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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.
- ItemFood safety, a global challenge(Basel : MDPI, 2015) Uyttendaele, Mieke; Franz, Eelco; Schlüter, OliverTo provide more food and make use of precious water and nutrient resources, communities increasingly value sustainable food production. However, this should be done safely to maximize public health gains and environmental benefits. Food safety is being challenged nowadays by the global dimensions of food supply chains, the need for reduction of food waste and efficient use of natural resources such as clean water. Food safety deals with safeguarding the own national food supply chain from the introduction, growth or survival of hazardous microbial and chemical agents. But within a larger international context, borders are fading and surely this is the case for foodstuffs which are an important globally traded commodity. There is great divergence in the degree of organization, infrastructure, teaching capacity across countries and food protection (food quality, food preservation, food safety) needs to be tackled globally. This special issue assembled topics in food safety, with case studies of food safety concerns from various parts of the world, research on risk factors in agricultural production of fresh produce, use of water and water treatment technologies in food production, and outlooks on food safety for vulnerable persons. The main conclusion throughout all papers is that ensuring food safety of the food supply chain is a continuous challenge and needs our attention.
- 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.
- ItemImpact of surface structure and feed gas composition on Bacillus subtilis endospore inactivation during direct plasma treatment(Lausanne : Frontiers Media, 2015) Hertwig, Christian; Steins, Veronika; Reineke, Kai; Rademacher, Antje; Klocke, Michael; Rauh, Cornelia; Schlüter, OliverThis study investigated the inactivation efficiency of cold atmospheric pressure plasma treatment on Bacillus subtilis endospores dependent on the used feed gas composition and on the surface, the endospores were attached on. Glass petri-dishes, glass beads, and peppercorns were inoculated with the same endospore density and treated with a radio frequency plasma jet. Generated reactive species were detected using optical emission spectroscopy. A quantitative polymerase chain reaction (qPCR) based ratio detection system was established to monitor the DNA damage during the plasma treatment. Argon + 0.135% vol. oxygen + 0.2% vol. nitrogen as feed gas emitted the highest amounts of UV-C photons and considerable amount of reactive oxygen and nitrogen species. Plasma generated with argon + 0.135% vol. oxygen was characterized by the highest emission of reactive oxygen species (ROS), whereas the UV-C emission was negligible. The use of pure argon showed a negligible emission of UV photons and atomic oxygen, however, the emission of vacuum (V)UV photons was assumed. Similar maximum inactivation results were achieved for the three feed gas compositions. The surface structure had a significant impact on the inactivation efficiency of the plasma treatment. The maximum inactivation achieved was between 2.4 and 2.8 log10 on glass petri-dishes and 3.9 to 4.6 log10 on glass beads. The treatment of peppercorns resulted in an inactivation lower than 1.0 log10. qPCR results showed a significant DNA damage for all gas compositions. Pure argon showed the highest results for the DNA damage ratio values, followed by argon + 0.135% vol. oxygen + 0.2% vol. nitrogen. In case of argon + 0.135% vol. oxygen the inactivation seems to be dominated by the action of ROS. These findings indicate the significant role of VUV and UV photons in the inactivation process of B. subtilis endospores.
- 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.
- ItemA comparison of carbon footprint and production cost of different pasta products based on whole egg and pea flour(Basel : MDPI, 2016) Nette, Antonia; Wolf, Patricia; Schlüter, Oliver; Meyer-Aurich, AndreasFeed and food production are inter alia reasons for high greenhouse gas emissions. Greenhouse gas emissions could be reduced by the replacement of animal components with plant components in processed food products, such as pasta. The main components currently used for pasta are semolina, and water, as well as additional egg. The hypothesis of this paper is that the substitution of whole egg with plant-based ingredients, for example from peas, in such a product might lead to reduced greenhouse gas emissions (GHG) and thus a reduced carbon footprint at economically reasonable costs. The costs and carbon footprints of two pasta types, produced with egg or pea protein, are calculated. Plant protein–based pasta products proved to cause 0.57 kg CO2 equivalents (CO2eq) (31%) per kg pasta less greenhouse gas emissions than animal-based pasta, while the cost of production increases by 10% to 3.00 €/kg pasta.