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Bioaccessibility and anti-inflammatory activity in Caco-2 cells of phytochemicals from industrial by-products of coffee (Coffea arabica L.)
(2025) Jiménez-Gutiérrez, Milena; Zielinski, Christian; Esquivel, Patricia; Frank, Jan; Irías-Mata, Andrea; Jiménez-Aspee, Felipe
Coffee by-products are rich in nutrients and bioactive compounds in free soluble form and bound to cell wall components. These compounds undergo chemical changes during gastrointestinal digestion, affecting their bioaccessibility and bioactivity. This study is the first to investigate coffee by-products from industrial wet processing to evaluate the impact of simulated gastrointestinal digestion on their phytochemical composition and subsequent anti-inflammatory activity in Caco-2 cells. Digestion significantly reduced the stability and solubility of main compounds; however, digested bioaccessible by-products still exhibited anti-inflammatory properties, reducing IL-6, IL-8, and TNF-α levels. Correlation analysis identified rutin, quercetin-3-glycoside, caffeine and 5-caffeoylquinic acid as strongly linked to cytokine suppression, suggesting key roles and possible synergies. These results highlight the potential of coffee by-products as functional ingredients targeting intestinal inflammation. Future work should confirm in vivo efficacy, optimize extraction at scale, and address regulatory requirements to support industrial application and promote circular economy benefits.
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
Background: 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.
Decoding the aroma of Jägermeister liqueur through sensory-directed flavor analysis combined with solvent-assisted flavor evaporation and headspace-stir bar sorptive extraction
(2025) Zhu, Lin; Lin, Zexin; Zheng, Yan; Liang, Jiaqi; Li, Yupan; Kramp, Sarah; Zhang, Youfeng; Xiang, Can; Chen, Leyin; Rigling, Marina; Hannemann, Lea; Oellig, Claudia; Zhang, Yanyan
Jägermeister liqueur is one of the most famous herbal liqueurs worldwide, distinguished by its unique anise-like, bitter, and caramel-like flavor. This study comprehensively analyzed its aroma components using sensory-directed flavor analysis combined with gas chromatography-mass spectrometry and gas chromatography-sulfur chemiluminescence detector. Results identified eugenol (clove-like, OAV = 1260), anethole (anise-like, OAV = 723), p-anisaldehyde (almond-like, OAV = 97), linalool (flowery, OAV = 25), and terpinen-4-ol (apple-like, OAV = 119) as key aroma-active compounds in Jägermeister. Two sulfur-containing compounds with meaty and caramel-like aromas were detected; however, their OAVs were below 1. Ethers (53.2 mg/L), phenolics (27.8 mg/L), and terpenoids (10.5 mg/L) were the most abundant compounds in Jägermeister, while esters were present at relatively low concentrations (254 μg/L). A comparative analysis revealed that Jägermeister exhibits a unique aroma profile among ten European herbal liqueurs, particularly enriched in caramel-like and licorice-like notes, which showed positive correlations with key aroma compounds such as vanillin. This work not only provides the first systematic aroma deconstruction of Jägermeister, but also offers new insights into the compositional patterns and classification of European herbal liqueurs, contributing to quality control, product authentication, and flavor optimization.
Effect of liquefaction temperature and enzymatic treatment on bioethanol production from mixed waste baked products
(2025) Almuhammad, Mervat; Kölling, Ralf; Einfalt, Daniel
This study investigates the effect of different liquefaction temperatures (50–70 °C) and four commercial enzyme formulations on glucose release and subsequent ethanol yield, using mixed waste baked products as a substrate. Among the enzymes tested, Amylase GA 500 proved to be superior in the hydrolysis of starch at lower temperatures (50 °C and 55°C). At higher liquefaction temperatures (65 °C and 70°C) all four enzyme preparations showed comparable activity. The highest glucose concentration (205.7 g/L) and the highest ethanol yield (92 g/L) were achieved with Amylase GA 500 at 65 °C. Its superior performance is attributed to the synergistic activity of α-amylase and glucoamylase, which facilitates efficient starch hydrolysis. Crucially, we discovered that the liquefaction temperature profoundly affects fermentation speed independently of the initial glucose concentration or the enzyme preparation used for starch hydrolysis. This novel mechanistic insight suggests that higher temperature treatment either makes an additional factor crucial for yeast fermentation available or depletes/destroys an inhibitor present in the complex waste bakery product matrix. These findings highlight the critical role of temperature and enzyme formulation in optimizing bioethanol production from bakery waste, supporting the development of more sustainable and efficient waste-to-biofuel processes.
Effect of packaging and storage conditions on the pasting and functional properties of pretreated yellow-fleshed cassava flour
(2024) Ekeledo, Esther; Abass, Adebayo; Müller, Joachim
Cassava is highly susceptible to post harvest physiological deterioration which makes it necessary to initiate processing so as to extend the shelf life. In order to improve and enhance the nutritional characteristics of the processed cassava flour, this research was carried out so as to evaluate the adequate packaging materials and storage conditions necessary for safe storage and good flour quality. Pasting properties of food/flour is an indication of the different applicability of starch-based food ingredients in product development. The effect of packaging materials (cylindric polyvinyl containers and aluminum ziplock pouch bags) on quality attributes of pretreated yellow-fleshed cassava flour (YFCF) samples stored in two storage conditions a (cooling chamber at 5 ◦ C and 30 % relative humidity and; in a climate chamber at 30 ◦C and 50 % relative humidity) was investigated for 8 weeks. Flour samples from each package type were evaluated for water absorption capacity, pasting and oil absorption capacity fortnightly. The treated initial flour sample before storage-sulfured (BSS) had the highest peak viscosity (891 RVU). The low peak time at the end of storage in non-sulfured flours packed in aluminum pouch bags and stored at 5 ◦C is an evidence of time and energy saving capacity. The water absorption capacity of non-sulfured flour samples packed in cylindric polyvinyl containers and the sulfured flour sample packed in an aluminum pouch bag at 30 ◦C increased with storage duration. The aluminum ziplock pouch bags showed excellent storage quality and retained better pasting property. The climatic storage condition revealed better keeping quality. The use of sodium metabisulphite revealed its suitability as a pretreatment tool.
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
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.
The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes
(2024) Hiller, Eric; Off, Manuel; Hermann, Alexander; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Lilge, Lars; Hausmann, Rudolf
Background The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. Conclusions Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.
Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii
(2024) Bechtel, Anna; Seitl, Ines; Pross, Eva; Hetzel, Frank; Keutgen, Mario; Fischer, Lutz
The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.
Structure formation in fruit preparations by fruit fermentates produced with exopolysaccharide-forming lactic acid bacteria
(2025) Festini, Silvan; Zipori, Dor; Wallisch, Marc; Weiss, Agnes; Neidhart, Sybille; Schmidt, Herbert; Jekle, Mario
Fruit preparations are intermediate food products that are primarily used in the dairy industry for the production of fruit yogurt or frozen desserts. Typically, they are stabilized by added hydrocolloids like pectins. The objective of this study was to investigate the potential replacement of conventional stabilizers by structure-forming fermentates produced by exopolysaccharides (EPS)-forming lactic acid bacteria (LAB). Peach puree was selected as fermentation matrix. Prior to 72 h of incubation, it was inoculated with either the heterofermentative LAB strain Levilactobacillus brevis TMW 1.2112 or the homofermentative LAB strain Pediococcus parvulus strain LTH 1110, both being known to produce EPS in form of β-D-glucan. The lyophilized fermentates were applied as stabilizers to produce strawberry fruit preparations. Flow curves, viscoelastic behaviour and shear stability were measured to investigate the effect of fermentate incorporation on the rheological properties of the products. A fermentatively induced effect was observed in terms of a 1.3-fold increase in viscosity of strawberry model fruit preparations with 10 % fermentate of Lv. brevis TMW 1.2112 compared to the addition of the same dose of fermentate blank. Further, increasing the fermentate blank dose from 10 % to 15 % resulted in a 2.4-fold viscosity increase of the model fruit preparations. High shear stability was found in all model strawberry fruit preparations. However, fermentation had no clear benefit in terms of viscoelastic behaviour and shear stability of the fruit preparations. Although the fermentatively induced thickening potential was limited, production of viscosity-increasing peach fermentate with minor changes in the sugar and amino acid profiles of the fruit proved to be feasible.
Towards sustainable biointelligent food design: structuring potential of plant-based materials exemplified using apricot seed oil oleogels and bigels through 3D food printing
(2025) Reinmuth, Evelyn; Fahmy, Ahmed Raouf; Ribette, Olivia; Jekle, Mario
Background/Introduction: Biointelligence in the approach of food additive manufacturing represents a significant advancement, enabling the reverse engineering and design of foods. Legislation restricting trans-fats has accelerated research into alternatives, but ingredients like saturated and trans fats play key roles in food quality and functionality. Oleogels are a promising replacement. Food additive manufacturing introduces a biointelligent approach, combining biological and technical components with information technology to optimize food design. This study investigates 3D printing of oleogel and bigel systems using apricot seed oil, aiming to assess their significance, applicability, and printability as sustainable alternatives to trans fats for innovative, resource-efficient food production. Methods: Apricot seed oil, rich in antioxidants and polyunsaturated fatty acids, was processed into plant-based oleogels and bigels. The material systems were incorporated into 3D printed food structures. Material characterization and techno-functional analysis were conducted to evaluate the suitability of apricot seed oil for structuring 3D printed foods and controlling food texture. Results: Adjusting the type and concentration of oil-gelator mixtures enabled tailored texture and lipid distribution to fit consumer preferences. Sustainability impacts were assessed at intermediate processing steps, demonstrating the value of holistic evaluations beyond technical factors. Discussion: Biointelligent 3D printing offers a platform to optimize sensory and sustainability qualities in food design. The integration of apricot seed oil into novel food matrices enables versatile nutritional product development, supporting researchers and industry stakeholders in advancing consumer-centric, sustainable production and consumption practices.

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