Institut für Lebensmittelwissenschaft und Biotechnologie
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Publication Influence of different storage atmospheres in packaging on color stability of beef(2024) Krell, Johannes; Poveda‐Arteaga, Alejandro; Weiss, Jochen; Witte, Franziska; Terjung, Nino; Gibis, Monika; Krell, Johannes; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Poveda‐Arteaga, Alejandro; DIL, German Institute of Food Technology, Quakenbrück, Germany; Weiss, Jochen; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Witte, Franziska; DIL, German Institute of Food Technology, Quakenbrück, Germany; Terjung, Nino; DIL, German Institute of Food Technology, Quakenbrück, Germany; Gibis, Monika; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, GermanyThe influence of storage atmosphere on the color development and myoglobin (Mb) redox state of beef was investigated. Beef samples were packaged in 6 different atmospheres including different degrees of vacuum, levels of oxygen, nitrogen, and a mixture with 20% CO2 and stored at 2°C for 14 days. Over this time, color and reflection of the packaged samples were measured. The used method allows quick, easy, and non‐invasive measurement of the packaged samples, without using time consuming chemical assays. The method could be implemented in beef production lines, with potential for automatization. The data was used to illustrate the L*a*b* values for insights regarding qualitative color changes. Quantitative color changes were analyzed by calculation of color difference ΔE2000. Additionally, the relative levels of the deoxymyoglobin (DMb), oxymyoglobin (OMb) and metmyoglobin (MMb) were calculated from reflection spectra. The most important findings are: there is a strong correlation (rsp = 0.80 to 0.99 with one exception at rsp = 0.69 (high vacuum), p ≤ 0.05) between a* values and relative OMb levels. Storage atmospheres containing high oxygen concentrations lead to an attractive meat color, but a decreased overall color and Mb stability (ΔE = 5.02 (synthetic air) and ΔE = 2.23 (high oxygen) after 14 days of storage). Vacuum packaged samples are most stable in regards of color and Mb stability (ΔE = 1.79 (high vacuum) and ΔE = 1.63 (low vacuum) after 14 days of storage), but lack in the vibrant red color desired for sale. The experiments showed that color measurement can be a fast, non‐invasive marker for meat quality.Practical ApplicationIn this research article, six different storage atmospheres are compared regarding their influence on color stability and color quality of beef during storage in packaging. The results suggest which atmospheres to use in various sales‐related scenarios. The method described can easily be applied in the meat industry to quickly monitor changes during storage and wet‐aging without damaging the meat or opening the meat packages.Publication Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain(2025) Hermann, Alexander; Hiller, Eric; Hubel, Philipp; Biermann, Lennart; Benatto Perino, Elvio Henrique; Kuipers, Oscar Paul; Hausmann, Rudolf; Lilge, Lars; Hermann, Alexander; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Hiller, Eric; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, 70599 Stuttgart, Germany;; Biermann, Lennart; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Kuipers, Oscar Paul; Department of Molecular Genetics, University of Groningen, 9747 AG Groningen, The Netherlands;; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Fouillaud, MireilleBackground: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis . This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved.Publication Protein interactions between the bacteriophage T4 tail tip and the inner membrane of Escherichia coli(2025) Wenzel, Sabrina; Kuhn, AndreasThe infection process of bacteriophage T4 represents a fascinating series of orchestrated events, involving highly coordinated conformational changes and molecular interactions. While the initial stages, such as adsorption, host receptor recognition, and tail sheath contraction, are well-characterized (Islam et al., 2019; Leiman et al., 2004), the molecular mechanism underlying the translocation of the T4 genome across the inner host membrane remains poorly understood. The transport of the T4 DNA across bacterial membranes is particularly challenging due to its hydrophilic, polyanionic nature and the need to traverse the hydrophobic barriers of bacterial membranes. This step is critical for infection success, as it must occur without disrupting the host cell’s membrane potential or metabolic energy supply, which are essential for phage propagation. The central spike complex, comprising gp5Cβ and gp5.4, is the first component to enter the periplasm upon contraction of the T4 phage tail. This study aimed to investigate the interactions between the T4 phage tail tip proteins gp27, gp5, and gp5.4 and periplasm and inner membrane components of Escherichia coli. The first part of this study examined the behavior of the T4 central spike complex, specifically the gp5Cβ-gp5.4 component, after penetrating the outer membrane of E. coli. The spike complex, composed of three gp5Cβ and one gp5.4 subunit, punctures the outer membrane during tail contraction and enters the periplasm. To explore potential interactions with periplasmic components, in vitro binding assays using purified gp5Cβ-gp5.4 complexes and E. coli spheroplasts were performed. Results indicated that the spike complex lacked significant affinity for membrane bilayers, consistent with its structural properties, which lack hydrophobic or amphipathic regions. Cross-linking experiments with spheroplasts revealed a transient interaction between the spike complex and the periplasmic chaperone PpiD. This interaction was confirmed using proteoliposomes reconstituted with purified PpiD, which showed enhanced binding of the spike complex compared to control liposomes. PpiD is a periplasmic chaperone anchored to the inner membrane and is known for its role in stabilizing unfolded proteins. It is hypothesized that PpiD transiently interacts with the spike complex, stabilizing it or facilitating its positioning near the inner membrane for subsequent stages of infection. The biological relevance of PpiD was assessed using an efficiency of plating (EOP) assay, which revealed a reduction in infection efficiency in PpiD deletion mutants, with plaque formation decreasing to approximately 80% of wild type levels. This suggests that while PpiD is not essential for infection, it provides a supportive role, potentially complemented by other periplasmic chaperones. The second part of the study focused on the tail tip protein gp27 and its interactions with the inner membrane. Gp27, which forms a trimeric structure associated with gp5, is proposed to play a crucial role in forming a channel for DNA translocation. To investigate its potential binding to inner membrane proteins, T4 phage particles carrying His-tagged gp27 were constructed, and cross-linking experiments were performed in vivo. These studies identified several host proteins that interact with gp27, including DamX and PpiD. DamX, a cell division protein involved in peptidoglycan remodeling and septal ring stabilization, was identified as a key binding partner of gp27. Cross-linking and affinity purification confirmed this interaction, and subsequent infection assays demonstrated a significant reduction in T4 infection efficiency to 60% in DamX deletion mutants. These findings underscore the critical role of DamX in facilitating phage DNA translocation across the inner membrane. The study suggests that DamX may provide a structural or functional scaffold, stabilizing the phage-host interface during infection. PpiD, which was also identified in the first part of the study, was co-purified with gp27, highlighting its involvement in multiple stages of the infection process. Its role in stabilizing the phage at the inner membrane or facilitating structural rearrangements required for DNA translocation warrants further investigation. In addition to identifying these interactions, the study explored the functional implications of DamX and PpiD during infection. Complementary experiments demonstrated that both proteins are not only important for initial binding but may also be involved in facilitating the structural transitions required for successful DNA translocation.Publication Experimental investigation of CO2 uptake in CO2 hydrates formation with amino acids as kinetic promoters and its dissociation at high temperature(2022) Srivastava, Shubhangi; Kollemparembil, Ann Mary; Zettel, Viktoria; Claßen, Timo; Gatternig, Bernhard; Delgado, Antonio; Hitzmann, Bernd; Srivastava, Shubhangi; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, Germany; Kollemparembil, Ann Mary; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, Germany; Zettel, Viktoria; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, Germany; Claßen, Timo; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, Germany; Gatternig, Bernhard; German Engineering Research and Development Center LSTME Busan, Busan, Republic of Korea; Delgado, Antonio; German Engineering Research and Development Center LSTME Busan, Busan, Republic of Korea; Hitzmann, Bernd; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, GermanyThe dissociation of CO2 gas hydrates (GH) with amino acid kinetic promoters and without promoters was studied at a high temperature of 90 °C for a period of 20 min to understand the percentage of CO2 gas and to select the best promoter that aids CO2 gas entrapment along with stability at a high temperature. The possibility of using four hydrophobic food grade amino acids, namely cysteine, valine, leucine, and methionine, and one surfactant, lecithin, as kinetic promoters for CO2 GH has been studied. The amino acids were added 0.5 g (wt%), and lecithin was added 5 g for the GH production. Furthermore, the amino acids leucine and methionine gave some positive results, therefore, these amino acids were carried further for the experimentation purpose in the production of CO2 GH. Also, a combinational use of these amino acids was studied to investigate the effect on % CO2 retention in comparison to the normal GH. From the results, 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.Publication Effect of oil-water colloidal states in liquid feeds on extrudability and textural attributes of high-moisture meat alternatives(2024) Stehle, Florian; Woern, Carlos; Tan, Nicholle Kirsten; Weiss, Jochen; Grossmann, Lutz; Stehle, Florian; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; Woern, Carlos; Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, USA; Tan, Nicholle Kirsten; Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, USA; Weiss, Jochen; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; Grossmann, Lutz; Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, USAThe incorporation of lipids in the extrusion process to produce composite protein–lipid high-moisture meat alternatives is a major challenge due to slip conditions induced by the oil phase. This study investigates the impact of non-emulsified and emulsified liquid feeds – using soy protein isolate and Quillaja saponin as two different emulsifiers – at 6% oil content on the extrudability, visual appearance, textural and structural properties of a soy-based meat alternative. Homogenization pressures from 20 MPa to 140 MPa were used to achieve d4,3 droplet sizes ranging from 1053 nm to 117 nm, respectively. The emulsions stabilized by soy protein isolate exhibited a larger droplet size at low pressures and a smaller droplet size at higher pressures compared to the emulsions containing Quillaja saponin (1053 nm vs. 659 nm at 20 MPa and 117 nm vs. 243 nm at 140 MPa, respectively). The addition of any kind of lipid feed resulted in a lower specific mechanical energy input compared to the standard with no oil. Non-emulsified oil reduced the directional protein fiber formation and enhanced the protein cross-linking into bulk strips, which resulted in significantly lower mechanical anisotropy compared to the standard. Emulsions stabilized by Quillaja saponin were able to resemble the degree of anisotropy with the smallest mean oil droplet size (243 nm) yielding a slightly higher anisotropic index than the control. Microstructural analyses revealed embedded oil droplets between protein fibers, which increased the visual fibrousness. However, only minor changes in the color measurements were observed among all treatments. The results demonstrate the potential of using emulsified liquid feeds to manufacture high-moisture meat alternatives with an incorporated oil phase by extrusion processing without losing the anisotropic character due to oil slip.Publication Importance of the 5’ untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007(2025) Senger, Jana; Schulz, Adriana; Seitl, Ines; Heider, Martin; Fischer, Lutz; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Schulz, Adriana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Seitl, Ines; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Heider, Martin; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, GermanyThe production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the PaprE and P43 promoters and the corresponding 5’ untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii. Expression controlled by the PaprE promoter yielded a significantly higher activity of 2515 µkat/L, compared to 56 µkat/L with the P43 promoter. Modifications on the PaprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve β-galactosidase production. Since the aprE 5’ untranslated region contributes to a high mRNA stability, it was incorporated into the P43 construct to determine whether mRNA stability is responsible for the differences observed in β-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher β-galactosidase production of 2756 µkat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the β-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.Publication A research note: effect of pH on meat iridescence in precooked cured pork(2022) Ruedt, Chiara; Gibis, Monika; Weiss, Jochen; Ruedt, Chiara; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Gibis, Monika; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Weiss, Jochen; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, GermanyObjective: The objective of this study was to investigate the effect of pH change of cooked cured pork M. longissimus thoracis et lumborum on iridescence intensity and extent (= percentage of iridescent area) since interaction with light may be related to pH-induced alterations in microstructure. Muscles were injected with brines of different pH values, cooked, sliced perpendicular to muscle fiber direction, and visually evaluated by a panel of 20 experienced panelists. Results: Muscles with lowest pH (5.38) showed the lowest iridescence score of 4.63 (p < 0.05). Iridescence was greatest in muscles with normal (5.78) and high pH (6.03, respectively 6.59), but did not differ significantly (p > 0.05). Iridescence was positively correlated (p < 0.01) with pH and water content, and negatively correlated (p < 0.01) with cooking loss. Hence, hydration state and light scattering from microstructure may be important factors that determine the degree of iridescence in cooked meat products.Publication Assessing the capabilities of 2D fluorescence monitoring in microtiter plates with data-driven modeling for secondary substrate limitation experiments of Hansenula polymorpha(2023) Berg, Christoph; Herbst, Laura; Gremm, Lisa; Ihling, Nina; Paquet-Durand, Olivier; Hitzmann, Bernd; Büchs, Jochen; Berg, Christoph; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Herbst, Laura; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Gremm, Lisa; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Ihling, Nina; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, Germany; Paquet-Durand, Olivier; Department of Process Analytics & Cereal Science, Institute for Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Hitzmann, Bernd; Department of Process Analytics & Cereal Science, Institute for Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Büchs, Jochen; AVT - Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Aachen, GermanyBackground: Non-invasive online fluorescence monitoring in high-throughput microbioreactors is a well-established method to accelerate early-stage bioprocess development. Recently, single-wavelength fluorescence monitoring in microtiter plates was extended to measurements of highly resolved 2D fluorescence spectra, by introducing charge-coupled device (CCD) detectors. Although introductory experiments demonstrated a high potential of the new monitoring technology, an assessment of the capabilities and limits for practical applications is yet to be provided. Results: In this study, three experimental sets introducing secondary substrate limitations of magnesium, potassium, and phosphate to cultivations of a GFP-expressing H. polymorpha strain were conducted. This increased the complexity of the spectral dynamics, which were determined by 2D fluorescence measurements. The metabolic responses upon growth limiting conditions were assessed by monitoring of the oxygen transfer rate and extensive offline sampling. Using only the spectral data, subsequently, partial least-square (PLS) regression models for the key parameters of glycerol, cell dry weight, and pH value were generated. For model calibration, spectral data of only two cultivation conditions were combined with sparse offline sampling data. Applying the models to spectral data of six cultures not used for calibration, resulted in an average relative root-mean-square error (RMSE) of prediction between 6.8 and 6.0%. Thus, while demanding only sparse offline data, the models allowed the estimation of biomass accumulation and glycerol consumption, even in the presence of more or less pronounced secondary substrate limitation. Conclusion: For the secondary substrate limitation experiments of this study, the generation of data-driven models allowed a considerable reduction in sampling efforts while also providing process information for unsampled cultures. Therefore, the practical experiments of this study strongly affirm the previously claimed advantages of 2D fluorescence spectroscopy in microtiter plates.Publication Effect of partial condensation (dephlegmation) in fruit brandy distillation equipment on the composition of apple brandies(2025) Yagishita, Manami; Reber, Oliver; Alter, Daniela; Kölling, Ralf; Einfalt, Daniel; Chinnici, FabioFruit brandy equipment commonly uses partial condensation (dephlegmation) to generate reflux in the distillation column. Here, we examined the effect of dephlegmation on the composition of fruit brandies in both lab-scale and large-scale settings. In lab-scale experiments, the dephlegmator led to a pronounced enrichment of ethanol in the distillate due to preferred condensation of water, while the concentration of flavor compounds was differentially affected. Some compounds were enriched in the distillate, some were depleted, and some were unaffected by dephlegmation compared with the control without a dephlegmator. Large-scale fruit brandy equipment relying exclusively on dephlegmation was compared as standard with an enrichment section containing three trays. In the equipment relying on dephlegmation, tail components such as fusel alcohols were less well separated from the middle run, which led to a reduced yield of clean spirit in the middle run. In triangle tests, the spirits from the two devices could be clearly differentiated, but there was no clear preference for one spirit or the other. This study provides for the first time detailed data on the influence of dephlegmators on the behavior of flavor compounds during fruit brandy distillation.Publication Recombinant production of bovine αS1-casein in genome-reduced Bacillus subtilis strain IIG-Bs-20-5-1(2025) Biermann, Lennart; Tadele, Lea Rahel; Benatto Perino, Elvio Henrique; Nicholson, Reed; Lilge, Lars; Hausmann, Rudolf; Biermann, Lennart; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Tadele, Lea Rahel; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Benatto Perino, Elvio Henrique; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Nicholson, Reed; Motif FoodWorks, Inc., 27 Drydock Ave, Boston, MA 02210, USA;; Lilge, Lars; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Hausmann, Rudolf; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Fouillaud, MireilleBackground: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine αS1-casein protein. Methods: A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine αS1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant αS1-casein production. After increasing the molecular abundance of the recombinant αS1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine αS1-casein. Results: Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant αS1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mgcasein/gCDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native αS1-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of B. subtilis for the recombinant production of bovine αS1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.Publication Structure elucidation and characterization of novel glycolipid biosurfactant produced by Rouxiella badensis DSM 100043T(2025) Harahap, Andre Fahriz Perdana; Conrad, Jürgen; Wolf, Mario; Pfannstiel, Jens; Klaiber, Iris; Grether, Jakob; Hiller, Eric; Vahidinasab, Maliheh; Salminen, Hanna; Treinen, Chantal; Perino, Elvio Henrique Benatto; Hausmann, Rudolf; Harahap, Andre Fahriz Perdana; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Conrad, Jürgen; Department of Organic Chemistry (130b), Institute of Chemistry, University of Hohenheim, Garbenstr. 30, 70599 Stuttgart, Germany; (J.C.); (M.W.); Wolf, Mario; Department of Organic Chemistry (130b), Institute of Chemistry, University of Hohenheim, Garbenstr. 30, 70599 Stuttgart, Germany; (J.C.); (M.W.); Pfannstiel, Jens; Mass Spectrometry Unit, Core Facility Hohenheim, University of Hohenheim, Ottilie-Zeller-Weg 2, 70599 Stuttgart, Germany; (J.P.); (I.K.); Klaiber, Iris; Mass Spectrometry Unit, Core Facility Hohenheim, University of Hohenheim, Ottilie-Zeller-Weg 2, 70599 Stuttgart, Germany; (J.P.); (I.K.); Grether, Jakob; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Hiller, Eric; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Vahidinasab, Maliheh; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Salminen, Hanna; Department of Food Material Science (150g), Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 21/25, 70599 Stuttgart, Germany;; Treinen, Chantal; Cellular Agriculture, TUM School of Life Sciences, Technical University of Munich, 85354 Freising, Germany;; Perino, Elvio Henrique Benatto; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Hausmann, Rudolf; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; (A.F.P.H.); (J.G.); (E.H.); (M.V.); (E.H.B.P.); Serianni, Anthony S.Microbial biosurfactants have become increasingly attractive as promising ingredients for environmentally friendly products. The reasons for this are their generally good performance and biodegradability, low toxicity, production from renewable raw materials, and benefits for the environment perceived by consumers. In this study, we investigated the chemical structure and properties of a novel glycolipid from a new biosurfactant-producing strain, Rouxiella badensis DSM 100043 T . Bioreactor cultivation was performed at 30 °C and pH 7.0 for 28 h using 15 g/L glycerol as a carbon source. The glycolipid was successfully purified from the ethyl acetate extract of the supernatant using medium pressure liquid chromatography (MPLC). The structure of the glycolipid was determined by one- and two-dimensional ( 1 H and 13 C) nuclear magnetic resonance (NMR) and confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). NMR analysis revealed the hydrophilic moiety as a glucose molecule and the hydrophobic moieties as 3-hydroxy-5-dodecenoic acid and 3-hydroxydecanoic acid, which are linked with the glucose by ester bonds at the C2 and C3 positions. Surface tension measurement with tensiometry indicated that the glucose–lipid could reduce the surface tension of water from 72.05 mN/m to 24.59 mN/m at 25 °C with a very low critical micelle concentration (CMC) of 5.69 mg/L. Moreover, the glucose–lipid demonstrated very good stability in maintaining emulsification activity at pH 2–8, a temperature of up to 100 °C, and a NaCl concentration of up to 15%. These results show that R. badensis DSM 100043 T produced a novel glycolipid biosurfactant with excellent surface-active properties, making it promising for further research or industrial applications.Publication Consumption of yeast-fermented wheat and rye breads increases colitis and mortality in a mouse model of colitis(2022) Zimmermann, Julia; De Fazio, Luigia; Kaden-Volynets, Valentina; Hitzmann, Bernd; Bischoff, Stephan C.; Zimmermann, Julia; Department of Nutritional Medicine/Prevention, University of Hohenheim, Stuttgart, Germany; De Fazio, Luigia; Department of Medical and Surgical Science (DIMEC), University of Bologna, Bologna, Italy; Kaden-Volynets, Valentina; Department of Nutritional Medicine/Prevention, University of Hohenheim, Stuttgart, Germany; Hitzmann, Bernd; Department of Process Analytics and Cereal Science, University of Hohenheim, Stuttgart, Germany; Bischoff, Stephan C.; Department of Nutritional Medicine/Prevention, University of Hohenheim, Stuttgart, GermanyBackground: Cereals are known to trigger for wheat allergy, celiac disease and non-celiac wheat sensitivity (NCWS). Inflammatory processes and intestinal barrier impairment are suspected to be involved in NCWS, although the molecular triggers are unclear. Aims: We were interested if different bread types influence inflammatory processes and intestinal barrier function in a mouse model of inflammatory bowel disease. Methods: Epithelial caspase-8 gene knockout (Casp8 ΔIEC ) and control (Casp8 fl ) mice were randomized to eight groups, respectively. The groups received different diets for 28 days (gluten-free diet, gluten-rich diet 5 g%, or different types of bread at 50 g%). Breads varied regarding grain, milling and fermentation. All diets were isocaloric. Results: Regardless of the diet, Casp8 ΔIEC mice showed pronounced inflammation in colon compared to ileum, whereas Casp8 fl mice were hardly inflamed. Casp8 fl mice could tolerate all bread types. Especially yeast fermented rye and wheat bread from superfine flour but not pure gluten challenge increased colitis and mortality in Casp8 ΔIEC mice. Hepatic expression of lipopolysaccharide-binding protein and colonic expression of tumor necrosis factor-α genes were inversely related to survival. The bread diets, but not the gluten-rich diet, also decreased colonic tight junction expression to variable degrees, without clear association to survival and inflammation. Conclusions: Bread components, especially those from yeast-fermented breads from wheat and rye, increase colitis and mortality in Casp8 ΔIEC mice highly susceptible to intestinal inflammation, whereas control mice can tolerate all types of bread without inflammation. Yet unidentified bread components other than gluten seem to play the major role.Publication Fed-batch bioreactor cultivation of Bacillus subtilis using vegetable juice as an alternative carbon source for lipopeptides production: a shift towards a circular bioeconomy(2024) Gugel, Irene; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Hiller, Eric; Marchetti, Filippo; Costa, Stefania; Pfannstiel, Jens; Konnerth, Philipp; Vertuani, Silvia; Manfredini, Stefano; Hausmann, Rudolf; Gugel, Irene; Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy, (S.V.);; Vahidinasab, Maliheh; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstrasse 12, 70599 Stuttgart, Germany; (E.H.B.P.);; Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstrasse 12, 70599 Stuttgart, Germany; (E.H.B.P.);; Hiller, Eric; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstrasse 12, 70599 Stuttgart, Germany; (E.H.B.P.);; Marchetti, Filippo; Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy, (S.V.);; Costa, Stefania; Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy, (S.V.);; Pfannstiel, Jens; Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, Ottlie-Zeller-Weg 2, 70599 Stuttgart, Germany; Konnerth, Philipp; Department of Conversion Technology of Biobased Resources, University of Hohenheim, Garbenstrasse 9, 70599 Stuttgart, Germany;; Vertuani, Silvia; Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy, (S.V.);; Manfredini, Stefano; Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy, (S.V.);; Hausmann, Rudolf; Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstrasse 12, 70599 Stuttgart, Germany; (E.H.B.P.);; Gudiña, EduardoIn a scenario of increasing alarm about food waste due to rapid urbanization, population growth and lifestyle changes, this study aims to explore the valorization of waste from the retail sector as potential substrates for the biotechnological production of biosurfactants. With a perspective of increasingly contributing to the realization of the circular bioeconomy, a vegetable juice, derived from unsold fruits and vegetables, as a carbon source was used to produce lipopeptides such as surfactin and fengycin. The results from the shake flask cultivations revealed that different concentrations of vegetable juice could effectively serve as carbon sources and that the fed-batch bioreactor cultivation strategy allowed the yields of lipopeptides to be significantly increased. In particular, the product/substrate yield of 0.09 g/g for surfactin and 0.85 mg/g for fengycin was obtained with maximum concentrations of 2.77 g/L and 27.53 mg/L after 16 h, respectively. To conclude, this study provides the successful fed-batch cultivation of B. subtilis using waste product as the carbon source to produce secondary metabolites. Therefore, the consumption of agricultural product wastes might be a promising source for producing valuable metabolites which have promising application potential to be used in several fields of biological controls of fungal diseases.Publication Editorial: Microbial biosurfactants: updates on their biosynthesis, production and applications(2024) Hausmann, Rudolf; Déziel, Eric; Soberón-Chávez, GloriaPublication Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440(2025) Grether, Jakob; Dittmann, Holger; Willems, Leon; Schmiegelt, Tabea; Benatto Perino, Elvio Henrique; Hubel, Philipp; Lilge, Lars; Hausmann, Rudolf; Grether, Jakob; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Dittmann, Holger; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Willems, Leon; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Schmiegelt, Tabea; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, Ottilie-Zeller-Weg 2, 70599, Stuttgart, Germany; Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, GermanyBackground: In biomanufacturing of surface-active agents, such as rhamnolipids, excessive foaming is a significant obstacle for the development of high-performing bioprocesses. The exploitation of the inherent tolerance of Pseudomonas putida KT2440, an obligate aerobic bacterium, to microaerobic conditions has received little attention so far. Here low-oxygen inducible promoters were characterized in biosensor strains and exploited for process control under reduction of foam formation by low aeration and stirring rates during biosynthesis of rhamnolipids. Results: In this study, homologous promoters of P. putida inducible under oxygen limitation were identified by non-targeted proteomic analyses and characterized by fluorometric methods. Proteomics indicated a remodeling of the respiratory chain and the regulation of stress-related proteins under oxygen limitation. Of the three promoters tested in fluorescent biosensor assays, the promoter of the oxygen-sensitive cbb3-type cytochrome c oxidase gene showed high oxygen-dependent controllability. It was used to control the gene expression of a heterologous di-rhamnolipid synthesis operon in an auto-inducing microaerobic two-phase bioprocess. By limiting the oxygen supply via low aeration and stirring rates, the bioprocess was clearly divided into a growth and a production phase, and sources of foam formation were reduced. Accordingly, rhamnolipid synthesis did not have to be controlled externally, as the oxygen-sensitive promoter was autonomously activated as soon as the oxygen level reached microaerobic conditions. A critical threshold of about 20% oxygen saturation was determined. Conclusions: Utilizing the inherent tolerance of P. putida to microaerobic conditions in combination with the application of homologous, low-oxygen inducible promoters is a novel and efficient strategy to control bioprocesses. Fermentation under microaerobic conditions enabled the induction of rhamnolipid production by low oxygen levels, while foam formation was limited by low aeration and stirring rates.Publication Oral processing of anisotropic food structures: A modelling approach to dynamic mastication data(2024) Oppen, Dominic; Weiss, JochenMaterials that have been generated through a directionally oriented growth process often exhibit anisotropic properties. Plant materials such as tubers and roots or animal matter used to produce products such as steaks or pasta filata are characterized by an alignment of molecules, aggregates or cells in certain dimensions leading to differing properties depending on direction. Such an anisotropic property behavior is important for a wide range of quality attributes such as texture, appearance, stability and even aroma and taste. Especially the former is of critical importance to consumer liking and acceptance of foods. Structure-texture relationships have already been established for certain foods. For anisotropic foods though, a determination of such relationships is difficult, since the comminution of foods during chewing causes complex changes to the underlying anisotropic structure elements that are not easily measurable using conventional mechanical texture analysis tests such as cutting, shearing or compression. On the other hand, sensory tests using panels are very time consuming and often do not reveal structural causes for texture like or dislike by consumers. The lack of availability of suitable analytical techniques that allow for a description of texture properties relevant to mastication hampers especially the development of meat substitutes that are currently trending. The aim of this work was therefore to characterize changes to anisotropic structures induced by chewing (henceforth referred to as "oral processing") using a novel measurement approach that records kinematic and electromyographic properties of the chewing process. The kinematics of jaw movement were recorded using a 3D motion tracking system. Muscle activity was recorded using an electromyograph. From the measured data, characteristics for individual chews were calculated, which were represented in a linear mixed model as a function of the food structure. Section I provides the scientific basis for this work through a preface and a literature review. Grown and manufactured anisotropic foods are identified and described. A general overview of the production, phase phenomena and characterization methods for anisotropic food materials is given. Section II contains the oral processing experiments. In Chapter III, the focus was put on the impact of fiber length of grown structures on mastication behavior. Meat model systems with different microstructures but the same composition were produced. The model systems with anisotropic and isotropic microstructures were comminuted to different sizes, and the fiber length was inferred from the length of the particles, taking into account the particle size effect of chewing. The results indicate that longer fibers cause greater jaw movement and muscle activity. For instance, estimate peak muscle activity of anisotropic samples is 58.2857 µV higher (p=0.0156) compared to isotropic samples. Chapter IV describes minced meat products in which certain phase volumes were replaced by a finely comminuted meat mass. The aim of the study was to find detection limits beyond which an increase or decrease in muscle fiber cells does not lead to a further adjustment of the mastication properties. In the study, a transition point was identified at around 50 % of batter-like substances. Food models with more than 50 % of batter-like substance showed a smaller change in mastication parameters. The effect was more pronounced with higher proportions of fibrous material. Chapter V dealt with the topic of meat substitutes. A simple model of meat substitutes was used to test whether the effects found in anisotropic animal-based products can also be found in plant-based products. Hydrocolloid gels with different phase volumes of wet textured plant protein were produced. Similar effects for the animal-based products were observed, although the correlation was not as strong. It was hypothesized that a large part of the effect was due to the weak binding ability of hydrocolloid gels. Thus, the anisotropic particles could not be held together with a low proportion of the outer hydrocolloid gel and required less muscle activity despite a higher content of structured phase. Section III assessed alternative data evaluation strategies to the linear mixed model. The aim of the study in Chapter VI was to anticipate the model products from Chapter III using a classification approach. Algorithms of three categories were trained with the data set of the chewing processes. Two approaches were used to evaluate whether the algorithms could either resolve each individual food model with variations in microstructure (anisotropy) and macrostructure (particle size) or in microstructure only. For both approaches, the algorithms performed significantly better compared to a random guessing. The best classification results were achieved by the boosted ensemble learner "XGBoost", which assigned 96.617 % of all bites to the corresponding food microstructure. Furthermore, it was demonstrated that standardized and normalized oral processing data are almost not subject-dependent. In addition, feature importance analysis confirmed that lateral jaw movement is a good indicator of the presence of anisotropic food material and, with a weight of 0.39205, is the most important feature for classifying samples according to their structure. In summary, this work was able to show that the dynamic characteristics of mastication change depending on anisotropic properties. In general, modeling of mastication characteristics has never been conducted before and represents a promising advance over mean-based evaluation. The machine learning approach is also new in the field of oral processing and proved to be promising. For future research, it is proposed to correlate the dynamic features with sensory texture data to obtain direct correlations between chewing characteristics and texture attributes.Publication Ingenious wheat starch/Lepidium perfoliatum seed mucilage hybrid composite films: Synthesis, incorporating nanostructured Dy₂Ce₂O₇ synthesized via an ultrasound-assisted approach and characterization(2025) Zinatloo-Ajabshir,Sahar; Yousefi, Alireza; Jekle, Mario; Sharifianjazi, FariborzIn this study, Dy₂Ce₂O₇ nanostructures were fabricated using an environmentally friendly, ultrasound-assisted method. These nanostructures were then incorporated into a blend of wheat starch (WS) and Lepidium perfoliatum seed mucilage (LPSM), along with sodium montmorillonite (Na-MMT) nanoparticles. The composite films were produced through a casting method, combining these components to enhance the films' structural and functional properties. FTIR results confirmed the chemical interactions between the NPs and the biopolymeric matrix of the nanocomposites. SEM surface morphology and XRD crystallography results indicated that up to a 1 % weight ratio, the dispersion of Dy₂Ce₂O₇ in the nanocomposite matrix was uniform, while at higher percentages, due to nanoparticle aggregation, crystallinity increased. Interestingly, the elongation of nanocomposites containing Dy₂Ce₂O₇ increased, while their tensile strength and elastic modulus decreased. More than 92 % of UV radiation in the 240–360 nm range was absorbed with the inclusion of 1 % wt. Dy₂Ce₂O₇, and the water vapor permeability (WVP) significantly decreased. Among the Dy₂Ce₂O₇-based nanocomposites, TGA results showed that the WS/LPSM/MMT/Dy1 % sample had the highest thermal stability. Overall, based on the results of this study, the WS/LPSM/MMT/Dy1 % sample was introduced as a composite film with suitable physicochemical and mechanical properties for food and pharmaceutical packaging.