Institut für Lebensmittelwissenschaft und Biotechnologie

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Process, structure and function relationship in ground meat
(2023) Berger, Lisa Marie; Weiss, Jochen
Ground beef has enjoyed high popularity with consumers because it is convenient to use and facilitates a rapid preparation of a large variety of different meals. In the production of ground meat, the particle size of the meat is systematically reduced, and the cell structures are partially disintegrated. Ideally, the original cellular meat or fat structure is preserved as much as possible so that important quality attributes are optimized. However, the effect of varying conditions and parameters in modern processes on the quality of ground meat has not yet been investigated in detail. According to the current German “Leitsätze für Fleisch und Fleischerzeugnisse”, hamburgers must not contain more than 20 Vol.% of non-intact cell structures to be sold without further declaration. Therefore, this work aimed to identify process, structure, and function relationships in ground meat production to facilitate a gentler processing of in particular hamburgers. To investigate these effects systematically, a standardized production method for hamburgers was developed and a pilot plant scale meat grinder was set up with the possibility to record process-relevant data. The relationship between the structure and functionality of ground meat was investigated using a model system with increasing amounts of added meat batter to simulate changes in meat structure due to cell disintegration. A new term, i.e., the amount of non-intact cells (ANIC), was introduced to quantify the amount of disintegrated meat cells during processing. It was shown that changes in the structure due to a higher or lower ANIC resulted in altered physicochemical and functional properties of the ground meat system. The effect of frozen meat content and temperature on the structure and function of hamburgers was investigated to verify the above-obtained correlation to an application-relevant setting. As the specific cutting resistance is significantly higher in frozen than in chilled meat, it was assumed, that the impact on the ground meat’s structure and function differed accordingly. Indeed, this could be verified. In hamburger manufacturing, it is common practice to re-fed imperfectly molded patties, e.g., in a frozen, coarsely crushed state. In contrast to those findings, the use of up to 20 % re-fed material in hamburger manufacturing did not result in any noticeable differences as neither the specific mechanical energy input (SME) nor the ANIC was significantly changed. It was thus demonstrated, that some raw material variations can have an impact on both structure and function of hamburgers. Especially, temperature effects and associated changes in the cutting resistance of the raw material had the strongest influence on structure and function of ground meat. However, if structural differences were found, they were not sufficient to manifest in differences in sensory evaluation. This means that the consumer perception and thus the quality of the hamburger was not influenced. The process parameters and their impact on the structure and function of hamburgers were studied by investigating the impact of the four main processing steps pre-grinding, mixing, grinding, and forming. An increased ANIC was determined with progressive processing, whereby the grinding steps accounted for the strongest increase. Mixing and forming were of minor importance for structural and functional changes. By varying the cutting set parameters, the influence of the cutting set compositions on the structure and function of hamburgers was assessed. The SME and the ANIC increased if more cutting levels were used due to higher shear stress applied to the meat. However, the hole plate properties did cause no or only negligible changes in the ANIC and SME. Although an impact of the cutting set composition on the structure could be found, no or only marginal effects on the function and the sensory and optical quality of the hamburgers were found. It can therefore be concluded that the shear forces acting on the meat during grinding have the strongest influence on the structure and function of beef. By reducing the acting shear forces, the grinding can be designed to be gentler resulting in lower ANIC. Despite the influence on the process-control (SME, pressure, torque) and the structural parameters (ANIC), it needs to be emphasized that the influence on the function and quality of the hamburgers is small in application-relevant ranges. In application-relevant ranges this relationship is only slightly pronounced. Comparable results were found, as raw material variations only partially caused structural, functional, and quality effects in the hamburgers. This in turn means that changes in structure cannot always be linked to a shift in perceived quality. In order to carry out an integrated evaluation of the product, structural parameters and quality parameters must be defined, assessed separately, and merged into a combined overall sample assessment.
Evaluation and method development for the biosynthesis of microbial lipopeptides by bacillus species
(2023) Vahidinasab, Maliheh; Hausmann, Rudolf
Microbial lipopeptides are secondary metabolites produced by bacteria and single-celled microorganisms. They consist of a cyclic or linear peptide chain linked to a lipid residue. Due to their high-foaming biosurfactant properties, they have various industrial applications such as in detergents, food emulsifiers, bioremediation, and enhanced oil recovery. Additionally, they possess other functional properties such as antifungal activity, making them an environmentally friendly alternative to synthetic fertilizers and fungicides. Bacillus species produce cyclic lipopeptides known for their potent antifungal activity, which makes them a potential source of bio-fungicides in agriculture. However, the production titer of wild-type Bacillus species does not meet industrial needs. Thereby, genetic modification of producer strains and bioprocess engineering can help increase the production of lipopeptides. Nevertheless, the regulation and basis of biosynthesis for Bacillus lipopeptides are still not completely understood, and ongoing research aims to enhance their production. In general, three main lipopeptide families, including surfactins, iturins, and fengycins are produced by different Bacillus species. Among these, surfactin as the strong biosurfactant is the most extensively studied lipopeptide produced by Bacillus species. The focus of this doctoral thesis was mainly to evaluate the biosynthesis of iturin and fengycin families, which are strong antimicrobial lipopeptides produced by Bacillus subtilis and Bacillus velezensis. This involved developing strains through genetic engineering and enhancing the lipopeptide titer by evaluating the cultivation medium. Initially, the entire genome of the bacteria used in this thesis was examined in terms of lipopeptide biosynthesis, and the structure and yield of the different produced lipopeptides were analyzed. Regarding the lipopeptide producer derivatives of the domesticated laboratory model strain B. subtilis 168 and B. subtilis 3NA, a spore deficient strain appropriate for bioreactor cultivation, surfactin is the lipopeptide with the highest yield, while plipastatin which is a member of fengycin family, is produced in lower quantities. In the present thesis, the biosynthesis of plipastatin by B. subtilis BMV9 as the lipopeptide producer derivative of strain 3NA was evaluated. The study aimed to convert BMV9 to a constitutive plipastatin mono-producer strain. In this sense, overexpressing plipastatin biosynthesis operon using the stronger constitutive Pveg promoter led to a five-fold increase in plipastatin production. Interestingly, it was observed that deletion of srfAA-AD operon in BMV9 and the constructed constitutive plipastatin producer strain has not improved plipastatin production. Therefore, it can be stated that presumably the biosynthesis of plipastatin may be positively influenced in a post-transcriptional manner by the surfactin synthetase or some of its subunits. However, the regulatory mechanism behind this effect remained unknown and requires further research. Another attempt to enhance the plipastatin biosynthesis in strain BMV9 was repairing the degQ expression. One main genome characterization of strains with B. subtilis 168 and 3NA background is that the pleiotropic degQ gene expression, which is known to have a positive effect on plipastatin biosynthesis, is silenced due to a mutation in the promoter area. However, while repair of degQ expression in BMV9 increased the plipastatin production, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) has not significantly increased the plipastatin yield. To further evaluate the impact of degQ expression on surfactin and plipastatin biosynthesis, two strains of B. subtilis were selected: JABs24, a lipopeptide producer derived from the 168 strain, and DSM10T, the wild-type strain expressing native degQ. The findings demonstrated that surfactin biosynthesis is negatively affected by DegQ-associated DegU regulation, while increased plipastatin biosynthesis is achieved in the presence of native degQ expression. In addition to production of lipopeptides, the DegU regulatory system also plays a role in the formation of secretory proteases. A comparison of extracellular protease activities between JABs24 and DSM10T showed that degQ expression led to DSM10T having five times higher protease activity than JABs24. Interestingly, production of extracellular proteases has not affected the stability of both plipastatin and surfactin during cultivation, suggesting that lipopeptides are less targeted by extracellular proteases. The identification of proficient wild-type strains is critical to the advancement of bio-fungicide in agriculture. Therefore, the subsequent approach of this thesis centered on the production of microbial lipopeptide by wild-type B. velezensis strains. Here, the lipopeptide productivity and antifungal ability of B. velezensis UTB96 was higher than B. velezensis FZB42, as a well-established strain for biocontrol of plant pathogens in agriculture. Furthermore, addition of certain amino acids stimulated lipopeptide production, and using a bioreactor system resulted in enhancement of lipopeptide production, especially iturin A by UTB96. Overall, the doctoral thesis evaluates the biosynthesis of antimicrobial lipopeptides produced by B. subtilis and B. velezensis. The study involves genetic engineering such as promoter exchange, deletion of genes involved in competing biosynthetic pathways and cultivation medium development with amino acid supplementation to enhance the lipopeptide titer. The thesis also identifies B. velezensis UTB96 as a promising candidate for further research to be used as a wild-type antifungal agent in agriculture.
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.
Plant protein gels as binders in meat product analogues
(2023) Herz, Eva Maria; Weiss, Jochen
In response to concerns about the environmental, ethical, and health impacts of meat consumption, plant-based meat analogues have become an important development in the food industry. To obtain prodcts with similar texture and nutritional properties, three major components of meat products (fibrous meat particles, adipose tissue, and myofibrillar meat proteins) need to be replicated. Furthermore, different binding mechanisms, such as heat, acid, and enzyme induction, and drying, are used to create coherent matrices for plant-based meat analogues. In Chapter 2, the study focuses on the use of soy protein gels as binders, with a particular emphasis on a combination of transglutaminase (TG) induced gels. The results indicate that TG-induced soy protein gels offer promising binding strength for meat analogues. Chapter 3 explores a combination of TG and slowly acidifying glucono-delta-lactone (GDL) as a binder, showing that this approach results in acidic gels with enhanced textural properties, making it suitable for acidic meat analogue products like fermented sausages. Chapter 4 applies previously studied soy protein gels as binders for sausage analogues. The research indicates that the choice of binder content influences the cohesiveness and hardness of the sausage analogues, with drying having a significant impact on hardness. In Chapter 5, hydrated gluten is used as a binder, leading to increased cohesiveness and springiness with rising binder content. It emphasizes the importance of adhesive properties between the binder and other particles in achieving desirable meat analogue texture. Overall, the thesis underscores that plant protein suspensions can serve as effective binders for meat analogue products, provided they exhibit both sufficient hardening through network formation and adhesive properties to ensure cohesiveness. It also discusses various formulation and process-based approaches to modulate the texture of meat analogue products.
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.
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.
Characterization of the aroma properties in fragrant rapeseed oil and aroma variation during critical roasting phase
(2023) Zhang, Youfeng; Zhang, Yanyan
Rapeseed oil is one of the third most-produced vegetable oil in the world, which is appreciated for its characteristic flavor and high nutritional value. Fragrant rapeseed oil (FRO) produced by a typical roasting process is popular for its characteristic aroma, which has an annual consumption exceeding 1.5 million tons. However, the changes in aroma blueprint of FRO during the typical roasting processing are still unclear, which challenges rapeseed oil quality and consumer acceptance. Accordingly, the aim of this work was to investigate the aroma characteristics and their precursors pyrolysis behavior of FRO to provide a basis and guidance for the control of FRO aroma quality during production processing. First, a systematic review on summarizing, comparing, and critiquing the literature regarding the flavor of rapeseed oil, especially about employed analysis techniques (i.e., extraction, qualitative, quantitative, sensorial, and chemometric methods), identified representative/off-flavor compounds, and effects of different treatments during the processes (dehulling, roasting, microwave, flavoring with herbs, refining, oil heating, and storage) was performed. One hundred and thirty-seven odorants found in rapeseed oil from literature are listed, including aldehydes, ketones, acids, esters, alcohols, phenols, pyrazines, furans, pyrrolines, indoles, pyridines, thiazoles, thiophenes, further S-containing compounds, nitriles, and alkenes, and possible formation pathways of some key aroma-active compounds are also proposed. Nevertheless, some of these compounds require further validation (e.g., nitriles) due to lack of recombination experiments in the previous work. To wrap up, advanced flavor analysis techniques should be evolved toward time-saving, portability, real-time monitoring, and visualization, which aims to obtain a “complete” flavor profile of rapeseed oil. Aparting from that, studies to elucidate the influence of key roasting processing on the formation of aroma-active compounds are needed to deepen understanding of factors resulting in flavor variations of rapeseed oil. Following, a systematic comparison among five flavor trapping techniques including solid-phase microextraction (SPME), SPME-Arrow, headspace stir bar sorptive extraction (HSSE), direct thermal desorption (DTD), and solvent-assisted flavor evaporation (SAFE) for hot-pressed rapeseed oil was conducted. Besides, methodological validation of these five approaches for 31 aroma standards found in rapeseed oil was conducted to compare their stability, reliability, and robustness. For the qualification of the odorants in hot-pressed rapeseed oil, SAFE gave the best performance, mainly due to the high sample volumes, but it performed worse than other methods regarding linearity, recovery, and repeatability. SPME-Arrow gave good performances in not only odorant extraction but also quantification, which is considered most suitable for quantifying odorants in hot-pressed rapeseed oil. Taking cost/performance ratio into account, SPME is still an efficient flavor extraction method. Multi-method combination of flavor capturing techniques might also be an option of aroma analysis for oil matrix. Afterwards, by application of the Sensomics approach the key odorants in representative commercial FRO samples were decoded. On the basis of the aroma blueprint, changes of overall aroma profiles of oils and their key odorants were studied and compared in different roasting conditions. To better simulate industrial conditions, high temperatures (150-200 ºC) were used in our roasting study, which was rarely studied before. Identification and quantitation of the key odorants in FRO were well performed by means of the Sensomics concept. Glucosinolate degradation products were a special kind of key odorants existing in rapeseed oil. Most of the odorants showed first rising and then decline trends as the roasting process progressed. Aroma profile results showed that high-temperature-short time and low-temperature-long time conditions could have similar effects on the aroma profiles of roasted rapeseed oils, which could provide a reference for the time cost savings in industrial production. To gain the fundamental knowledge of the aroma formation in FRO, the thermal degradation behavior of progoitrin (the main glucosinolate of rapeseed) and the corresponding generated volatile products were investigated in liquid (phosphate buffer at a pH value of 5.0, 7.0, or 9.0) and solid phase systems (sea sand and rapeseed powder). The highest thermal degradation rate of progoitrin at high temperatures (150-200 ºC) was observed at a pH value of 9.0, followed by sea sand and then rapeseed powder. It could be inferred that bimolecular nucleophilic substitution reaction (SN2) was mainly taken place under basic conditions. The highest degradation rate under basic conditions might result from the high nucleophilicity of present hydroxide ions. Under the applied conditions in this study, 2,4-pentadienenitrile was the major nitrile formed from progoitrin during thermal degradation at high temperature compared to l-cyano-2-hydroxy-3-butene, which might be less stable. The possible formation pathways of major S-containing (thiophenes) and N-containing (nitriles) volatile (flavor) compounds were proposed. Hydrogen sulfide, as a degradation product of glucosinolates, could act as a sulfur source to react further with glucose to generate thiophenes. Overall, the present work comprehensively documented the effects of thermal conditions and matrices on the aroma characteristics, aroma profiles, and key odorants of hot-pressed rapeseed oil, which could provide data and theoretical basis for the flavor control of FRO under thermal treatment at actual production temperatures (150-200 °C).
Application of nature-inspired optimization algorithms to improve the production efficiency of small and medium-sized bakeries
(2023) Babor, Md Majharul Islam; Hitzmann, Bernd
Increasing production efficiency through schedule optimization is one of the most influential topics in operations research that contributes to decision-making process. It is the concept of allocating tasks among available resources within the constraints of any manufacturing facility in order to minimize costs. It is carried out by a model that resembles real-world task distribution with variables and relevant constraints in order to complete a planned production. In addition to a model, an optimizer is required to assist in evaluating and improving the task allocation procedure in order to maximize overall production efficiency. The entire procedure is usually carried out on a computer, where these two distinct segments combine to form a solution framework for production planning and support decision-making in various manufacturing industries. Small and medium-sized bakeries lack access to cutting-edge tools, and most of their production schedules are based on personal experience. This makes a significant difference in production costs when compared to the large bakeries, as evidenced by their market dominance. In this study, a hybrid no-wait flow shop model is proposed to produce a production schedule based on actual data, featuring the constraints of the production environment in small and medium-sized bakeries. Several single-objective and multi-objective nature-inspired optimization algorithms were implemented to find efficient production schedules. While makespan is the most widely used quality criterion of production efficiency because it dominates production costs, high oven idle time in bakeries also wastes energy. Combining these quality criteria allows for additional cost reduction due to energy savings as well as shorter production time. Therefore, to obtain the efficient production plan, makespan and oven idle time were included in the objectives of optimization. To find the optimal production planning for an existing production line, particle swarm optimization, simulated annealing, and the Nawaz-Enscore-Ham algorithms were used. The weighting factor method was used to combine two objectives into a single objective. The classical optimization algorithms were found to be good enough at finding optimal schedules in a reasonable amount of time, reducing makespan by 29 % and oven idle time by 8 % of one of the analyzed production datasets. Nonetheless, the algorithms convergence was found to be poor, with a lower probability of obtaining the best or nearly the best result. In contrast, a modified particle swarm optimization (MPSO) proposed in this study demonstrated significant improvement in convergence with a higher probability of obtaining better results. To obtain trade-offs between two objectives, state-of-the-art multi-objective optimization algorithms, non-dominated sorting genetic algorithm (NSGA-II), strength Pareto evolutionary algorithm, generalized differential evolution, improved multi-objective particle swarm optimization (OMOPSO) and speed-constrained multi-objective particle swarm optimization (SMPSO) were implemented. Optimization algorithms provided efficient production planning with up to a 12 % reduction in makespan and a 26 % reduction in oven idle time based on data from different production days. The performance comparison revealed a significant difference between these multi-objective optimization algorithms, with NSGA-II performing best and OMOPSO and SMPSO performing worst. Proofing is a key processing stage that contributes to the quality of the final product by developing flavor and fluffiness texture in bread. However, the duration of proofing is uncertain due to the complex interaction of multiple parameters: yeast condition, temperature in the proofing chamber, and chemical composition of flour. Due to the uncertainty of proofing time, a production plan optimized with the shortest makespan can be significantly inefficient. The computational results show that the schedules with the shortest and nearly shortest makespan have a significant (up to 18 %) increase in makespan due to proofing time deviation from expected duration. In this thesis, a method for developing resilient production planning that takes into account uncertain proofing time is proposed, so that even if the deviation in proofing time is extreme, the fluctuation in makespan is minimal. The experimental results with a production dataset revealed a proactive production plan, with only 5 minutes longer than the shortest makespan, but only 21 min fluctuating in makespan due to varying the proofing time from -10 % to +10 % of actual proofing time. This study proposed a common framework for small and medium-sized bakeries to improve their production efficiency in three steps: collecting production data, simulating production planning with the hybrid no-wait flow shop model, and running the optimization algorithm. The study suggests to use MPSO for solving single objective optimization problem and NSGA-II for multi-objective optimization problem. Based on real bakery production data, the results revealed that existing plans were significantly inefficient and could be optimized in a reasonable computational time using a robust optimization algorithm. Implementing such a framework in small and medium-sized bakery manufacturing operations could help to achieve an efficient and resilient production system.
Production of CO₂ gas hydrates with its application in wheat bread making process
(2023) Srivastava, Shubhangi; Hitzmann, Bernd
The basic requirements necessary for gas hydrate (GH) formation are low temperature, high pressure, the presence of guest molecules, and the desired amounts of water molecules. The most common guest molecules used for the GH are ethane, methane, butane, propane, nitrogen, and carbon dioxide. Hydrate based technological applications almost always require rapid hydrate formation along with high gas uptake to be economically viable. One possible approach to achieving the same is the introduction of particular additives into the system. These additives are known as hydrate promoters. In recent times, amino acids have emerged as a highly effective class of promoters, and unlike surfactants, they promise a clean mode of kinetic action, i.e., no foam formation. Hence, the first part of the thesis dealt with the optimisation of GH formation with the application of amino acid promoters. The optimisation of the GH production was performed with different combinations of promoter ingredients namely cysteine, valine, leucine, and methionine. The amino acids leucine and methionine gave some positive results with the application of promoters for the production of GH therefore, these two amino acids were carried further for the experimentation purpose in the production of GH. Also, a combinational use of these amino acids (leucine and methionine) was studied to investigate the effect on percentage CO₂ retention in comparison to the normal water GH. The conventional baker’s yeast, Saccharomyces cerevisiae, remains the popular leavening agent in the bread baking industry. Carbon dioxide required for the rising of dough is produced by the metabolism of yeast with the consumption of sugars in the dough, which is a time and energy-consuming process. This research attempts to utilize carbon dioxide gas hydrate as a leavening agent in bread. Despite plentiful experiments on CO₂ gas hydrates in other fields, there is still an urge to carry out more analysis to elucidate various applications of GH in baking and positively validate its sustainability. The temperature stability of GH is important while baking due to the exposure to high temperatures during the various steps involved. In order to effectively use CO₂ GH as a leavening agent in the baking industry, a concise evaluation of the formation of CO₂ GH and its gas containment capacity should be adequately analysed and documented. Also, the effect of CO₂ GH properties by the addition of promoters should be taken into consideration as baking involves higher temperatures, and stabilising the GH at higher temperatures is an important criterion in the context of baking different products. Hence, the effect of a higher temperature of 90 ℃; on the CO₂ gas entrapment of the produced GH with promoters was studied. It was observed that the stability of GH decreases with an increase in temperature, but the addition of promoters, especially leucine + methionine + lecithin increased the CO₂ uptake during GH formation. Another part of the thesis was the application of GH in the baking bread with/without promoters and the study of physio chemical properties of bread. By varying the percentage of gas hydrates from 10-60 %, analysis of the performance of CO₂ GH as a leavening agent during baking was done. The effectiveness of GH bread was evaluated by comparing its characteristics to those of standard bread made with yeast. Also, a comparative evaluation was made for bread with and without promoters GH as leavening agents in terms of different physio chemical characteristics of the bread, such as moisture analysis, volume analysis, pore analysis, texture profile analysis, and baking loss. The results show that the bread with 20 % and 40 % GH obtained the best results in terms of volume and pore size. The next part of the thesis dealt with a comparative analysis of the partial replacement of yeast with CO₂ GH as leavening agents in bread baking. By partially eliminating the yeast, variations of bread dough were produced by the addition of GH in different percentages (20-70 %). The effectiveness of GH on bread manufacture was evaluated by comparing its characteristics to that of standard bread made with yeast. Once the bread was baked, the texture profile, volume, moisture content, and pore size were recorded to compare the leavening effect of GH with the standard recipe when partial addition of yeast was done. The best results combinations with respect to specific volume, pore analysis and hardness were obtained with 70 % GH + 50 % yeast and 70 % GH + 75 % yeast, respectively. As the final part of the thesis, the influence of additives on wheat bread baked with promoter induced CO₂ GH as leavening agents was studied. The additives used for the study were ascorbic acid (AC), egg white (EW), and rice flour (RF). These additives were added to the GH bread containing different amounts of GH (40, 60, and 70 % GH). Also, a combination of these additives in a wheat GH bread recipe was studied for each respective percentage of GH. Based on the results of the study, it was found that 70 % GH+ AC+EW+RF wheat bread was found to be the best in terms of textural analysis, pore size analysis, and other physiochemical parameters. Therefore, this research study will help us in understanding the application of GH in the bread baking process with replacement of conventional baking agents such as yeast.
Foam management in distillation plants
(2022) Heller, Daniel; Kölling, Ralf
Foam formation occurs for various substrates during distillation processes. A concrete prediction of the foam formation can only be approximated due to the physical, chemical, and biochemical complexity of the influencing factors. Foam formations affect both the design and the operation of distillation plants, due to various undesirable negative effects of foams on the process and the product. In this work, foam formations under boiling conditions in distillation plants of the spirit industry were investigated and different foam control methods for a holistic foam management system were developed. It was intended to make the distillation process more foam-resilient, and less subjected to foam-induced process disruption. To investigate foam formation in distillation processes lab-scale experiments and experiments on a column still were carried out. In the first step for a foam management system, the inhibition of foams by modification of substrate properties was investigated. In experiments various physical and rheological parameters of mashes as well as other foam-relevant parameters were determined. The aim was to derive a possible link with foam formation. It was shown that the viscosity and viscosity-determining compounds of the substrate have a significant influence on the foaming behavior of mashes. Rye mash was used as the demonstration medium in these experiments. In rye mash the compound pentosan was, in particular, influencing the viscosity. The experiments demonstrated, that the degradation of pentosans prior to distillation resulted in a decrease in viscosity and reduced foam accumulation. Next to foam-promoting substrate properties, foam-promoting operating conditions were investigated. The aim was to link passive process parameters to foam formations. On a laboratory scale, the foam formation in rye mashes was investigated as a function of passive process parameters and operating conditions, respectively, during distillation. It was demonstrated, that foam formations only occurred in a narrow temperature range of 89.5 – 98.2 °C. Additionally, foam formations were significantly lower with reduced energy input. The findings of the lab scale experiments were applied to develop foam-resilient heating profiles for distillations in the column still. In addition, it was focused on the separation effectiveness and economic efficiency of the new heating profiles, particularly with regard to process duration and the quality of the distillates obtained. Promising foam-resilient heating profiles were transferred to different substrates and their effectiveness was tested. Based on the findings, recommendations for distilleries for a foam-resilient distillation process could be derived, as well as predictions regarding effects on the product quality and process effectiveness. As the last step, active measures for foam destruction utilizing ultrasound were investigated. Ultrasound was introduced into the column at the level of the foam retention device of the distillation unit. The introduction of ultrasound into the column at the level of the foam retention device resulted in a reduction of foams. The observed decrease in foams was attributed to ultrasound-induced drainage of the liquid phase and subsequent destruction of the foam. However, also limitations of the method were found, e.g. limited area of effect. Further research is needed to validate the results and overcome these limitations. Overall, it was shown that foam management, which is not based on chemical defoamers, is possible in foam formation under boiling conditions in distillation processes. Several proposed measures, including inhibition, reduction, and destruction of foams were proposed. By combining them a holistic foam management is feasible.
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.
Erklärung der Strukturbildung und Trocknungskinetik von Einzeltropfen im Ultraschall-Levitator
(2022) Hülsmann, Ramona; Kohlus, Reinhard
In recent years, acoustic levitation has been increasingly used to investigate individual particles during the drying process. In this process, a droplet is positioned in a standing ultrasonic field in such a way that non-contact observation can take place. Within the scope of this work, an experimental setup for a levitator was designed and the drying and levitation behavior of various substances was investigated.
Teff-based functional probiotic beverage processed with selected probiotic bacteria
(2022) Alemneh, Sendeku Takele; Hitzmann, Bernd
The recent consumers’ demand have moved from the primary role of food to the healthier action of biologically active food components. For this purpose, production of probiotic functional foods through a fermentation process is the current particular interest. Dairy-based products have been used for probiotics delivery since a very long time; however, due to the drawbacks associated with them such as milk lactose indigestibility, the prevalence of cholesterol related to dairy products, and allergy to milk protein are limited their further utilization for probiotics delivery. Alternatively, cereals are becoming the favorite choices to using as fermentable substrates for the growth and delivery of probiotics. Also, vegetarianisms are increasing through time because of medical reasons. Whole grain cereals are readily available with important nutrient sources of phytochemicals, and other bioactive compounds. Cereals have functioned as an encapsulation materials to improve probiotic stability and their bioactive prebiotics selectively stimulate the growth of probiotics present in the gastrointestinal tract. Particularly, teff is a gluten free and its nutritional value is attractive with high dietary fiber. Amino acids find in teff are well balanced and contains high lysine content. Teff is a good source of essential fatty acids, fiber, minerals, and phytochemicals such as polyphenols and phytates. Consequently, the first primary objective of this research was to compare the quality attributes of whole grain teff flours grown in Ethiopia and South Africa for their proximate composition (moisture, protein, ash, fat, fiber, and carbohydrate), mineral contents (calcium, zinc, and iron), profiles of eighteen amino acids, pasting and thermal properties, and functional properties (water absorption capacity, oil absorption capacity, and swelling power), falling number and color. The proximate composition was examined using the methods of the European Commission Regulation (152/2009). Atomic spectrometer, ion-exchange chromatography, Rapid Visco-Analyzer, and Differential Scanning Calorimetry were used respectively to measure minerals, amino acids, pasting, and thermal properties. Correlation of the measured attributes were analyzed by Pearson correlation and principal component analysis. Significant (p < 0.05) differences were observed in most of the measured attributes between the two teff varieties; however, several significant (p < 0.01) correlations were obtained among the measured attributes by Pearson correlation and principal component analysis. The measured contents of moisture, protein, and zinc in South African teff variety were observed higher than the one grown in Ethiopia. However, much higher calcium and iron contents were found in Ethiopian teff variety. Ethiopian teff variety had showed higher values of foam stability, water absorption capacity, oil absorption capacity, and swelling power as compared to South African teff variety. Results from thermal and pasting properties showed that onset, peak and end temperatures, trough, final, and setback viscosities, as well as peak time, pasting temperature were observed higher in case of South African teff variety. The second primary objective was to examine the suitability of teff made substrates for their potential for the growing and delivering of selected probiotic strains of Lactiplantibacillus plantarum A6 (LPA6) and Lacticaseibacillus rhamnosus GG (LCGG). Single and co-culture fermentations were performed without pH adjustment. In 24 h fermentation with single strain of LPA6, cell count was increased to 8.35 log cfu/mL. Titratable acidity (TA) and pH were measured between 0.33 and 1.4 g/L, and 6.3 and 3.9, respectively. For the investigation of optimum fermentation process variables, Nelder-Mead simplex method was applied and found the optimum values for time and inoculum respectively as 15 h and 6 log cfu/mL. Afterwards, co-culture fermentation was performed by using the optimized process variables. As a result of co-culture fermentation, glucose was progressively consumed while lactic acid and acetic acid were produced. Cell counts of LPA6 and LCGG were able to grow to 8.42 and 8.25 log cfu/mL, respectively, which are a good counts as compared to the minimum required probiotics level of 6 log cfu/mL at consumption time. Findings showed that similar pH and TA values were attained in short time during co-culture fermentation compared to single culture fermentation. Also, without any addition teff substrate was found to be suitable for the growing and delivering of the tested probiotic strains of LPA6 and LCGG. Another focus of this research was to apply two-dimensional fluorescence spectroscopy for the on-line supervision of the fermentation process of teff-based substrate inoculated with LPA6 and LCGG. The fluorescence spectra were measured by using BioView sensor. Analysis of the fermentation process by using the conventional methods such as high performance liquid chromatography for determination of glucose and lactic acid, and using agar plate count for determination of cell counts are time consuming, labor intensive and costly methods. As an alternative the application of fluorescence spectroscopy coupled with partial least square regression and artificial neural network was applied for the on-line quantitative analysis of cell counts of LPA6 and LCGG, glucose, and lactic acid. For the prediction of cell counts of LPA6 and LCGG, the percentage errors of prediction were determined in the range of 2.5-4.5 %. Also, for lactic acid prediction, the percentage error was 7.7 %; however, percentage error for glucose prediction showed a rather high error value. This part of study verified that a two-dimensional fluorescence spectroscopy combined with partial least square regression and artificial neural network can be applied during fermentation process to predict cell counts of LPA6 and LCGG, and content of lactic acid with low uncertainty. Finally, this study was focused on the effect of refrigerator storage on the physicochemical characteristics and viability of LPA6 and LCGG in a teff-based probiotic beverage. As well as a 9-point hedonic scale was applied for sensory test of the beverage. For these determinations, a teff-based probiotic beverage was produced through the fermentation of whole grain teff flour inoculated with co-culture strains of LPA6 and LCGG. Then, the beverage was stored in refrigerator (4-6 ℃) for 25 days. Samples were taking every five days including the first day of storage to quantify cell counts of LPA6 and LCGG, pH, TA, glucose, acetic acid, lactic acid, and maltose. Over the storage time, cell counts of LPA6 and LCGG were decreased from 8.45 and 8.15 log cfu/mL to 8.28 and 7.86 log cfu/mL, respectively. While cell counts were decreased during storage, their cell counts are still observed above the minimum suggested level of 6-7 log cfu/mL at the time of consumption. Lactic acid, acetic acid, glucose, and maltose as well as TA were increased with reduction of pH over the storage time. Metabolic activities observed over the storage time indicated presence of active enzymes that were produced during fermentation process. As examined the beverage, E. coli, Pseudomonas aeruginosa, coagulase-positive Staphylococci, presumptive Bacillus cereus, Salmonella spp., and Listeria monocytogenes weren’t detected. Sensory test attributes of color, appearance, aroma, and taste of the beverage were observed between 6.2 and 6.9, which are in the accepted range. Six and above average score values of the sensory test attributes are considered to be accepted by the panelists. Overall, it could be possible to say the proposed aim for the production of a teff-based probiotic functional beverage was accomplished successfully.
New approaches in salami manufacture with in-situ exopolysaccharide-forming starter cultures
(2021) Velasco Cucaita, Lina Maria; Weiss, Jochen
Lactic acid bacteria have always been of great importance in the production of fermented sausages such as salami, as they contribute not only to microbial stability but also to acidity and flavor profiles of such products. Recently, exopolysaccharide (EPS)-forming starter cultures have attracted the interest of the food industry. EPS have water-binding, gelling, viscosity-increasing, as well as emulsifying properties and, due to these technofunctionalities, can contribute to the improvement of existing products as well as to new product developments. However, compared to hydrocolloids, which have similar functionalities, in-situ formed EPS do not have to be legally declared as ingredients on a package. Initial studies looking at the use of such cultures in spreadable, short-ripened raw sausages showed that the use of EPS-forming starter cultures can lead to a significant improvement in the spreadability of fat-reduced tea sausage and deeper acidified onion mettwurst (pH 5.1 instead of 5.6). However, no study to date has comprehensively addressed the use of in-situ EPS-forming starter cultures in sliceable, raw fermented sausage products such as salami, which differ significantly from spreadable raw sausage products in terms of product matrix. Since growth kinetics and acidification depend on the microorganism and the food matrix used, the growth and acidification behavior of selected homo- and heteropolysaccharide (HePS)-forming lactic acid bacteria as a function of different sugar concentrations (2.5 - 10 g/kg) was initially investigated. This was done to obtain an indication of the sugar concentration required in the raw sausage mass to achieve a target pH of 4.8-5.3 in the final product. Subsequently, the performance of two HePS-producing strains L. plantarum TMW 1.1478, and 1.25; and the two homopolysaccharide-producing lactic acid bacteria L. curvatus TMW 1.624 and L. sakei TMW 1.411 was investigated in a raw sausage model system (inoculation concentration 106 CFU/g), which, in addition to 25% pork back fat, 75% lean pork meat, also contained ascorbic acid (0.5 g/kg), nitrite curing salt (28 g/kg), and dextrose or sucrose (5 g/kg). Thereby, the strains to be used were specifically analyzed with regard to their suitability for EPS-formation under typical fermentation conditions prior to use in salami production. The latter was done qualitatively by confocal laser microscopy (CLSM), followed by semi-quantitative data interpretation using MATLAB. The results showed that all selected strains were able to produce EPS in the raw sausage model matrix. There, EPS were located on the surfaces of the proteins. Since presence of HePS, which are more complex in terms of chemical structure and are often charged, can lead to changes in the organization of protein matrices even when used in very small amounts due to e.g. electrostatic interactions, sausages were subsequently prepared with a HePS-forming (L. plantarum 1.1478) and a non-EPS-forming starter culture (L. sakei 1.2037; control). Moreover, the influence of different inoculation concentrations (107 and 109 CFU/g) on fermentation and associated HePS-formation, as well as their effect on quality parameters of the final products, were investigated. The selection of inoculation concentrations was governed by the hypothesis that higher inoculation concentrations could lead to a higher in-situ formed HePS amount in the raw sausage matrix and therefore to enhanced structural and thus organoleptic relevant effects. For this purpose, pork meat and fat-based raw sausages were prepared by adding and mixing spices, 0.5 g/kg Na-ascorbate, 5 g/kg sugar, the appropriate starter culture (107-109 CFU/g), and in the end 28 g/kg nitrite curing salt. Afterwards, the mass was filled, fermented (24 °C), smoked, and dried to a weight loss of 31%. In addition to pH and bacterial plate counts, the formed EPS were detected by CLSM and the influence of the formed HePS on the texture of the raw sausages was analyzed by texture profile analysis (at 16, 23, 27, and 31% weight loss) and further evaluated in a sensory evaluation for the attributes of consistency and taste. Although no significant differences were found with respect to the detected HePS and the inoculation concentration used, dependencies emerged with respect to product quality. Raw sausages produced with the HePS-producing starter culture L. plantarum 1.1478 were significantly (p < 0.05) softer than the corresponding control samples. This effect was more pronounced the higher the inoculation concentrations, which was also reflected in the sensory evaluation of samples. Semi-quantitative data interpretation of the CLSM images revealed that the HePS were predominantly formed during the first 72 h of fermentation at 24 °C, until the final pH of 4.95 ± 0.05 was reached. Although there was no clear preference in the sensory analysis performed, raw sausages with a firmer consistency are generally preferred in Germany. Accordingly, the use of an EPS-forming culture could, depending on the market, also have a negative impact on product properties. To gain a better understanding of the observed results and the influence of process conditions on in-situ HePS-formation and its effects on the quality of sliceable raw fermented sausages, the temperatures of the fermentation phase were varied in a further study. In addition to the 24 °C already examined, an additional incubation temperature of 16 °C, commonly used in the production of raw sausages, and a low temperature incubation of 10 °C were chosen, since increased stress conditions are often associated with increased EPS formation. Raw sausages inoculated with L. plantarum 1.1478 or L. sakei 1.2037 (108 CFU/mL) were fermented at 10, 16, or 24 °C within the first 7 days and then dried under the same conditions (14 °C, controlled relative humidity) until a weight loss of 31% was reached. Microbial growth, pH, and weight loss development were monitored, EPS detected with CLSM, and products further characterized by texture profile analysis and a sensory test. Here, texture profile analysis was performed not only from the final product, but also after 21% and 26% weight loss to better understand the influence of the in-situ produced HePS. Differences were found depending on the starter culture used as well as on the fermentation temperature. Products manufactured with the non-EPS-forming strain L. sakei 1.2037 reached the target weight loss of 31% slightly faster than products manufactured with the HePS-former L. plantarum 1.1478. In both products, the final weight loss of 31% was reached faster at an initial fermentation temperature of 24 °C than at the lower fermentation temperatures. A correlation of temperatures with the amount of HePS formed could not be conclusively proven using semi-quantitative data analysis of CLSM images because matrix effects complicated the determination. However, texture profile analysis results showed a difference between products fermented at 24 °C and those fermented at cooler temperatures. In addition, significant (p < 0.05) differences were again observed between products with (softer) and without (harder) HePS-forming starter cultures at weight losses at or above 21%. These results were confirmed in the final sensory evaluation of the products (pH 4.89 - 5.01; 31% weight loss). In summary, the results of this thesis show that the use of a HePS-forming starter culture in sliceable raw fermented sausage can induce specific structural and textural changes. HePS-formation and associated quality attributes may be modulated via the inoculation concentration and control of processing parameters such as fermentation temperature. The texture softening observed in the present work, can be positively or negatively associated with the product depending on the target country and market. Taken together, results of this work underline the importance of a suitable starter culture selection for the production of fermented sausages.
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.
Exploiting novel strategies for the production of surfactin in Bacillus subtilis cultures
(2021) Hoffmann, Mareen; Hausmann, Rudolf
Biosurfactants are synthesized by various microorganisms. These surface-active molecules are a promising alternative to petrochemically and oleochemically produced surfactants. Advantageously, biosurfactants are reported to be better biodegradable and less toxic. The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays one interesting biosurfactant. Many studies report on the outstanding physico-chemical characteristics and add on benefits such as antimicrobial properties. Hence, surfactin has the potential to be used in a variety of industrial sectors. Nevertheless, processes ensuring both robustness and high titers are rare, especially as conventional aerobic bioreactor cultivations share one major disadvantage, namely excessive foaming. To approach industrial processes, different methods are applied, which can be categorized in three practices. These are (1) media and process parameter optimization, (2) strain engineering, and (3) investigating novel process strategies. For the latter category, the anaerobic growth by nitrate respiration poses an interesting foam-free alternative. In this sense, the anaerobic cultivation of B. subtilis to produce surfactin coupled with the three afore mentioned practices was addressed in this thesis targeting at a foam-free surfactin production process. In the 1st publication, the genome reduced strain B. subtilis IIG-Bs20-5-1, a derivative of the laboratory strain 168 able to synthesize surfactin, was evaluated with respect to its suitability as surfactin producer at various temperatures under both aerobic and anaerobic conditions. It was hypothesized that a deletion of 10% of the genome, e.g., non-essential genes synthesizing prophages or the antibiotic bacilysin, saves metabolic resources and hence results in increased surfactin titers. Strains B. subtilis JABs24, a 168 derivative able to synthesize surfactin but without genome reduction, and the surfactin producer B. subtilis DSM 10T served for comparison. Although strain IIG-Bs20-5-1 was superior regarding specific growth rate µ and biomass yield YX/S, the strain was inferior with respect to surfactin titers, product related yields YP/S and YP/X, and specific productivity q. Indeed, compared to others in literature described strains, B. subtilis JABs24 was emphasized as promising target strain for further process development, reaching high surfactin titers of 1147 mg/L aerobically and 296 mg/L anaerobically as well as exceptionally high product yields YP/X under anaerobic conditions. Accordingly, iterative process optimization was hypothesized to improve anaerobically achieved surfactin titers. However, several aspects to consider of anaerobic growth of B. subtilis by nitrate respiration were described in the 2nd publication. Amongst others, increasing ammonium concentrations, resulting from nitrate reduction to ammonium via nitrite, were shown to have no impact on growth of strain JABs24, but surfactin titers and expression of nitrate reductase promoter PnarG were reduced. Nitrite was shown to peak within the first hours of cultivation and concentrations up to 10 mmol/L resulted in prolonged lag-phases. Moreover, acetate accumulated drastically during the time course of cultivation independent of glucose availability, thus decreasing the glucose flux into biomass. Acetate additionally influenced both specific growth rate µ and PnarG expression negatively. Concluding, the general feasibility of anaerobic fed-batch cultivations to synthesize surfactin was shown, but several aspects must be addressed in future works to make this strategy an equated process with aerobic cultivations. In the 3rd publication, a self-inducible surfactin synthesis process was presented where expression of the surfactin operon in B. subtilis JABs24 was induced under oxygen limited conditions. The native promoter of the srfA operon PsrfA was replaced by anaerobically inducible nitrate reductase promoter PnarG and nitrite reductase promoter PnasD. Shake flask cultivations with varying oxygen availabilities demonstrated that both PnarG and PnasD can serve as auto-inducible promoters. At high oxygen availability, surfactin was not produced in the promoter exchange strains. At lowest oxygen availability, the strain carrying PnarG reached lower surfactin titers than the native JABs24 strain, although expression levels of PnarG and PsrfA were similar. However, strain B. subtilis MG14 with PsrfA::PnasD reached 1.4-fold higher surfactin titers with 696 mg/L and an exceptionally high YP/X of 1.007 g/g with overall lower foam levels. Though, bioreactor cultivations have illustrated that the anaerobic induction must be performed slowly as to avoid cell lysis, resulting in so-defined aerobic-anaerobic switch processes. With further appropriate process optimization, a simple and robust surfactin production process with highly reduced or even no foam formation can be achieved employing strain B. subtilis MG14.
HybridMeat - products from animal and plant sources
(2022) Ebert, Sandra Gabriele; Weiss, Jochen
Consumer diversification and concerns about insufficient protein supply and global malnutrition demand for an exploitation of alternative protein sources such as plant proteins. While manufacturers have made substantial progress in industrially scaled extraction processes and structuring of plant proteins e.g. by extrusion, there is still a lack of information on their fundamental functional and organoleptic properties and interactions with other ingredients in traditional formulations. As a result, food product developers are facing a lot of challenges and are often forced to base their work on trial-and-error rather than mechanistically guided approaches. This is in particular the case for foods where complex raw material requirements and production processes make the manufacture of products with high acceptance and shelf stability not trivial. This includes the design of hybrid meat products that are composed of mixtures of meat and plant proteins. There, traditional meat products are often set as a benchmark, making the performance of such mixed products mostly unsatisfactory. Establishing composition material property functionality relationships may be a first step to overcome these obstacles. Therefore, a variety of plant proteins was assessed for their composition, physicochemical properties, and techno functionalities to gain an understanding of their suitability for the formulation of hybrid meat products. This included their dispersibility, the miscibility of select plant protein fractions with solubilized meat proteins at varying pH and mixing ratios, and the characterization of their odor-active compounds. The latter included powdered as well as extruded plant proteins due to their increasing relevance in the manufacture of hybrid meat and analogue products. Following this, plant proteins were screened in terms of their performance in hybrid meat formulations and during traditional manufacture with a special focus on dry cured products in order to define feasible protein sources and application thresholds. The first part of this thesis showed that aqueous solubility, native pH, and appearance of a variety of 26 plant protein powders from carbohydrate and vegetable oil production correlated with purity and the extraction process. Solubility ranged from as low as 4 % to as high as 100 % based on the protein concentration and prevalence of select protein fractions. For example, large amounts of prolamins (wheat) or glutelins (rice, pumpkin) resulted in low values, while high shares of albumins and globulins promoted moderate to high solubility in sunflower, pea, and potato proteins. A highly soluble (100 %) small molecular weight fraction (< 24 kDa) of the latter was subsequently screened for its particle size and electrostatic and hydrophobic properties as compared to solubilized water and salt soluble meat proteins and the miscibility of both proteins was assessed at pH 3.0 to 7.0 and at select mixing ratios. Phase behavior of mixtures started to change below the isoelectric point (pI) of salt soluble meat proteins (pH ~ 5.5), which was identified as a defining boundary value. Here, one-phase/co-soluble systems (pH > pI) transitioned to two-phased/aggregated ones mediated by interactions (pH ≤ pI) in between individual meat and meat and potato proteins. This resulted in dense, irregularly shaped meat-potato heteroprotein particles, that deviated from the characteristic assembly of pure meat proteins into regular, anisotropic aggregates. A perturbing effect of potato proteins on the structural, organized association of meat proteins below their pI was found. Protein-protein interactions were based on both electrostatics and hydrophobics as shown by variations in surface charge, hydrophobicity, and particle size if sole potato/meat and mixtures were compared. For example, particle size of solubilized meat proteins increased from 18.0 ± 2.9 µm (pH 3.0) to 26.8 ± 9.0 µm (pH 3.0) in 50:50 mixtures. FTIR results confirmed alterations as a function of mixing ratio and pH. Image analysis of microstructures revealed a shift from elongated regular networks towards more disorder and irregularity along with a lower degree of branching. Besides solubility, organoleptic properties influence the suitability of plant proteins as food ingredients. Therefore, odor active compounds of two pea isolates were analyzed by gas chromatography mass spectrometry-olfactometry (GC MS O) after direct immersion stir bar sorptive extraction (DI SBSE), and results were compared to those of their respective extrudates to define changes during dry and wet extrusion. Twenty-four odor-active compounds were found, whereof nine represented major (off-) flavor contributors in peas: hexanal, nonanal, 2 undecanone, (E)-2-octenal, (E, Z)-3,5-octadiene-2-one, (E, E) 2,4 decadienal, 2 pentyl furan, 2-pentyl-pyridine, and γ-nonalactone. The quantity of these nine volatiles was affected distinctively by extrusion. Hexanal was reduced from 3.29 ± 1.05 % (Isolate I) to 0.52 ± 0.02 % (Wet Extrudate I) and (E,Z)-3,5-Octadiene-2-one and (E,E)-2,4-decadienal decreased by 1.5- and 1.8-fold when powdered and dry texturized pea proteins were compared. As a result of the perturbing effect of soluble potato proteins and the higher amount of off flavors in pea isolates compared to their extrudates, use of plant powders as additives was rejected in favor of extruded ones for all subsequent studies. As the focus of this work was the development of dry cured hybrid meat products, the effect of various amounts of extrudates on the traditional formulation and manufacture of this product class was assessed. This included the susceptibility of extrudates towards acid-induced pH changes as compared to pork meat, as well as their behavior in a traditional acidification and drying processes. To that purpose, pork meat and six wet extrudates from peas, pumpkin, or sunflower seeds were analyzed in their proximate composition and subjected to titration starting from the same pH value and using the same acid concentrations. It was shown that wet texturized pumpkin and sunflower proteins had the highest buffering capacity (BC), especially between pH 7.0 and pH 4.5, while pea protein extrudates and pork meat were more prone to acidification and similar in buffering capacity with an average of 881 ± 5 mmol H+/(kg*ΔpH). The obtained data was then used to relate BC with the compositional elements of extrudates such as minerals, proteins, select amino acid, and non–protein nitrogen. These findings on varying susceptibility towards acids were extended by studies on a minced meat model systems containing pork meat, curing salt, and various amounts (0 to 100 wt%) of wet extrudates and the chemical acidifier Glucono delta-lactone (GDL). It was shown, that increasing concentrations of plant extrudates resulted in a linear increase of the initial (pH0h), intermediate (pH6h), and final (pH48h) pH of minced meat model systems. A sufficient acidification to common target pH values in dry cured meat products (pH ~ 5.0) could be achieved with acidifier amounts of 1.0 wt% up at no more than 15 wt% of extrudates. A mathematical model was proposed to correlate pH, time, acidifier, extrudate concentration, and plant protein origin to aid in the adjustments of formulations at higher extrudate contents, and to describe thresholds of feasible extrudate and acidifier concentrations. The calculated concentrations were then implemented to manufacture dry cured hybrid sausages where meat was partially replaced by 12.5, 25, 37.5, and 50 % of pumpkin seed extrudates. All recipes reached the target pH value with an accuracy of pH 5.0 ± 0.06 thereby validating the proposed mathematical correlations. Hybrid recipes with up to 25 % of extrudates were comparable to the traditional all-meat formulation in both the drying behavior and the distribution of moisture and free water. However, higher meat replacement levels promoted distinct changes in drying behavior and product texture where chewiness, hardness, and cohesiveness decreased by up to 70 %. In conclusion, plant protein functionality differs profoundly from the one of meat proteins, and this functionality also depends on the respective protein source as well as the applied extraction process. Their structuring by extrusion provides beneficial organoleptic changes and eases their incorporation in hybrid formulations. The fundamental characterization of plant proteins in terms of their proximate composition and (physico)chemical properties may be used to establish mathematical correlations to estimate the effect of these novel ingredients in hybrid meat products. Thus, the obtained results offer a valuable basis that manufacturers can draw upon not only to create new foods within this product class but also to broaden and facilitate the application of plant proteins on a large scale.
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.
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.
Self-learning modules for spectra evaluation
(2022) Sadeghi Vasafi, Pegah; Hitzmann, Bernd
Monitoring milk processing is an essential task as it affects the quality and safety of the final product. The aim of this investigation was to develop and analyse the self-learning system for the supervision of the processing of milk. In the self-learning evaluation module, several algorithms for data analysis of near infrared (NIR) and Raman spectra was implemented for the prediction of sample quality and safety. In the first part of this thesis, the use of NIR spectroscopy for controlling milk processing was investigated. For this reason, a high-quality quartz flow cell with a 1 mm pathlength including temperature controlling option for liquids was implemented. For sample preparation, UHT-milk with 1.5 % fat content was measured at 5 °C and considered as the reference milk. Samples with various changes such as added water and cleaning solution, different fat content and temperature as well as milks from various suppliers were investigated as the modified samples. A data set from reference and modified samples was obtained with NIR measurements. In this study, first Savitzky-Golay derivative with second polynomial order and window size of 15 was applied. It was compared with the usefulness of raw spectrum and also the combination of raw and first derivative spectrum. For the self-learning sector, an autoencoder neural network was employed. Within this thesis, it was shown that the trained autoencoder using first derivative spectra was capable to detect 5 % added water and 9 % cleaning solution in the milk. However, by using the combination spectra, the difference of 0.1 % in fat concentration was perfectly recognized. These two procedures were able to detect milks from different suppliers and difference of 10 °C in the measurement temperature. Another part of this work was done using Raman spectroscopy. The aim of this part was to check if the previous result can be improved. In this step, the circulation method was again employed the same as in the previous part. However, because of the heat introduced to the sample by the laser using in Raman spectroscopy and the length of plastic tubes which can be affected by the temperature of the laboratory, the measurement temperature was kept at 10 °C. 1.5 % fat UHT milk was utilized as the reference sample. Milks with various changes such as different fat contents, various measurement temperatures and added water or cleaning solution were investigated as the modified samples. In this investigation, not only the autoencoder but also some chemometric models were utilized with the purpose of anomaly detection. Principal component analysis (PCA) was investigated to check if the various samples can becategorized separately. In addition, two chemometric modelling techniques such as principal component regression and Gaussian process regression were tested to check the ability for change detection. By using the results obtained by PCA, a sufficient categorization of various samples was not achieved. While the PCR did not present a promising prediction as the related R2 was 0.7, Gaussian process regression with R2 of 0.97 predicted the changes almost perfectly. The trained autoencoder and Gaussian process regression both were able to define 5 % water and cleaning solution, difference of 0.1 % fat content, and variation of 5 °C in the measurement temperature. In comparison between the autoencoder and Gaussian process regression, it should be mentioned that the Gaussian process regression was capable to determine more abnormal signals than the autoencoder, however, it must be trained with all the possible changes. In contrast, the autoencoder can be trained once just with reference signals and used in online monitoring properly. As the final part and to detect which type of anomalies happened during the milk processing, several classification approaches such as linear discriminant analysis, decision tree, support vector machine, and k nearest neighbour were utilized. While decision trees and linear discriminant analysis failed to effectively characterize the various types of anomalies, the k nearest neighbor and support vector machine presented promising results. The support vector machine presented an accuracy of 81.4 % for test set, while the k nearest neighbor showed an accuracy of 84.8 %. As a result, it is reasonable to assume that both algorithms are capable of classifying various groups of data accurately. It can help the milk business figure out whats going wrong during the processing of milk. In general, Raman spectroscopy produced better findings than NIR spectroscopy, because the typical absorption bands of milk components in NIR spectrometers may be impacted by high water absorption combined with substantial light scattering by fat globules. Additionally, the autoencoder as self-learning system was capable of correctly detecting changes during milk processing, however, classification algorithms can aid in obtaining more details.

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