Antje Fröhling

Impact of cold plasma on Citrobacter freundii in apple juice: Inactivation kinetics and mechanisms

Various studies have shown that cold plasma is capable of inactivating microorganisms located on a variety of food surfaces, food packaging materials and process equipment under atmospheric pressure conditions; however, less attention has been paid to the impact of cold plasma on microorganisms in liquid foodstuffs. The present study investigates cold plasmas ability to inactivate Citrobacter freundii in apple juice. Optical emission spectroscopy (OES) and temperature measurements were performed to characterise the plasma source. The plasma-related impact on microbial loads was evaluated by traditional plate count methods, while morphological changes were determined using scanning electron microscopy (SEM). Physiological property changes were obtained through flow cytometric measurements (membrane integrity, esterase activity and membrane potential). In addition, mathematical modelling was performed in order to achieve a reliable prediction of microbial inactivation and to establish the basis for possible industrial implementation. C. freundii loads in apple juice were reduced by about 5 log cycles after a plasma exposure of 480s using argon and 0.1% oxygen plus a subsequent storage time of 24h. The results indicate that a direct contact between bacterial cells and plasma is not necessary for achieving successful inactivation. The plasma-generated compounds in the liquid, such as H2O2 and most likely hydroperoxy radicals, are particularly responsible for microbial inactivation.

Occurrence and genetic diversity of Arcobacter spp. in a spinach-processing plant and evaluation of two Arcobacter-specific quantitative PCR assays

Some species of the genus Arcobacter are considered to be emerging food pathogens. With respect to recent vegetable-borne outbreaks, the aim of this work was to investigate the occurrence and diversity of Arcobacter within the production chain of a spinach-processing plant by a combination of cultivation and molecular methods. Samples including spinach, water, and surface biofilm were taken over a period of three years from the entire processing line. Ten 16S rRNA (rrs) gene clone libraries were constructed and analysed using amplified rRNA gene restriction analysis (ARDRA). Approximately 1200 clones were studied that resulted in 44 operational taxonomic units (OTUs). Sequences with high similarities to Arcobacter cryaerophilus (13% of clones, 3 OTUs), A. ellisii (4%, 6 OTUs), A. suis (15%, 3 OTUs), and the type strain of A. nitrofigilis (1%, 7 OTUs) were identified. This represents the first report of the detection of the recently described species A. ellisii, A. suis and, in addition, A. venerupis from alternative habitats. A total of 67% of the clones (22 OTUs) could not be assigned to a genus, which indicated the presence of uncharacterised Arcobacter species. For the cultivation-independent detection of Arcobacter, two genus-specific quantitative PCR (qPCR) assays were developed and tested on 15 Arcobacter species. When these assays were applied to samples from the spinach-processing plant, they showed positive results for up to 35% of the samples and supported the conclusion that there is a considerable risk for the transfer of pathogenic Arcobacter species on vegetables, which was also verified by a cultivation approach.

Characterization of the cultivable microbial community in a spinach-processing plant using MALDI-TOF MS.

A better and regular control of the production chain of fresh fruits and vegetables is necessary, because a contamination of the product by human- and phyto-pathogenic microorganisms may result in high losses during storage and poses a threat to human health. Therefore, detailed knowledge about the occurrence and the diversity of microorganisms within single processing steps is required to allow target-oriented produce safety control. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successfully used to identify bacterial colonies. Bacteria can be identified with high accuracy by comparing them with generated spectra of a reference database. In this study, spinach and wash water samples were taken of the complete process line of a spinach-washing plant. Bacteria in the samples were grown on plate-count, Arcobacter selective, marine and blood agar. In total, 451 colonies were evaluated by MALDI-TOF MS, 16S rRNA gene sequence and phylogenetic analysis. 50% of the detected species belonged to the class of Gammaproteobacteria. Firmicutes were present with 22%. Mostly, the detected species showed 16S rRNA gene sequence dissimilarities larger than 1% to known reference species and, hence, could not be assigned to a distinct species. However, many isolated species belonged to genera which contain pathogenic or opportunistic pathogenic bacteria. In addition, the bacterial diversity on the spinach surface increased after the first washing step indicating a process-borne contamination of the spinach.

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

BackgroundThe 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.ResultsSix 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.ConclusionsThe 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.

Impact of different water activities (aw) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores

Much 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.

Comparative study on the high pressure inactivation behavior of the Shiga toxin-producing Escherichia coli O104:H4 and O157:H7 outbreak strains and a non-pathogenic surrogate

Enterohemorrhagic Escherichia coli strains cause each year thousands of illnesses, which are sometimes accompanied by the hemolytic uremic syndrome, like in the 2011 outbreak in Germany. For preservation thermal pasteurization is commonly used, which can cause unwanted quality changes. To prevent this high pressure treatment is a potential alternative. Within this study, the 2011 outbreak strain O104:H4, an O157:H7 and a non-pathogenic strain (DSM1116) were tested. The cells were treated in buffer (pH 7 and pH 5) and carrot juice (pH 5.1) in a pressure temperature range of 0.1-500 MPa and 20-70 °C. Flow cytometry was used to investigate the pressure impact on cell structures of the strain DSM1116. Both pathogenic strains had a much higher resistance in buffer and carrot juice than the non-pathogenic surrogate. Further, strains cultivated and treated at a lower pH-value showed higher pressure stability, presumably due to variations in the membrane composition. This was confirmed for the strain DSM1116 by flow cytometry. Cells cultivated and treated at pH 5 had a stronger ability to retain their membrane potential but showed higher rates of membrane permeabilization at pressures <200 MPa compared to cells cultivated and treated at pH 7. These cells had the lowest membrane permeabilization rate at around 125 MPa, possibly denoting that variations in the fatty acid composition and membrane fluidity contribute to this stabilization phenomenon.

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

Since 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.

Non-destructive mobile monitoring of microbial contaminations on meat surfaces using porphyrin fluorescence intensities

A non-destructive mobile system for meat quality monitoring was developed and investigated for the possible application along the whole production chain of fresh meat. Pork and lamb meat was stored at 5 °C for up to 20 days post mortem and measured with a fluorescence spectrometer. Additionally, the bacterial influence on the fluorescence signals was evaluated by different experimental procedures. Fluorescence of NADH and different porphyrins could be correlated to the growth of diverse bacteria and hence used for contamination monitoring. The increase of porphyrin fluorescence started after 9 days p.m. for pork and after 2 days p.m. for lamb meat. Based on the results, a mobile fluorescence system was built and compared with the laboratory system. The corrected function of the meat slices showed a root mean square error of 1156.97 r.u. and a mean absolute percentage error of 12.59%; for lamb the values were 470.81 r.u. and 15.55%, respectively. A mobile and non-invasive measurement system would improve the microbial security of fresh meat.

Respiration and Storage Quality of Fresh-Cut Apple Slices Immersed in Sugar Syrup and Orange Juice

Storing fresh-cut apple slices in suitable fruit juice or sugar syrup is a general practice. However, application of this approach is mainly based on empirical knowledge, while systematic and comprehensive analyses of the relevant effects of this storage technique on keeping quality-related physiological properties of fresh-cut products is still missing. Hence, the aim of this study was to evaluate the impacts of complete immersion of fresh-cut apples in sugar syrup and fruit juice solution on respiratory behaviour and other relevant quality attributes (colour, tissue strength, and soluble solid and acidity). Sugar syrup and pure orange juice showed a high potential to store and protect fresh-cut apples. Results showed that only pure orange juice positively affected the produce quality by preventing browning effects. In addition, sugar syrup of 13.4–20% most effectively prevented browning of apple slices and guaranteed high product quality retention during storage. The application of different liquid media provides a practical means to prevent browning and maintain product quality.

Screening of microbial communities associated with endive lettuce during postharvest processing on industrial scale

In this study, the composition of the microbial community on endive lettuce (Cichorium endivia) was evaluated during different postharvest processing steps. Microbial community structure was characterized by culture-dependent and culture-independent methods. Endive lettuce was sampled exemplarily at four different stages of processing (raw material, cut endive lettuce, washed endive lettuce, and spin-dried (ready to pack) endive lettuce) and analysed by plate count analysis using non-selective and selective agar plates with subsequent identification of bacteria colonies by matrix-assisted laser desorption/ionization time-of light mass spectrometry (MALDI-TOF MS). Additionally, terminal-restriction fragment length polymorphism (TRFLP) analysis and 16S rRNA gene nucleotide sequence analysis were conducted. The results revealed structural differences in the lettuce microbiomes during the different processing steps. The most predominant bacteria on endive lettuce were detected by almost all methods. Bacterial species belonging to the families Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, and Moraxellaceae were detected in most of the examined samples including some unexpected potentially human pathogenic bacteria, especially those with the potential to build resistance to antibiotics (e.g., Stenotrophomonas maltophilia (0.9 % in cut sample, 0.4 % in spin-dried sample), Acinetobacter sp. (0.6 % in raw material, 0.9 % in cut sample, 0.9 % in washed sample, 0.4 % in spin-dried sample), Morganella morganii (0.2 % in cut sample, 3 % in washed sample)) revealing the potential health risk for consumers. However, more seldom occurring bacterial species were detected in varying range by the different methods. In conclusion, the applied methods allow the determination of the microbiomes structure and its dynamic changes during postharvest processing in detail. Such a combined approach enables the implementation of tailored control strategies including hygienic design, innovative decontamination techniques, and appropriate storage conditions for improved product safety.