Institut für Lebensmittelwissenschaft und Biotechnologie
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Publication Barley-sorghum craft beer production with Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima yeast strains(2020) Einfalt, DanielThe use of different yeast strains contributes to obtain insights into beer products with diverse sensory characteristics. In this study, three yeast species of different genera were selected to evaluate their fermentation performance and sensory profile for barley-sorghum beer production. Baley-sorghum wort was produced with 12.5°P and fermented with Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima yeast strains. Differences were observed in terms of fermentation time and ability to ferment maltose. S. cerevisiae attenuated initial maltose concentration within 72 h, while M. pulcherrima and T. delbrueckii performed fermentation within 120 and 192 h, respectively. Both yeast strains simultaneously produced 11% and 23% lower ethanol concentrations, compared to S. cerevisiae with 37.9 g/L. Wort fermented with T. delbrueckii showed residual maltose concentration of 19.7 ± 4.1 g/L, resulting in significantly enhanced beer sweetness. S. cerevisiae produced significantly increased levels of higher alcohols, and obtained the highest scores for the sensory attribute body perception. Beer produced with T. delbrueckii contained significantly lower fermentative 2,3-butanediol and 2-methyl-1-butanol volatiles; this beer also showed reduced body perception. Beer conditioned with T. delbrueckii was significantly preferred over M. pulcherrima. Besides S. cerevisiae with high fermentative power, T. delbrueckii and M. pulcherrima were found to have reduced maltose fermenting abilities and provide significantly different sensory attributes to barley-sorghum beers.Publication Characterization of the aroma profile of food smoke at controllable pyrolysis temperatures(2023) Rigling, Marina; Höckmeier, Laura; Leible, Malte; Herrmann, Kurt; Gibis, Monika; Weiss, Jochen; Zhang, YanyanSmoking is used to give food its typical aroma and to obtain the desired techno-functional properties of the product. To gain a deeper knowledge of the whole process of food smoking, a controllable smoking process was developed, and the influence of wood pyrolysis temperature (150–900 °C) on the volatile compounds in the smoking chamber atmosphere was investigated. The aroma profile of smoke was decoded by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Subsequently, the correlations in the most important substance classes, as well as in individual target components, were investigated by the Pearson test. Phenols and lactones showed an increase over the entire applied temperature range (rT = 0.94 and rT = 0.90), whereas furans and carbonyls showed no strict temperature dependence (rT < 0.6). Investigations on single aroma compounds showed that not all compounds of one substance class showed the same behavior, e.g., guaiacol showed no significant increase over the applied pyrolysis temperature, whereas syringol and hydoxyacetone showed a plateau after 450 °C, and phenol and cyclotene increased linear over the applied temperature range. These findings will help to better understand the production of aroma-active compounds during smoke generation in order to meet consumers preferences.Publication Correction: Schubert et al. Microencapsulation of bacteriophages for the delivery to and modulation of the human gut microbiota through milk and cereal products. Appl. Sci. 2022, 12, 6299(2023) Schubert, Christina; Fischer, Sabina; Dorsch, Kathrin; Teßmer, Lutz; Hinrichs, Jörg; Atamer, ZeynepPublication Effect of storage temperature on volatile marker compounds in cured loins fermented with Staphylococcus carnosus by brine injection(2020) Bosse, Ramona; Wirth, Melanie; Weiss, Jochen; Gibis, MonikaIn this study, the influence of low (5 °C), intermediate (15 °C) and high (25 °C) storage temperatures on the profile of volatile compounds of North European cured loins fermented with Staphylococcus carnosus strains was investigated. In this context, proteolytic activity, bacterial growth, key volatile compounds and sensory attributes were studied. In conclusion, storage temperature significantly affected the volatile marker compounds. A multiple regression indicated significant effects of seven volatile compounds (acetophenone, benzaldehyde, butanone, 3-methylbutanal, 1-octen-3-ol, nonanal and pentanone) on the overall odor (R2 = 95.9%) and overall flavor (R2 = 81.1%). The sum of the marker volatiles aldehydes, ketones and alcohol increased with rising temperatures and the highest amounts of the odor active 3-methylbutanal up to 155 and 166 ng/g meat were detected in high temperature-stored loins. Moreover, the addition of S. carnosus strain LTH 3838 showed maximum effect at 5 °C-storage temperature in comparison to the control.Publication Effects of fingerroot (Boesenbergia pandurata) oil on microflora as an antimicrobial agent and on the formation of heterocyclic amines in fried meatballs(2024) Soikam, Panida; Rachtanapun, Chitsiri; Suriyarak, Sarisa; Weiss, Jochen; Gibis, MonikaThis study aimed to determine the antibacterial activity of the essential oil of fingerroot (Boesenbergia pandurata) (EOF) as a natural preservative in ground meat and its effect on the formation of heterocyclic amines (HAs) in pan-fried meatballs. EOF was applied either by adding it to ground pork or marinating pork in it before grinding. In addition, the antibacterial activity of EOF was tested. Aerobic mesophilic total viable count (TVC), lactic acid bacteria (LAB), and Enterobacteriaceae bacteria were monitored. The results show that EOF exhibited strong antibacterial activity when added at concentrations of 1.0 and 2.5 wt%. Antimicrobial activity against TVC, LAB, and especially Enterobacteriaceae bacteria was observed at all EOF concentrations (0.25, 0.5, 1.0, and 2.5 wt%). A 2.5% concentration of EOF applied by marinating trimmings can extend the shelf-life of ground pork to 18 days, while 2.5% EOF applied via addition can extend the shelf-life to 15 days, compared with 3 days for the control sample. After frying the meatballs, the inhibitory effect on the formation of heterocyclic amines was only significant for MeIQx with the highest addition of EOF (2.5 wt%). Significant increases in the concentrations of all other HAs were determined by adding EOF (2.5 wt%).Publication Effects of Ugali maize flour fortification with chia seeds (Salvia hispanica L.) on its physico-chemical properties and consumer acceptability(2024) Chemutai, Susan; Mburu, Monica; Njoroge, Daniel; Zettel, ViktoriaThe study investigated the effect of incorporating whole chia seeds (WCS) and defatted chia seed flour (DCF) into whole maize meal for ugali preparation. Both were incorporated at substitution levels of 3%, 6%, and 9% separately, and the resulting treatments subjected to laboratory analysis. In addition, ugali samples were prepared from all the resulting flour formulations and subjected to consumer acceptability assessment. Incorporation of both DCF and WCS resulted in increased water absorption capacity (ranging from 0.78 to 0.98 g/mL), swelling index (ranging from 0.15 to 3.25 mL/g), and swelling capacity (ranging from 2.46 to 5.74 g/g). WCS decreased the bulk density and oil absorption capacity. DCF, however, resulted in an increase in bulk density and oil absorption capacity. Both DCF and WCS lowered the lightness (L*) of the products. Proximate composition ranged from 4.78 to 7.46% for crude fat, 7.22% to 9.16% for crude protein, and 1.74 to 4.27% for crude fiber. The obtained results show the potential of chia seeds as a good fortificant of maize flour since it resulted in nutritionally superior products (crude ash, crude protein, crude fat, and energy value) when compared to control. The freshly prepared ugali samples were generally acceptable to the panelists up to 9% WCS and 6% DCF substitution levels.Publication Evaluation of an external foam column for in situ product removal in aerated surfactin production processes(2023) Treinen, Chantal; Claassen, Linda; Hoffmann, Mareen; Lilge, Lars; Henkel, Marius; Hausmann, RudolfIn Bacillus fermentation processes, severe foam formation may occur in aerated bioreactor systems caused by surface-active lipopeptides. Although they represent interesting compounds for industrial biotechnology, their property of foaming excessively during aeration may pose challenges for bioproduction. One option to turn this obstacle into an advantage is to apply foam fractionation and thus realize in situ product removal as an initial downstream step. Here we present and evaluate a method for integrated foam fractionation. A special feature of this setup is the external foam column that operates separately in terms of, e.g., aeration rates from the bioreactor system and allows recycling of cells and media. This provides additional control points in contrast to an internal foam column or a foam trap. To demonstrate the applicability of this method, the foam column was exemplarily operated during an aerated batch process using the surfactin-producing Bacillus subtilis strain JABs24. It was also investigated how the presence of lipopeptides and bacterial cells affected functionality. As expected, the major foam formation resulted in fermentation difficulties during aerated processes, partially resulting in reactor overflow. However, an overall robust performance of the foam fractionation could be demonstrated. A maximum surfactin concentration of 7.7 g/L in the foamate and enrichments of up to 4 were achieved. It was further observed that high lipopeptide enrichments were associated with low sampling flow rates of the foamate. This relation could be influenced by changing the operating parameters of the foam column. With the methodology presented here, an enrichment of biosurfactants with simultaneous retention of the production cells was possible. Since both process aeration and foam fractionation can be individually controlled and designed, this method offers the prospect of being transferred beyond aerated batch processes.Publication Evaluation of an oxygen‐dependent self‐inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD(2021) Hoffmann, Mareen; Braig, Alina; Fernandez Cano Luna, Diana Stephanie; Rief, Katharina; Becker, Philipp; Treinen, Chantal; Klausmann, Peter; Morabbi Heravi, Kambiz; Henkel, Marius; Lilge, Lars; Hausmann, RudolfA novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter PsrfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters PnarG and PnasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfA expression. As hypothesized, activity of PnarG and PnasD increased with lower oxygen levels and surfactin was not produced by PsrfA::PnarG as well as PsrfA::PnasD mutant strains under conditions with highest oxygen availability. PnarG showed expressions similar to PsrfA at lowest oxygen availability, while maximum value of PnasD was more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall YP/X of 1.007 g/g. This value also surpassed the YP/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.Publication Formation of key aroma compounds during 30 weeks of ripening in gouda-type cheese produced from pasteurized and raw milk(2024) Duensing, Philipp W.; Hinrichs, Jörg; Schieberle, PeterGouda-type cheeses were produced on a pilot-scale from raw milk (RM-G) and pasteurized milk (PM-G). Sixteen key aroma compounds previously characterized by the sensomics approach were quantitated in the unripened cheeses and at five different ripening stages (4, 7, 11, 19, and 30 weeks) by means of stable isotope dilution assays. Different trends were observed in the formation of the key aroma compounds. Short-chain free fatty acids and ethyl butanoate as well as ethyl hexanoate continuously increased during ripening but to a greater extent in RM-G. Branched-chain fatty acids such as 3-methylbutanoic acid were also continuously formed and reached a 60-fold concentration after 30 weeks, in particular in PM-G. 3-Methylbutanal and butane-2,3-dione reached a maximum concentration after 7 weeks and decreased with longer ripening. Lactones were high in the unripened cheeses and increased only slightly during ripening. Recent results have shown that free amino acids were released during ripening. The aroma compounds 3-methylbutanal, 3-methyl-1-butanol, and 3-methylbutanoic acid are suggested to be formed by microbial enzymes degrading the amino acid l-leucine following the Ehrlich pathway. To gain insight into the quantitative formation of each of the three aroma compounds, the conversion of the labeled precursors (13C6)-l-leucine and (2H3)-2-keto-4-methylpentanoic acid into the isotopically labeled aroma compounds was studied. By applying the CAMOLA approach (defined mixture of labeled and unlabeled precursor), l-leucine was confirmed as the only precursor of the three aroma compounds in the cheese with the preferential formation of 3-methylbutanoic acid.Publication Improving the colloidal stability of pectin–phycocyanin complexes by increasing the mixing ratio(2024) Buecker, Stephan; Gibis, Monika; Bartmann, Laura; Bussler, Sara; Weiss, JochenIn the food industry, the phycobiliprotein phycocyanin acts as a color pigment or the functional part of the superfood “Spirulina.” It is industrially extracted from Arthrospira platensis. Current scientific research is focusing on finding complex partners with the potential to stabilize phycocyanin against its sensitivity toward heating and pH changes. Less attention is paid to the factors that influence complexation. This study focuses on the mixing ratio of phycocyanin with pectin. Phycocyanin concentration was fixed, and the mixing ratios ranged from 0.67 to 2.50 (pectin:phycocyanin). All samples were analyzed for their color, size, microscopic structure, zeta potential, and sedimentation stability before and after heating at 85°C. It was found that increasing the pectin content fostered the initial interactions with the protein and chromophore, resulting in a color shift from blue to turquoise. The size of the complexes decreased from several micrometers to nanometers with increasing pectin concentration. Those smaller complexes that were formed at a mixing ratio of 2.5 showed a higher colloidal stability over a period of ∼2 days. It is suggested that at a low mixing ratio (0.67), phycocyanin cannot be completely entrapped within the complexes and attaches to the complex surface as well. This results in aggregation and precipitation of the complexes upon heating. With increasing aggregation and consequently size as well as density of the complexes, sedimentation was accelerated.Publication Influence of milk pasteurization on the key aroma compounds in a 30 weeks ripened pilot-scale gouda cheese elucidated by the sensomics approach(2024) Duensing, Philipp W.; Hinrichs, Jörg; Schieberle, PeterGouda cheese was produced from pasteurized milk and ripened for 30 weeks (PM-G). By application of gas chromatography/olfactometry and an aroma extract dilution analysis on the volatiles isolated by extraction/SAFE distillation, 25 odor-active compounds in the flavor dilution (FD) factor range from 16 to 4096 were identified. Butanoic acid, 2- and 3-methylbutanoic acid, and acetic acid showed the highest FD factors, and 2-phenylethanol, δ-decalactone, and δ-dodecalactone were most odor-active in the neutral-basic fraction. Quantitations by stable isotope dilution assays followed by a calculation of odor activity values (OAVs) revealed acetic acid, 3-methylbutanoic acid, butanoic acid, and butane-2,3-dione with the highest OAVs. Finally, an aroma recombinate prepared based on the quantitative data well agreed with the aroma profile of the PM-G. In Gouda cheese produced from raw (nonpasteurized) milk (RM-G), qualitatively the same set of odor-active compounds was identified. However, higher OAVs of butanoic acid, hexanoic acid, and their corresponding ethyl esters were found. On the other hand, in the PM-G, higher OAVs for 3-methylbutanoic acid, 3-methylbutanol, 3-methylbutanal, and butane-2,3-dione were determined. The different rankings of these key aroma compounds clearly reflect the aroma differences of the two Gouda-type cheeses. A higher activity of lipase in the RM-G and higher amounts of free l-leucine in PM-G on the other side were responsible for the differences in the concentrations of some key aroma compounds.Publication Microscopic analysis of gluten network development under shear load—combining confocal laser scanning microscopy with rheometry(2023) Vidal, Leonhard Maria; Ewigmann, Hans; Schuster, Clemens; Alpers, Thekla; Scherf, Katharina Anne; Jekle, Mario; Becker, ThomasA comprehensive in‐situ analysis of the developing gluten network during kneading is still a gap in cereal science. With an in‐line microscale shear kneading and measuring setup in a conventional rheometer, a first step was taken in previous works toward fully comprehensible gluten network development evaluation. In this work, this setup was extended by an in‐situ optical analysis of the evolving gluten network. By connecting a laser scanning microscope with a conventional rheometer, the evaluation of the rheological and optical protein network evolution was possible. An image processing tool for analyzing the protein network was applied for evaluating the gluten network development in a wheat dough during the shear kneading process. This network evaluation was possible without interruption or invasive sample transfer comparing it to former approaches. The shear kneading system was able to produce a fully developed dough matrix within 125% of the reference dough development time in a classical kneader. The calculated network connectivity values from frequency testing ranged over all samples was in good agreement with traditional kneaded wheat dough just over peak consistency.Publication Online process state estimation for Hansenula polymorpha cultivation with 2D fluorescence spectra-based chemometric model calibrated from a theoretical model in place of offline measurements(2023) Babor, Majharulislam; Paquet-Durand, Olivier; Berg, Christoph; Büchs, Jochen; Hitzmann, BerndThe use of 2D fluorescence spectra is a powerful, instantaneous, and highly accurate method to estimate the state of bioprocesses. The conventional approach for calibrating a chemometric model from raw spectra needs a large number of offline measurements from numerous runs, which is tedious, time-consuming, and error-prone. In addition, many process variables lack direct signal responses, which forces chemometric models to make predictions based on indirect responses. In order to predict glycerol and biomass concentrations online in batch cultivation of Hansenula polymorpha, this study substituted offline measurements with simulated values. The only data from cultivations needed to generate the chemometric model were the 2D fluorescence spectra, with the presumption that they contain sufficient information to characterize the process state at a measurement point. The remainder of the evaluation was carried out with the aid of a mathematical process model that describes the theoretical interferences between process variables in the system. It is shown that the process model parameters, including microbial growth rate, the yield of biomass from glycerol, and lag time can be determined from only the spectra by employing a model-based calibration (MBC) approach. The prediction errors for glycerol and biomass concentrations were 8.6% and 5.7%, respectively. An improved model-based calibration (IMBC) approach is presented that calibrates a chemometric model for only biomass. Biomass was predicted from a 2D fluorescence spectrum in new cultivations, and glycerol concentration was estimated from the process model utilizing predicted biomass as an input. By using this method, the prediction errors for glycerol and biomass were reduced to 5.2% and 4.7%, respectively. The findings indicate that model-based calibration, which can be carried out with only 2D fluorescence spectra gathered from prior runs, is an effective method for estimating the process state online.Publication Oral processing, rheology, and mechanical response: Relations in a two‐phase food model with anisotropic compounds(2023) Oppen, Dominic; Weiss, JochenFood‐material poses a challenging matrix for objective material scientific description that matches the consumers' perception. With eyes on the emerging structured food materials from alternative protein sources, objectively describing perceived texture characteristics became a topic of interest to the food industry. This work made use of the well‐known methodologies of jaw tracking and electromyography from the field of “food oral processing" and compared outcomes with mechanical responses to the deformation of model food systems to meat alternatives. To enable transferability to meat alternative products, an anisotropic structuring ingredient for alternative products, high‐moisture texturized vegetable protein (HM‐TVP), was embedded in an isotropic hydrocolloid gel. Data of the jaw movement and muscle activities exerted during mastication were modeled in a linear mixed model and set in relation to characteristic values obtained from small‐ and large‐strain deformation. For improvement of the model fit, this work makes use of two new data‐processing strategies in the field of oral processing: (i) Muscle activity data were set in relation to true forces and (ii) measured data were standardized and subjected to dimensional reduction. Based on that, model terms showed decreased p‐values on various oral processing features. As a key outcome, it could be shown that an anisotropic structured phase induces more lateral jaw movement than isotropic samples, as was shown in meat model systems.Publication Rapid acidification and off-flavor reduction of pea protein by fermentation with lactic acid bacteria and yeasts(2024) Zipori, Dor; Hollmann, Jana; Rigling, Marina; Zhang, Yanyan; Weiss, Agnes; Schmidt, HerbertPea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the “beany” off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.Publication Solidification of concentrated pea protein–pectin mixtures as potential binder(2023) Moll, Pascal; Salminen, Hanna; Stadtmüller, Lucie; Schmitt, Christophe; Weiss, JochenBACKGROUND: Binders in plant-based meat analogues allow different components, such as extrudate and fat particles, to stick together. Typically, binders then are solidified to transform the mass into a non-sticky, solid product. As an option for a clean- label binder possessing such properties, the solidification behavior of pea protein–pectin mixtures (250 g kg−1 , r = 2:1, pH 6) was investigated upon heating, and upon addition of calcium, transglutaminase, and laccase, or by combinations thereof. RESULTS: Mixtures of (homogenized) pea protein and apple pectin had higher elastic moduli and consistency coefficients and lower frequency dependencies upon calcium addition. This indicated that calcium physically cross-linked pectin chains that formed the continuous phase in the biopolymer matrix. The highest degree of solidification was obtained with a mixture of pea protein and sugar beet pectin upon addition of laccase that covalently cross-linked both biopolymers involved. All solidi- fied mixtures lost their stickiness. A mixture of soluble pea protein and apple pectin solidified only slightly through calcium and transglutaminase, probably due to differences in the microstructural arrangement of the biopolymers.Publication Stability of phenolic compounds, antioxidant activity and color parameters in colored-flesh potato chips(2023) Bravo, Catalina; Peña, Fabiola; Nahuelcura, Javiera; Vidal, Catalina; González, Felipe; Jiménez-Aspee, Felipe; Bustamante, Luis; Contreras, Boris; Ruiz, AntonietaPotato (Solanum tuberosum) chips are the most consumed snacks worldwide today. Colored potato chips prepared from potato cultivars with red and purple flesh are a novel alternative to traditional potato chips because of their higher phenolic compound content, such as anthocyanins and hydroxycinnamic acid derivatives (HCADs), which might make these chips healthier compared with traditional chips. There is little information on the stability of these compounds. In this study, the nutritional value of these chips was evaluated by determining phenolic profiles, antioxidant activity and color parameters with liquid chromatography diode array and mass spectrometry detection (HPLC-DAD-ESI-MS/MS) and spectrophotometric methods during storage for four months. Five anthocyanins and three HCADs were detected, with the latter compounds being the most abundant, with concentrations on average between the first (97.82 mg kg−1) and the last (31.44 mg kg−1) week of storage. Similar trends were observed in antioxidant activity and stability, with the CUPRAC method showing the highest response among all the methods employed. The color indices were stable throughout the storage time. Based on these results, colored-flesh potato chips are an optimal alternative for consumption because of their high retention of phenolic compounds and antioxidant activity during storage, providing potential benefits to human health.Publication Toward effects of hydrophobicity on biosurfactant production by Bacillus subtilis isolates from crude-oil-exposed environments(2024) Hashemi, Seyedeh Zahra; Fooladi, Jamshid; Vahidinasab, Maliheh; Hubel, Philipp; Pfannstiel, Jens; Pillai, Evelina; Hrenn, Holger; Hausmann, Rudolf; Lilge, LarsBackground: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.