Institut für Lebensmittelwissenschaft und Biotechnologie

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Chemical composition and consumer acceptability of oyster mushroom and sorghum-pearl millet based composite flours
(2024) Ndunge Charles, Alice; Mburu, Monica; Njoroge, Daniel; Zettel, Viktoria; Ndunge Charles, Alice; Institute of Food Bioresources Technology, Dedan Kimathi University of Technology, Nyeri, Kenya; Mburu, Monica; Institute of Food Bioresources Technology, Dedan Kimathi University of Technology, Nyeri, Kenya; Njoroge, Daniel; Institute of Food Bioresources Technology, Dedan Kimathi University of Technology, Nyeri, Kenya; Zettel, Viktoria; Department of Plant-based Foods, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany
Due to over-reliance on starchy staple foods in Kenya Micronutrient deficiency (MD) and Protein Energy Malnutrition (PEM) are the major nutritional concerns. Despite these foods being characterized by low nutrient densities, they are still used as main food ingredient especially in making porridges. Therefore, this study intended to develop composite flour of sorghum-pearl millet blends fortified with oyster mushroom and further evaluate its chemical composition and consumer acceptability. The sorghum and pearl millet were soaked in tap water then solar dried followed by milling into flour. Oyster mushrooms were also solar dried and milled. Subsequently, oyster mushroom was substituted to sorghum-pearl millet blends at 10–50 % to achieve the composite flours. Proximate composition of the samples was determined using standard methods, Inductively Coupled Plasma Optical Emission Spectroscopy and Ultra Performance Liquid Chromatography Ultraviolet Spectroscopy/Mass Spectrometry were used for mineral and vitamin analyses, respectively. In addition, consumer acceptability test was done on thin porridges was also carried out using untrained panelists. Protein content of the composite flours increased from 11.15–19.74 %, and there were significant increases in fiber (2.68–10.66 %) and ash (1.38 % to 5–79 %). Mineral content increased with Ca, P, Na, K, Mg, Zn and Fe as high as 286.25 mg/100 g, 2088.05 mg/100 g, 167.55 mg/100 g, 1833.05 mg/100 g, 136.75 mg/100 g, 4.22 mg/100 g and 9.46 mg/100 g respectively. An improvement of B1, B2, B3, B6 and B9 vitamin contents was also observed. Sensory analysis showed relatively moderate overall acceptance of thin porridges from the composite flours, although the acceptability decreased with the increase in oyster mushroom. Specifically, composite flour with 50% oyster mushroom was rich in most nutrients but it had the lowest scores for the sensory attributes. In conclusion, addition of oyster mushroom increased the overall nutritional composition of local cereal based staple foods an indication of potential solution to PEM and MD.
Flavor-boosting of Phaeodactylum tricornutum by fermentation with edible mushrooms
(2024) Rigling, Marina; Liang, Jiaqi; Entenmann, Isa; Frick, Konstantin; Schmid-Staiger, Ulrike; Xiang, Can; Kopp, Lena; Bischoff, Stephan C.; Zhang, Yanyan
Microalgae are a promising and sustainable source of nutritious food, especially for use in alternatives to fish and seafood. Among them, Phaeodactylum tricornutum (PT) stands out for its potential to revolutionize future diets with its rich nutrient profile and eco-friendly cultivation methods. However, its typically fishy and “brackish water” off-odor has been a significant deterrent. Using 13 basidiomycetes as starter cultures, the dynamic changes in the aroma were studied. To better understand the aroma development during fermentation, odor-active compounds were identified using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry–olfactometry. By submerged fermentation lasting 39 and 51 hours with Pleurotus citrinopileatus (PCI) and Pleurotus eryngii (PER), respectively, the unpalatable odor of PT was transformed into savory and seafood-like aromas, while retaining most of the valuable carotenoids (fucoxanthin and β-carotene were retained at 75 % and 90 %) and fatty acids (eicosapentaenoic acid and docosahexaenoic acid were preserved at 80 % of their initial concentrations). Throughout the fermentation process, key odorants responsible for the algae's initial green, grassy, and unpleasant odor were reduced, while compounds responsible for savory and seafood-like fragrances increased. A series of sulfur compounds, such as dimethyl disulfide, were found to be major contributors to the post-fermentation aroma.
Characterization of the key odorants in goji wines in three levels of sweetness by applications of sensomics approach
(2024) Zheng, Yan; Oellig, Claudia; Zhang, Youfeng; Liu, Yuan; Chen, Yanping; Zhang, Yanyan
The correlations and differences of the key odorants were systematically conducted among three sweetness of goji wines by the sensomics approach. After aroma (extract) dilution analysis, 67, 67, and 66 odorants were screened in sweet goji wine, semi-dry goji wine, and dry goji wine, in which, 63 odorants were identified in all goji wines. Determination of 53 odorants revealed a total of 30 odorants with the concentrations surpassing their olfactory thresholds. Overall, the odor activity values (OAVs) of ketones decreased, while esters, alcohols, phenols, and aldehydes increased with the decrease in sweetness in goji wine samples. Nevertheless, (E)-β-damascenone, trans- and cis-whisky lactones, and 3-methyl-2,4-nonanedione, evoked cooked apple-like, coconut-like, and hay-like odor impressions in goji wines and showed the highest OAVs. A reliable evaluation of the aroma contributions was executed as aroma recombinations and suggested a successful evaluation of key odorants in goji wines.
An extremely sensitive amylase activity assay and its application for the determination of the residual amylase activity in bread
(2025) Reichenberger, Katrin; Lutz-Wahl, Sabine; Kettner, Lucas; Fischer, Lutz; Reichenberger, Katrin; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Lutz-Wahl, Sabine; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Kettner, Lucas; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany
An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the α -amylase from Bacillus subtilis in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL −1 ), a GOD from Aspergillus niger (1550 nkat mL −1 ) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- ( α -amylase from B. subtilis ) and exo-acting amylase (maltogenic amylase from Geobacillus stearothermophilus ), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL −1 for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of α -amylase from B. subtilis in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.
Investigating different Bacillus subtilis expression systems for recombinant enzyme production
(2025) Senger, Jana; Fischer, Lutz
Enzymes play an indispensable role in the food industry by improving texture, color, shelf life, or nutritional value of food products. A prerequisite for the application of food enzymes is their high-yield and cost-effective production in a suitable host. The Gram-positive bacterium Bacillus subtilis is a promising host due to the organism's qualified presumption of safety status, its genetic accessibility, and short cultivation times. In addition, B. subtilis can efficiently secrete heterologous enzymes into the extracellular medium, which simplifies downstream processing. This thesis explored different B. subtilis expression systems for the recombinant production and secretion of the β-galactosidase (EC 3.2.1.23) from Paenibacillus wynnii (β-gal-Pw) and the protein-glutamine glutaminase (EC 3.5.1.44) from Bacteroides helcogenes (PGB). Both enzymes have a potential application in the food industry. The β-gal-Pw offers favorable kinetic properties for application in lactose-depleted dairy products. The PGB is used to modify the techno-functional properties of proteins, thereby improving their application in food products. The first study investigated the secretion of the cytoplasmic 120 kDa β-gal-Pw using B. subtilis SCK6. Different expression conditions were tested to find proficient conditions for secretion. Codon-optimization of the native β-gal-Pw gene and cultivation temperature reduction from 37°C to 30°C increased secretory β-gal-Pw production. Furthermore, two promoters and four signal peptides were tested in multiple combinations. Signal peptides of the Sec-pathway and the Tat-pathway enabled efficient secretion, which, however, depended on the specific combination of promoter and signal peptide used. The highest extracellular activity of 55 µkat/Lculture was achieved with the PaprE promoter and the Tat-signal peptide PhoD in shake flask cultivations. The subsequent bioreactor cultivation further improved secretory β-gal-Pw production by 1.4-fold and resulted in 21 mg/Lculture purified β-gal-Pw. The second study explored the intracellular β-gal-Pw production in the undomesticated strain B. subtilis 007. Great differences in production were observed by testing the PaprE and P43 promoter with each corresponding 5’ untranslated region (5’UTR). The use of the PaprE promoter led to an intracellular β-gal-Pw activity of 2515 µkat/Lculture, which was 45-fold higher compared to the P43 promoter. Further modification of the core PaprE promoter or the spacer sequence in the 5’UTR did not improve β-gal-Pw production. The 5’UTR in the P43 construct was replaced with the aprE 5’UTR, which significantly improved mRNA stability. In addition, β-gal-Pw production was enhanced from 56 µkat/Lculture to 2756 µkat/Lculture. The crucial role of the 5’UTR and the corresponding mRNA stability was subsequently confirmed by producing the β-glucosidase from Pyrococcus furiosus and the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus in B. subtilis 007. The third study focused on B. subtilis 007 for secretory and antibiotic-free PGB production. The genome of the undomesticated B. subtilis 007 was sequenced and provided the basis for multiple genomic integrations of the PGB expression cassette via CRISPR/Cas9. By selecting the specific integration sites, genes were simultaneously deleted to optimize the production strain and process. Four genes were targeted for the elimination of sporulation (sigF), foaming (sfp), motility (flgE), and α-amylase production (amyE). The first PGB expression cassette was integrated into sigF, which resulted in the expected asporogenic strain. An extracellular PGB activity of 4.1 µkat/Lculture was reached in bioreactor cultivations. The second expression cassette was integrated into sfp, which reduced foaming and increased the secretory PGB production by 1.3-fold. Since integration into the flgE locus did not enhance PGB production, the third PGB expression cassette was inserted into the amyE locus. The extracellular PGB activity of the respective strain was significantly increased from 5.4 µkat/Lculture to 9.5 µkat/Lculture.
Contamination-controlled upper gastrointestinal microbiota profiling reveals salivary-duodenal community types linked to opportunistic pathogen carriage and inflammation
(2025) Schmidt, Nina S.; Dörner, Elisabeth; Podlesny, Daniel; Bohlhammer, Elisabeth; Bubeck, Alena M.; Ruple, Hannah K.; Tetzlaff-Lelleck, Vivian; Sina, Christian; Schmidt, Herbert; Fricke, Florian W.
The upper gastrointestinal (uGI) microbiota has been implicated in infectious, metabolic, and immunological conditions, yet remains poorly characterized due to invasive sampling and low microbial biomass. We developed and validated a contamination-controlled 16S rRNA gene and transcript-based protocol to profile the murine and human uGI microbiota from low-biomass samples. We applied this protocol to murine esophageal, gastric, and duodenal tissues, and to human saliva, gastric, and duodenal aspirates from patients undergoing endoscopy for suspected food-related, mild GI symptoms. Our objective was to identify conserved compositional and structural uGI microbiota patterns and assess their clinical relevance in relation to pathogen burden and inflammation. In mice, we found evidence for transcriptionally inactive and active intestinal taxa along the uGI tract, supporting horizontal microbiota transfer. In humans, we identified two distinct, inversely correlated salivary microbiota types – one dominated by the Prevotella 7 genus – which were conserved in the duodenum. The Prevotella 7-dominated uGI microbiota type was associated with lower relative abundances of gastrointestinal and extraintestinal opportunistic pathogens. These patterns were reproducible in an independent cohort and associated with lower systemic TNF-α levels. Our findings suggest that noninvasive salivary microbiota profiling can stratify individuals based on uGI microbiota composition and inflammation-associated risk traits, offering new opportunities for clinical applications and translational studie
Effect of wet fractionation conditions and pulsed electric field on arabinoxylan and protein recovery from maize
(2025) Sukop, Ulrich; Hoefler, Katharina; Bender, Denisse; D’Amico, Stefano; Jekle, Mario; Schoenlechner, Regine; Domig, Konrad J.; Sukop, Ulrich; Department of Biotechnology and Food Science, Institute of Food Science, BOKU University, Muthgasse 18, 1190 Vienna, Austria; (U.S.); (K.J.D.); Hoefler, Katharina; Department for Feed Analysis and Quality Testing, Institute for Animal Nutrition and Feed, AGES–Austrian Agency for Health and Food Safety, Spargelfeldstraße 191, 1220 Vienna, Austria; (K.H.); (S.D.); Bender, Denisse; Department of Biotechnology and Food Science, Institute of Food Science, BOKU University, Muthgasse 18, 1190 Vienna, Austria; (U.S.); (K.J.D.); D’Amico, Stefano; Department for Feed Analysis and Quality Testing, Institute for Animal Nutrition and Feed, AGES–Austrian Agency for Health and Food Safety, Spargelfeldstraße 191, 1220 Vienna, Austria; (K.H.); (S.D.); Jekle, Mario; Department of Plant-based Foods, University of Hohenheim, Stuttgart, Garbenstraße 25, 70599 Stuttgart, Germany;; Schoenlechner, Regine; Department of Biotechnology and Food Science, Institute of Food Technology, BOKU University, Muthgasse 18, 1190 Vienna, Austria;; Domig, Konrad J.; Department of Biotechnology and Food Science, Institute of Food Science, BOKU University, Muthgasse 18, 1190 Vienna, Austria; (U.S.); (K.J.D.); Wang, Lili; Cao, Ruge
Maize wet fractionation by-products are primarily used as feed but offer potential for food applications. Arabinoxylans (AXs) and proteins are particularly valuable due to their network-forming properties, which depend on their molecular structure. This study assessed the effect of the steeping conditions (acid type and pH variation) combined with a pulsed electric field (PEF) as a strategy for recovering these polymers, while also evaluating their effect on the recovery yield, fraction composition, and key AX characteristics. The physical properties were studied in selected fractions to investigate the process-induced structural changes. Lactic acid and hydrochloric acid (pH 2.5) were most effective in enhancing AX and protein recovery in fiber-rich (FF) and protein-rich (PF) fractions, respectively, while acetic acid exhibited the lowest efficiency. However, bound polyphenols were best retained in the FF when lactic acid was used, indicating the lowest structural damage to AXs, compared to other acids and using a higher pH. Additional PEF pre-treatment significantly enhanced the release of proteins, dietary fiber, and fat from the FF while inducing physical modifications to the fractions (PF: higher protein unfolding, FF: improved water-binding, pasting when using PEF). These findings highlight the potential of optimizing the processing conditions to adjust the recovery of proteins and AXs from maize, while minimally affecting their functionality.
Characterization of volatile compounds and odorants in different sichuan pepper varieties in tallow hotpot
(2025) Li, Wenhua; Wang, Qiaojun; Huan, Huilin; Wu, Gangcheng; Jin, Qingzhe; Zhang, Youfeng; Wang, Xingguo; Li, Wenhua; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Wang, Qiaojun; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Huan, Huilin; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Wu, Gangcheng; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Jin, Qingzhe; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Zhang, Youfeng; Department of Flavor Chemistry, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany; Wang, Xingguo; State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; (W.L.); (Q.W.); (H.H.); (G.W.); (Q.J.); Noguera-Artiaga, Luis
Sichuan pepper plays a vital role in enhancing the flavor of hotpot. However, the specific flavor compounds involved are still unclear. In this study, the key aroma components of Sichuan pepper tallow hotpot were explored. Six aroma attributes were evaluated by quantitative descriptive sensory analysis (QDA). Gas chromatography–mass spectrometry (GC-MS) identified 56 compounds. Among them, a total of 27 aroma-active compounds were identified by gas chromatography–olfactometry (GC-O) and aroma extract dilution analysis (AEDA). Sixteen aroma-active compounds were determined using odor activity values (OAVs) ≥ 1. Linalool, linalyl acetate, D -limonene, sabinene, β -myrcene, eucalyptol, α -terpineol, terpinen-4-ol, acetic acid, ( E , E )-2,4-decadienal, ( E )-2-heptenal, and others were identified as the key aroma compounds. Chemometrics analysis indicated that the aroma of green Sichuan pepper tallow hotpot was green, and the aroma of different red Sichuan pepper tallow hotpots varied significantly. The research results serve as a foundation for the quality control and production of the hotpot industry.
A novel neotropical Bacillus siamensis strain inhibits soil-borne plant pathogens and promotes soybean growth
(2025) Moreira, Rodrigo F.; Pires, Elizabeth B. E.; Sousa, Odaiza F.; Alves, Giselly B.; Viteri Jumbo, Luis O.; Santos, Gil R.; Maia, Luís J.; Ribeiro, Bergmann M.; Smagghe, Guy; Perino, Elvio H. B.; Hausmann, Rudolf; Oliveira, Eugenio E.; Aguiar, Raimundo W. S.; Moreira, Rodrigo F.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Pires, Elizabeth B. E.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Sousa, Odaiza F.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Alves, Giselly B.; Departamento de Biotecnologia, Universidade Federal do Tocantins, Gurupi 77410-530, TO, Brazil; Viteri Jumbo, Luis O.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Santos, Gil R.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Maia, Luís J.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Ribeiro, Bergmann M.; Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília (UnB), Brasília 70910-900, DF, Brazil; Smagghe, Guy; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Perino, Elvio H. B.; 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, Germany; Oliveira, Eugenio E.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Aguiar, Raimundo W. S.; Programa de Pós-graduação em Biotecnologia, Universidade Federal do Tocantins (UFT), Gurupi 77402-970, TO, Brazil, (E.B.E.P.); (L.O.V.J.); (G.S.); Magalhães-Guedes, Karina Teixeira; Umsza-Guez, Marcelo Andrés
Soil-borne fungal pathogens such as Sclerotium spp., Rhizoctonia spp., and Macrophomina spp. pose significant threats to global agriculture, with soybean crops among the most severely affected due to damping-off disease. These pathogens cause substantial yield losses, making their management a critical concern. In this study, we investigated the potential of Bacillus siamensis BCL, a novel Neotropical strain, as an eco-friendly solution for managing Sclerotium , Rhizoctonia , and Macrophomina species. The strain exhibited strong antifungal activity, significantly inhibiting fungal growth in vitro, with the greatest suppression observed against Macrophomina spp., reaching up to 81%. In vivo assays further confirmed the biocontrol potential of B. siamensis . When applied at 10 6 colony-forming units (CFU)/mL, the strain reduced disease symptoms and improved plant growth parameters—including root length, shoot biomass, and leaf number—compared to untreated, infected controls. The protective effect varied by pathogen, with the most significant recovery in root length observed against Macrophomina spp. (85%) and Sclerotium spp. (78%). In preventive treatments, fermentation extracts of the B. siamensis strain suppressed disease progression, although they did not promote seedling growth. A genomic analysis of B. siamensis BCL revealed genes encoding antimicrobial secondary metabolites, including terpenes, fengycins, and surfactins. These findings highlight B. siamensis BCL as a promising candidate for sustainable crop protection and a valuable resource for developing novel antimicrobial strategies in agriculture.
Optimizing storage assignment, order picking, and their interaction in mezzanine warehouses
(2023) Lesch, Veronika; Müller, Patrick B.M.; Krämer, Moritz; Hadry, Marius; Kounev, Samuel; Krupitzer, Christian; Lesch, Veronika; University of Würzburg, Würzburg, Germany; Müller, Patrick B.M.; University of Applied Sciences Würzburg-Schweinfurt, Würzburg, Germany; Krämer, Moritz; io-consultants GmbH, Co. KG, Heidelberg, Germany; Hadry, Marius; University of Würzburg, Würzburg, Germany; Kounev, Samuel; University of Würzburg, Würzburg, Germany; Krupitzer, Christian; University of Hohenheim, Stuttgart, Germany
In warehouses, order picking is known to be the most labor-intensive and costly task in which the employees account for a large part of the warehouse performance. Hence, many approaches exist, that optimize the order picking process based on diverse economic criteria. However, most of these approaches focus on a single economic objective at once and disregard ergonomic criteria in their optimization. Further, the influence of the placement of the items to be picked is underestimated and accordingly, too little attention is paid to the interdependence of these two problems. In this work, we aim at optimizing the storage assignment and the order picking problem within mezzanine warehouse with regards to their reciprocal influence. We propose a customized version of the Non-dominated Sorting Genetic Algorithm II (NSGA-II) for optimizing the storage assignment problem as well as an Ant Colony Optimization (ACO) algorithm for optimizing the order picking problem. Both algorithms incorporate multiple economic and ergonomic constraints simultaneously. Furthermore, the algorithms incorporate knowledge about the interdependence between both problems, aiming to improve the overall warehouse performance. Our evaluation results show that our proposed algorithms return better storage assignments and order pick routes compared to commonly used techniques for the following quality indicators for comparing Pareto fronts: Coverage, Generational Distance, Euclidian Distance, Pareto Front Size, and Inverted Generational Distance. Additionally, the evaluation regarding the interaction of both algorithms shows a better performance when combining both proposed algorithms.
Almond-like aroma formation of acid whey by Ischnoderma benzoinum fermentation: potential application in novel beverage development
(2025) Hannemann, Lea; Klauss, Raphaela; Gleissle, Anne; Heinrich, Patrick; Braunbeck, Thomas; Zhang, Yanyan; Hannemann, Lea; Department of Flavor Chemistry, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, Stuttgart, Germany; Klauss, Raphaela; Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 21, Stuttgart, Germany; Gleissle, Anne; Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 21, Stuttgart, Germany; Heinrich, Patrick; Aquatic Ecology and Toxicology Group, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 504, Heidelberg, Germany; Braunbeck, Thomas; Aquatic Ecology and Toxicology Group, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 504, Heidelberg, Germany; Zhang, Yanyan; Department of Flavor Chemistry, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, Stuttgart, Germany
To address the sourish off-aroma of acid whey and enhance its upcycling, a new basidiomycete Ischnoderma benzoinum -mediated fermentation system was developed using pure acid whey as the sole substrate. A pleasant sweetish and marzipan-like odor was perceived after fermentation within 7 d at 24 °C in darkness, which was shaped from key contributors including 4-methoxybenzaldehyde (odor activity value (OAV) 878), 3-methylbutanal (OAV 511), 3,4-dimethoxybenzaldehyde (OAV 50), and benzaldehyde (OAV 28). The typical sweetish and almond-like odor persisted well after ultrahigh-temperature processing, though its intensity decreased slightly. Concurrently, the fermentation reduced lactose from 52 to 20 g/L but increased the contents of essential amino acids like threonine, leucine, and lysine. No significant cytotoxicity or genotoxicity differences were found between fermented and unfermented whey. Overall, the study highlights the capability of I. benzoinum fermentation to enhance the flavor of acid whey, offering a promising approach for creating nutritional and flavorful acid-whey-based products.
Smoldering smoke conditions affect contents of monochloropropanediols in Frankfurter-type sausages
(2025) Albert, Christopher; Leible, Malte; Döring, Maik; Jira, Wolfgang; Gibis, Monika; Albert, Christopher; Department of Safety and Quality of Meat, Max Rubner-Institut (MRI), E.-C.-Baumann-Straße 20, Kulmbach, Germany; Leible, Malte; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, Stuttgart, Germany; Döring, Maik; National Reference Centre for Authentic Food, Max Rubner-Institut (MRI), E.-C.-Baumann-Straße 20, Kulmbach, Germany; Jira, Wolfgang; Department of Safety and Quality of Meat, Max Rubner-Institut (MRI), E.-C.-Baumann-Straße 20, Kulmbach, Germany; Gibis, Monika; Department of Food Material Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, Stuttgart, Germany
Statistically designed smoldering smoke experiments with Frankfurter-type sausages were performed using 14 setups (two replicates, each). The target smoke generation temperatures (150, 300, 450, 600, 750, and 900 °C) were adjusted by air supply, and the actual temperatures were monitored over a smoking period of 3–15 min using three sensors placed in the ember bed. The contents of 3- and 2-monochloropropanediols (MCPD) in Frankfurters were analyzed and ranged between 2.7–60.6 and 0.2–1.7 μg/kg, respectively. The 3-MCPD/2-MCPD ratio ranged from 27 to 46 and was noticeably higher than reported for thermally processed foods. The MCPD levels were correlated with values derived from the temperature curves and the smoking time. For the first time, a correlation between the smoke generation temperature and the MCPD content was proven. Pearson’s correlations were obtained for the mean temperature of the three sensors over all time points with 0.921 (3-MCPD) and 0.947 (2-MCPD).
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, Germany
The 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.
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, Mireille
Background: 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.
Protein interactions between the bacteriophage T4 tail tip and the inner membrane of Escherichia coli
(2025) Wenzel, Sabrina; Kuhn, Andreas
The 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.
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, Germany
The 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.
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, USA
The 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.
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, Germany
The 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.
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, Germany
Objective: 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.
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, Germany
Background: 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.

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