum based on the principle of fluorescence. They were in the area of NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN. The 38 WLCs were used to predict the glucose, ethanol and biomass concentrations via partial least squares (PLS) regression. The best prediction from the PLS models with 38 WLCs had the percentage of root mean square error of prediction (pRMSEP) in the range of 3.1-6.3 %, which was not significantly different from the PLS models with the 120 variables. Therefore, the specific fluorescence sensor for yeast cultivations could be built with less filters, which would make it a low-cost device. The following plan of the research goal was to investigate the attribute of fluorophores inside cells in real time using a 2D fluorescence spectrometer. The considered intracellular fluorophores, such as NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN were observed during the yeast cultivations under three different conditions: batch, fed-batch with the glucose pulse during a glucose growth phase (GP) and fed-batch with the glucose pulse during an ethanol growth phase (EP) after a diauxic shift. With the help of principal component analysis, the different states of the yeast cultivations, particularly the glucose pulse during EP, can be recognized and identified from the on-line fluorescence spectra. On the other hand, the change of the fluorescence spectra in the fed-batch process with the glucose pulse during GP was not recognizable. Remarkably, the intensities of the fluorophores due to the glucose pulse during EP did not change in the same direction. The fluorescence intensities of NADH and riboflavin increased, but the intensity of tryptophan, pyridoxine and FAD/FMN decreased. The conversion between tryptophan and NADH intensities was quantified as a proportional factor. It was calculated from the ratio of the area of NADH and tryptophan fluorescence intensity after the glucose addition until depletion. The proportional factor was independent on various glucose concentrations with the coefficient of determination, R2 = 0.999. The correlative intensity changes of these fluorophores demonstrate a metabolic switch from ethanol to glucose growth phase. Based on the previous experiments, a closed-loop control has been implemented for yeast cultivations. 2D fluorescence spectroscopy was applied for an on-line monitoring and control of yeast cultivations to attain pure oxidative metabolism. A glucose concentration is an important factor in a fed-batch process of Saccharomyces cerevisiae. Therefore, it has to be controlled under a critical concentration to avoid overflow metabolism and to gain high productivity of biomass. The characteristic of the NADH intensity can effectively identify the metabolic switch between oxidative and oxidoreductive states. Consequently, the feed rates were regulated using the NADH intensity as a metabolic signal. With this closed-loop control of the glucose concentration, a biomass yield was obtained at 0.5 gbiomass/gglucose. Additionally, ethanol production could be avoided during the controlled feeding phase. The fluorescence sensor with the signal of the NADH intensity has potential to control a glucose concentration under the critical value in real time. The experiments carried out show that 2D fluorescence spectroscopy has great potential in on-line monitoring and process control of the yeast cultivations. Consequently, it is promising to build up a compact and economical fluorescence sensor with the specific wavelengths using light-emitting diodes and photodiodes. The sensor would be a cost-effective and miniaturized device for routine analysis, which could be advantageous to real-time bioprocess monitoring.
Development of software sensors for on-line monitoring of baker’s yeast fermentation process
(2021) Yousefi-Darani, Abdolrahim; Hitzmann, Bernd
Software sensors and bioprocess are well-established research areas which have much to offer each other. Under the perspective of the software sensors area, bioprocess can be considered as a broad application area with a growing number of complex and challenging tasks to be dealt with, whose solutions can contribute to achieving high productivity and high-quality products. Although throughout the past years in the field of software sensors and bioprocess, progress has been quick and with a high degree of success, there is still a lack of inexpensive and reliable sensors for on-line state and parameter estimation. Therefore, the primary objective of this research was to design an inexpensive measurement system for on-line monitoring of ethanol production during the backer’s yeast cultivation process. The measurement system is based on commercially available metal oxide semiconductor gas sensors. From the bioreactor headspace, samples are pumped past the gas sensors array for 10 s every five minutes and the voltage changes of the sensors are measured. The signals from the gas sensor array showed a high correlation with ethanol concentration during cultivation process. In order to predict ethanol concentrations from the data of the gas sensor array, a principal component regression (PCR) model was developed. For the calibration procedure no off-line sampling was used. Instead, a theoretical model of the process is applied to simulate the ethanol production at any given time. The simulated ethanol concentrations were used as reference data for calibrating the response of the gas sensor array. The obtained results indicate that the model-based calibrated gas sensor array is able to predict ethanol concentrations during the cultivation process with a high accuracy (root mean square error of calibration as well as the percentage error for the validation sets were below 0.2 gL-1 and 7 %, respectively). However the predicted values are only available every five minutes. Therefore, the following plan of the research goal was to implement an estimation method for continues prediction of ethanol as well as glucose, biomass and the growth rates. For this reason, two nonlinear extensions of the Kalman filter namely the extended Kalman filter (EKF) and the unscented Kalman filter (UKF) were implemented separately for state and parameter estimation. Both prediction methods were validated on three different cultivation with variability of the substrate concentrations. The obtained results showed that both estimation algorithms show satisfactory results with respect to estimation of concentrations of substrates 6 and biomass as well as the growth rate parameters during the cultivation. However, despite the easier implementation producer of the UKF, this method shows more accurate prediction results compared to the EKF prediction method. Another focus of this study was to design and implement an on-line monitoring and control system for the volume evaluation of dough pieces during the proofing process of bread making. For this reason, a software sensor based on image processing was designed and implemented for measuring the dough volume. The control system consists of a fuzzy logic controller which takes into account the estimated volume. The controller is designed to maintain the volume of the dough pieces similar to the volume expansion of a dough piece in standard conditions during the proofing process by manipulating the temperature of the proofing chamber. Dough pieces with different amounts of backer’s yeast added in the ingredients and in different temperature starting states were prepared and proofed with the supervision of the software sensor and the fuzzy controller. The controller was evaluated by means of performance criteria and the final volume of the dough samples. The obtained results indicate that the performance of the system is very satisfactory with respect to volume control and set point deviation of the dough pieces.
Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem
(2005) Dal Bello, Fabio; Hertel, Christian
Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations.
Effect of frozen to fresh meat ratio in minced pork on its quality
(2023) Tomasevic, Igor; Witte, Franziska; Kühling, Rike Elisabeth; Berger, Lisa M.; Gibis, Monika; Weiss, Jochen; Röser, Anja; Upmann, Matthias; Joeres, Eike; Juadjur, Andreas; Bindrich, Ute; Heinz, Volker; Terjung, Nino
The meat industry is typically using a mixture of fresh and frozen meat batters for minced meat production. Our goal was to find the exact threshold for fresh to frozen meat ratio capable of controlling the meat temperature during processing, but without having an adverse effect on the sensory quality of minced pork. To achieve this, the percentage of frozen meat used for the minced pork production was increased from 0% (control) to 50% (maximum) in 10% increments. To keep the minced meat temperature in control and make the processing resistant to fat smearing, the addition of 30% of frozen meat to the meat batter is sufficient. The soluble protein content, instrumental cutting force, and the sensory perceived firmness, juiciness, and inner cohesion were not affected by the addition of frozen meat. However, it has contributed to a significant increase of the drip loss and the amount of non-intact cells (ANIC). With the addition of frozen meat into the minced pork, the compliance to ANIC regulation by the German regulatory authorities is technologically (practically) almost impossible.
Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Entwicklung und Validierung schneller und selektiver Verfahren zum Nachweis von Salmonella enterica, Cronobacter spp. und Bacillus cereus in Milcherzeugnissen
(2014) Zimmermann, Jennifer; Schmidt, Herbert
The presence of pathogens is a serious problem in the food industry and contaminations of food with Bacillus cereus, Cronobacter spp. and Salmonella enterica are responsible for a large number of diseases worldwide. Milk products like milk, whey or cream powder are widely used in industry as an ingredient in other foods. Therefore it requires a fast and reliable identification of pathogenic microorganisms. The official methods according to § 64 LFGB or ISO/TS 22964 apply a common scheme of pre-enrichment, selective enrichment, detection and confirmation and take between three and six days. The aim of this work was the development and validation of a real-time PCR based method, which identifies the existence of the three pathogens in dairy products within 24 hours. The identification of B. cereus, Cronobacter spp. and S. enterica with the developed TaqMan real-time PCR was performed using specific genetic characteristics and an internal amplification control to eliminate false negative results. For B. cereus, the groEL gene, which codes for a heat shock protein, was selected as target. For the detection of Cronobacter spp. the ompA gene and for S. enterica the invA gene was chosen. Both genes are responsible for the invasion of the pathogens in the human epithelial cells. The adaptation of the method to the food matrix and an optimization of the enrichment time were affected by an artificial contamination of various dry dairy products. It was possible to detect 105 cfu/g C. sakazakii and S. Enteritidis cells with an initial concentration of 100 cfu/g in reconstituted powdered infant formula after enrichment of six hours. To simulate a natural contamination, powdered infant formula was contaminated with desiccated C. sakazakii cells in various concentrations and analyzed with the developed real-time PCR method. It was possible to detect an inoculum concentration of 0.01 CFU/g dry stressed C. sakazakii cells at low aw values (0.22). The new TaqMan real-time PCR is fast, reliable and specific for the clearly detection of the three major pathogenic microorganisms in milk products and was carried out within 24 hours.
Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.

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