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- ItemDevelopment of a flow-fluorescence in situhybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor(London : BioMed Central, 2013) Nettmann, Edith; Fröhling, Antje; Heeg, Kathrin; Klocke, Michael; Schlüter, Oliver; Mumme, JanBackground: The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. Results: Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. Conclusions: The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.
- ItemSchnelle Identifizierung von Mikroorganismen mittels MALDI-TOF MS(Darmstadt : KTBL, 2015) Fröhling, Antje; Erhard, Marcel; Muranyi, Peter; Schlüter, OliverSichere Lebensmittel von hoher Qualität stellen besonders bei leicht verderblichen Frischeprodukten eine Herausforderung für die Gestaltung der Nacherntekette dar. Da die Beprobung von Lebensmittelchargen in der Praxis meist anhand ausgewählter Indikatororganismen erfolgt, bleiben unerwartete, potenziell gefährliche Mikroorganismen häufig unentdeckt. Die Detektion dieser Bakterien ist jedoch von Interesse, um potenzielle Gefahren für den Verbraucher zu vermeiden. Am Beispiel von Mungobohnensprossen wurde die mikrobielle Diversität mittels Plattenzählverfahren und MALDI-TOF MS (matrix-assisted laser desorption/ionisation – time of flight mass spectrometry) ermittelt. Bei einer Gesamtkeimzahl zwischen 8 und 9 log KbE/g Sprossen konnten unter anderem Bakterien der Bacillus cereus Gruppe, Yersinia sp., Enterobacter spp., Klebsiella spp., Pantoea spp. und Pseudomonas spp. identifiziert werden.
- ItemImpact of cold atmospheric pressure plasma processing on storage of blueberries(Oxford [u.a.] : Wiley-Blackwell, 2020) Pathak, Namrata; Grossi Bovi, Graziele; Limnaios, Athanasios; Fröhling, Antje; Brincat, Jean-Pierre; Taoukis, Petros; Valdramidis, Vasilis P.; Schlüter, OliverThe current study aimed at investigating the impact of nitrogen (N)-generated cold atmospheric pressure plasma (CAPP) treatment on blueberries focusing on the overall impact on berry quality and microbial load along a storage period of 10 days. Blueberries were treated for 0 (control), 5, and 10 min. Assessment of fruit quality (°Bx, ascorbic acid, anthocyanins, titratable acidity, elasticity, and color parameters) and microbial analysis was performed. Results showed that CAPP treatment was more effective in inhibiting bacterial growth than fungal growth and during the subsequent storage, the quality parameters did not differ significantly from the control, under the same conditions. The study supports N-generated CAPP as a disinfection technique to reduce microbial load in blueberries without significantly impacting most quality parameters. Practical applications: Over the last decades, foodborne illness outbreaks around the world have been associated with berries. For that reason, due to the increasing consumption of berries it is paramount to study technologies that can eliminate pathogens responsible for such outbreaks. Cold atmospheric pressure plasma (CAPP) can be a promising technology to be used as an alternative to traditional decontamination methods of food. In this context, this study explored the effect and efficiency of this novel technology on reduction of native microflora and its impact on the physical and chemical properties of blueberries treated by nitrogen (N)-generated CAPP with subsequent storage of 10 days. Results of this work confirmed that such technology has high potential application for decontamination of berries without significantly impacting most quality parameters and thereby can be a potential technology for industrial applications. © 2020 The Authors. Journal of Food Processing and Preservation published by Wiley Periodicals LLC.
- ItemTowards efficient production of highly optically pure d-lactic acid from lignocellulosic hydrolysates using newly isolated lactic acid bacteria(New York, NY [u.a.] : Elsevier, 2022) Alexandri, Maria; Hübner, Dennis; Schneider, Roland; Fröhling, Antje; Venus, JoachimThis study presents the production of D-lactic acid with high enantiomeric purity using lignocellulosic hydrolysates from newly isolated lactic acid bacterial (LAB) strains. Six strains, 4 heterofermentative and 2 homofermentative, were investigated for their ability to grow and produce lactic acid on sugar beet pulp (SBP) hydrolysates, containing a mixture of hexose and pentose sugars. Among the strains tested, three were isolates designated as A250, A257 and A15, all of which belonged to the genus Leuconostoc. Only strain A250 could be reliably identified as Leuconostoc pseudomesenteroides based on cluster analysis of Maldi-ToF spectra. All strains produced D-lactic acid in the presence of SBP hydrolysates, but with varying optical purities. The homofermentative strains achieved higher D-lactic acid optical purities, but without assimilating the pentose sugars. Co-cultivation of the homofermentative strain Lactobacillus coryniformis subsp. torquens DSM 20005 together with the heterofermentative isolate A250 led to the production of 21.7 g/L D-lactic acid with 99.3 % optical purity. This strategy enabled the complete sugar utilization of the substrate. Nanofiltration of the SBP hydrolysate enhanced the enantiomeric purity of the D-lactic acid produced from the isolates A250 and A15 by about 5 %. The highest D-lactic acid concentration (40 g/L) was achieved in fed-batch cultures of A250 isolate with nanofiltered SBP, where optical purity was 99.4 %. The results of this study underline the feasibility of a novel isolate as an efficient D-lactic acid producer using lignocellulosic hydrolysates.
- ItemThermal Impact on the Culturable Microbial Diversity Along the Processing Chain of Flour From Crickets (Acheta domesticus)(Lausanne : Frontiers Media, 2020) Fröhling, Antje; Bußler, Sara; Durek, Julia; Schlüter, Oliver K.The role of insects for human consumption has lately increased in interest and in order to deliver safe and high-quality raw materials and ingredients for food and feed applications, processing of insects is a major pre-requisite. For edible insects a thermal treatment and appropriate storage conditions are recommended to minimize the microbiological risk and the impact of processing methods on the microbial contamination needs to be considered and determined. Based on standard process conditions for the production of Acheta domesticus flour, different heating treatments were used to reduce the microbial load of A. domesticus. In addition, the drying temperature and drying time were varied to determine whether the required residual moisture of <5% can be achieved more quickly with consistent microbial quality. The influence of the process conditions on the microbial community of A. domesticus along the processing chain was finally investigated under optimized process conditions. The total viable count was reduced from 9.24 log10 CFU/gDM to 1.98 log10 CFU/gDM along the entire processing chain. While Bacillaceae, Enterobacteriaceae, Enterococcaceae, and yeast and molds were no longer detectable in the A. domesticus flour, Staphylococcaceae and mesophilic spore forming bacteria were still found in the flour. The results indicate that the steaming process is essential for effectively increasing microbial safety since this processing step showed the highest inactivation. It is recommended to not only evaluate the total viable count but also to monitor changes in microbial diversity during processing to ensure microbial safety of the final product. © Copyright © 2020 Fröhling, Bußler, Durek and Schlüter.
- ItemImpact of different water activities (aw) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores(Lausanne : Frontiers Media, 2015) Sevenich, Robert; Reineke, Kai; Hecht, Philipp; Fröhling, Antje; Rauh, Cornelia; Schlüter, Oliver; Knorr, DietrichMuch research has been conducted to comprehend the mechanisms of high pressure (HP) inactivation of spores in aqueous systems but for food model systems these information are scarce. In these systems spores can interact with ingredients which then could possibly lead to retarded or reduced inactivation, which can cause a problem for the sterilization process. The protective mechanism of a reduced aw-value is still unclear. HP processing might prove valuable to overcome protective effects of solutes and achieve shorter process times for sterilization under HP. To gain insight into the underlying mechanisms five aw-values (0.9, 0.92, 0.94, 0.96, 1) were adjusted with two different solutes (NaCl, sucrose). Solutions were inoculated with spores of Bacillus amyloliquefaciens and treated at 105, 110, and 115°C at 600 MPa. Further a thermal inactivation was conducted at the same temperatures for a comparison with the HP data. Afterward, the influence of HP high temperature treatment on the inactivation, the dipicolinic acid (DPA)-release and membrane constitution was assessed by plate count, HPLC and flow cytometry (FCM). The results show that during HP treatments sucrose and salt both have a protective effect, in which the influence of sucrose on the retarded inactivation is higher. The threshold water activities (aw), which is 0.94, here salt and sucrose have a significant influence on the inactivation. The comparison of thermal (105–115°C) and HP and high temperature (600 MPa, 105–115°C) treated samples showed that the time needed to achieve a 4–5 log10 inactivation is reduced from 45 (aw = 1) to 75 (aw = 0.9) min at 105°C to 3 (aw = 1) to 15 (aw = 0.9) minutes at 600 MPa and 105°C. The release of DPA is the rate limiting step of the inactivation and therefore monitoring the release is of great interest. The DPA-release is slowed down in high concentrated solutions (e.g., sucrose, salt) in comparison to aw 1. Since there is a difference in the way the solutes protect the spore it could be seen as an inner spore membrane effect. Maybe as shown for vegetative microorganism the solutes can interact with membranes, e.g., the inner spore membrane. Flow cytometry (FCM) measurement data show a similar trend.
- ItemFlow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria(Lausanne : Frontiers Media, 2015) Fröhling, Antje; Schlüter, OliverSince heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters.