Airborne microbial re-contaminations are among the most frequent causes of food spoilage, contributing to food waste and economic losses. Cleanrooms can help to avoid bioaerosol re-contaminations, but are not suitable for open food processing environments. The present study assessed airborne microbial levels and their contamination routes within bakery production. Current airborne mold and total viable counts (TVCs) were evaluated over 8 months, indicating mold and TVCs ranging from 20 to 960 CFU/m3 and from 20 to 1600 CFU/m3, respectively. The relative contamination level of each source point towards the cooling zone was virtually reproduced. The overall aim of this study was to develop a tailored cost-effective and easy-to-apply partial protection strategy, based on filter fan units (FFUs). The FFU concept with an air velocity of 0.3 m/s resulted in a significant reduction of the relative contamination (99.94%). Hence, this study suggests an alternative FFU-based control strategy for airborne contaminations within open food processing areas.

Pulsed electric fields (PEF) can induce reversible or irreversible electroporation effects on bacterial cells resulting in sublethally or lethally injured cells. Hereafter, an alternative single-staining flow cytometry approach with SYTOXTM Green (SYTOX) is proposed, allowing for a straightforward and rapid screening of electroporation effects. SYTOX-staining indicated 38-63% of E. coli cells with intermediate cellular changes, likely being sublethally injured, at electric field intensities of 8 kV/cm-18 kV/cm. Compared to staining with propidium iodide, SYTOX showed a brighter fluorescence intensity, allowing for easier differentiation of subpopulations. The proportion of intermediate injury observed with SYTOX was distinctly higher than the sublethal damage detected with conventional plating on selective media (31-45%). It is, however, challenging to compare selective plating with the single-staining method based on membrane integrity, suggesting different analytical methods to indicate complex cellular states and sublethal effects.

As microbial contamination is persistent within the food and bioindustries and foodborne infections are still a significant cause of death, the detection, monitoring, and characterization of pathogens and spoilage microorganisms are of great importance. However, the current methods do not meet all relevant criteria. They either show (i) inadequate sensitivity, rapidity, and effectiveness; (ii) a high workload and time requirement; or (iii) difficulties in differentiating between viable and non-viable cells. Flow cytometry (FCM) represents an approach to overcome such limitations. Thus, this comprehensive literature review focuses on the potential of FCM and fluorescence in situ hybridization (FISH) for food and bioindustry applications. First, the principles of FCM and FISH and basic staining methods are discussed, and critical areas for microbial contamination, including abiotic and biotic surfaces, water, and air, are characterized. State-of-the-art non-specific FCM and specific FISH approaches are described, and their limitations are highlighted. One such limitation is the use of toxic and mutagenic fluorochromes and probes. Alternative staining and hybridization approaches are presented, along with other strategies to overcome the current challenges. Further research needs are outlined in order to make FCM and FISH even more suitable monitoring and detection tools for food quality and safety and environmental and clinical approaches.

The last decades have seen extensive scientific and technological improvements in many fields of microbiology and molecular biology. Correspondingly, flow cytometry—a rapid, precise and straightforward method for cultivation-independent detection of cells in liquids—has been a major topic in aquatic microbiology and drinking water analysis. Flow cytometry provides information at the single-cell level, including total cell counts, size measurements, nucleic acid content and bacterial viability and activity. While regulatory requirements for water testing rely on cultivation-based methods, flow cytometry can be considered a powerful tool to complement standard procedures. This article provides insights into the methodology and applicability of flow cytometry in the field of microbiological drinking water analysis and presents an overview on several case studies that cover a broad range of different objectives. The later are comprised of a study on flow cytometric characterization of Austrian drinking water resources, of an example for advanced data analysis methods of flow cytometric data, of a study on monitoring microbial regrowth within the distribution network, of an exemplary case of the application of online flow cytometry for high-frequency monitoring and of an introduction to the combination of flow cytometry and sequencing information. Finally, it is argued that due to the high microbiological variability of different water resources, unusual changes of flow cytometric parameters, rather than specific limits, could act as an indicator for further investigation. In this way flow cytometry can provide a good basis for risk assessments in water safety plans. The application of flow cytometry still remains utility-specific and a huge need for standardization of data analysis and interpretation exists in order to achieve a better cooperation of water utilities.

Biofilm characteristics of Microbacterium lacticum D84 (M. lacticum) and Staphylococcus capitis subsp. capitis (S. capitis) on polytetrafluoroethylene and AISI-304 stainless steel at early- (24, 48 h) and late-stage (144, 192 h) biofilm formation were investigated. M. lacticum biofilm structure was more developed compared to S. capitis, representing vastly mature biofilms with a strongly developed amorphous matrix, possibly extracellular polymeric substances (EPSs), at late-stage biofilm formation. S. capitis showed faster growth behavior but still resulted in a relatively flat biofilm structure. Strong correlations were found between several roughness parameters and S. capitis surface coverage (r ≥ 0.98), and between total surface free energy (γs) and S. capitis surface coverage (r = 0.89), while M. lacticum remained mostly unaffected. The pronounced ubiquitous biofilm characteristics make M. lacticum D84 a suitable model for biofilm research. Studying biofilm formation of these bacteria may help one understand bacterial adhesion on interfaces and hence reduce biofilm formation in the food industry.

Bacterial communities associated with the ripening process in artisanal wild boar and deer meat sausages were investigated by molecular barcoding using the 16S rRNA gene as a marker. A core microbiota shared by 83.54% of the samples indicated remarkable level of Lactobacillus sake/and Lactobacillus curvatus, accounting for 20.55% in initial and 70.48% in final products as well as spoilage-associated bacteria including Stenotrophomonas, Bacillus, Pseudomonas, Carnobacterium and Brochothrbc, with an average abundance 44.15% at the beginning and 13.98% at the end of the production. Of selected LAB isolates (n = 555), 43.83% were not suitable for food application due to the antibiotic resistance or the presence of the tric gene. Most of the strains designated as safe were able to grow at 25 degrees C even in the presence of 3.0 and 6.0% of NaCl or pH 4.5, but exposure to the same stressors resulted in growth reduction at 12 degrees C. Acidification and antimicrobial activity were found in 65.62% and 37.50% of strains, respectively. Most of the strains showed lipolytic and proteolytic activity, but only 9.37% were able to degrade sarcoplasmic proteins. These results give important information for the development of new starter formulation for the production of high quality game meat sausages.

Nanotechnology offers new possibilities to modify different aspects of biopolymer films in order to improve their performance in food packaging. The present study assessed the effect of nanocomposite films based on carboxymethyl cellulose (CMC), okra mucilage (OM), and ZnO nanoparticles (ZnO NPs) on the shelf-life of packed chicken breast meat stored at 4 degrees C. Treatments examined in the present study were the following: CMC, CMC/ZnO, CMC/OM30%/ZnO, CMC/OM40%/ZnO, and CMC/OM50%/ZnO. Total viable counts (TVC), Staphylococcus aureus counts, Lactic Acid Bacteria (LAB) counts, thiobarbituric acid (TBA), total volatile nitrogen (TVN), instrumental color (CIE L*, a*, b*), and sensory attribute were determined at a gap of 3-day interval for a period of 12 days. Irrespective of the film type, an increasing trend for all microorganisms was observed over the storage time. However, the growth was significantly inhibited by okra mucilage and ZnO nanoparticles used in the packaging system (P < 0.05). The increase of lipid oxidation and TVN were significantly restricted by incorporation of okra mucilage and ZnO NPs. Chicken samples packed with okra mucilage and ZnO NPs produced higher lightness (L*) values (P < 0.05) as compared to the CMC samples. It was concluded that CMC/OM50%/ZnO film had the best inhibitory effect on the microbial growth and chemical changes in the chicken breast meat and the samples packed with it recorded higher sensorial score under chilling storage as well.

In this study, the influence of meat batter composition and sausage diameter on the development of microbiota and sensory traits of traditional, spontaneously fermented wild boar meat sausages are evaluated. This research also demonstrates how principal component analysis (PCA) can be used to relate product sensory properties to particular microbial genotype and to select potential starter or adjunct culture. Generally, similar microbiological results were obtained in all types of products. The undesirable microbiota was either not detected at any sausage production stage or its number decreased below the detection limit in ripened sausages. The low growth rate of lactic acid bacteria (LAB) was consistent with the obtained pH and slow acidification rate. Although no differences in the composition of LAB species were noticed between sausage types (50S=50 % wild boar meat in small casing, 50L=50 % wild boar meat in large casing, 100S=100 % wild boar meat in small casing), a clear separation based on LAB genotypes could be observed. Upon quantitative descriptive analysis, significant differences in sensory attributes between sausage types were established. According to the PCA, the overall acceptability traits of sausages are closely linked to one Leuconostoc mesenteroides genotype (LM_4). Of all tested technological properties, LM_4 strains showed remarkable acidification ability, lowering the pH from pH=5.41 to 3.74, and pronounced proteolytic activity on skimmed milk as well as antagonistic activity against Staphylococcus aureus (DSM 20231) and Brochothrix thermosphacta (LMG 17208). Lipolytic and haemolytic activities were not detected, and all analyzed strains were susceptible to tested antibiotics and possessed no biogenic amine genes.

Spontaneously fermented wild boar and deer meat sausages produced in Croatia were physicochemically and microbiologically investigated at different time points of their production. Final products resulted with pH and a(w) values between 5.04-5.53 and 0.83-0.87, respectively. The histamine concentration was below 5.0 mg/kg whereas tyramine content ranged from 47.3 to 219.0 mg/kg. A total of 917 isolated lactic acid bacteria (LAB) were genotyped by rep-PCR and identified by 16S rRNA gene sequencing. Overall, Leuconostoc mesenteroides was identified as the most frequently isolated species (n = 259), followed by Lactobacillus sakei (n = 190) and Enterococcus casseliflavus (n = 106). Cluster analysis revealed a remarkable intraspecies diversity and a strong sausage-specific clustering of the LAB genotypes, indicating meat as a probable source of microbial diversity in sausages. 14.38% of LAB were positive for tyramine encoding gene and no other target biogenic amines genes were detected. Due to the elevated number of presumptive pathogens (E. coil. Enterobacteriaceae, B. cereus group, coliforms) in ready-to-eat sausages, 33.33% of products can be considered as inappropriate for human consumption.

Listeria monocytogenes is a food pathogen capable of growing at a broad temperature range from 50°C to refrigerator temperatures. A key requirement for bacterial activity and growth at low temperatures is the ability to adjust the membrane lipid composition to maintain cytoplasmic membrane fluidity. In this study, we confirmed earlier findings that the extents of fatty acid profile adaptation differed between L. monocytogenes strains. We were able to demonstrate for isolates from food that growth rates at low temperatures and resistance to freeze-thaw stress were not impaired by a lower adaptive response of the fatty acid composition. This indicated the presence of a second adaptation mechanism besides temperature-regulated fatty acid synthesis. For strains that showed weaker adaptive responses in their fatty acid profiles to low growth temperature, we could demonstrate a significantly higher concentration of isoprenoid quinones. Three strains even showed a higher quinone concentration after growth at 6°C than at 37°C, which is contradictory to the reduced respiratory activity at lower growth temperatures. Analyses of the membrane fluidity in vivo by measuring generalized polarization and anisotropy revealed modulation of the transition phase. Strains with increased quinone concentrations showed an expanded membrane transition phase in contrast to strains with pronounced adaptations of fatty acid profiles. The correlation between quinone concentration and membrane transition phase expansion was confirmed by suppression of quinone synthesis. A reduced quinone concentration resulted in a narrower transition phase. Expansion of the phase transition zone by increasing the concentration of non-fatty acid membrane lipids is discussed as an additional mechanism improving adaptation to temperature shifts for L. monocytogenes strains.IMPORTANCEListeria monocytogenes is a foodborne pathogen with an outstanding temperature range for growth. The ability for growth at temperatures close to the freezing point constitutes a serious contamination potential for cold stored food. The only known mechanism of the species for adaptation of membrane fluidity is modification of the membrane fatty acid composition. We were able to demonstrate that, at least for some strains, this adaptation mechanism is supported by regulation of the menaquinone concentration. The increase of this neutral membrane lipid is correlated with fluidization of the membrane under low-temperature conditions and therefore represents a fatty acid-independent mechanism for adaptation to low temperatures.

Traditional production of fermented dairy products in Montenegro is carried out without adding defined starter cultures. This way of production involves lactic acid bacteria (LAB) that are normally present in the raw milk and production environment. This autochthonous ("wild") fermentation microbiota represents a reservoir of unknown strains. In order to study the LAB diversity, 25 indigenous dairy products in Montenegro have been tested. Isolation was performed on microbial media M17 and MRS agar with or without supplementations under aerobic and anaerobic conditions at temperatures of 30?C, 37 ?C and 44?C. Identification of these isolates at species level was done by species-specific PCR and gene regions sequencing of representatives of each RAPD-cluster. RAPD-PCR was used to characterize the isolates at strain level. Nine Lactobacillus species, five Leuconostoc species, four Enterococcus species as well as strains of the species Lactococcus lactis, Pediococcus pentosaceus and Streptococcus thermophilus were detected. It can be concluded that a rich lactic acid bacteria diversity existed in the analyzed Montenegrin dairy products. Further examination of the isolates could lead to the development of autochthonous starter cultures that would contribute to a product that is characteristic for the geographical area and to which the local population is accustomed.

The viable but non-culturable (VBNC) state, as well as sublethal injury of microorganisms pose a distinct threat to food safety, as the use of traditional, culture-based microbiological analyses might lead to an underestimation or a misinterpretation of the product’s microbial status and recovery phenomena of microorganisms may occur. For thermal treatments, a large amount of data and experience is available and processes are designed accordingly. In case of innovative inactivation treatments, however, there are still several open points with relevance for the investigation of inactivation mechanisms as well as for the application and validation of the preservation processes. Thus, this paper presents a comprehensive compilation of non-thermal preservation technologies, i.e., high hydrostatic pressure (HHP), pulsed electric fields (PEFs), pulsed light (PL), and ultraviolet (UV) radiation, as well as cold plasma (CP) treatments. The basic technological principles and the cellular and molecular mechanisms of action are described. Based on this, appropriate analytical methods are outlined, i.e., direct viable count, staining, and molecular biological methods, in order to enable the differentiation between viable and dead cells, as well as the possible occurrence of an intermediate state. Finally, further research needs are outlined.

Surface-groundwater interactions play an important role in microbial community compositions of river bank filtrates. Surface water contaminations deriving from environmental influences are attenuated by biogeochemical processes in the hyporheic zone, which are essential for providing clean and high-quality drinking water in abstraction wells. Characterizing the flow regime of surface water into the groundwater body can provide substantial information on water quality, but complex hydraulic dynamics make predictions difficult. Thus, a bottom up approach using microbial community shifting patterns as an overall outcome of dynamic water characteristics could provide more detailed information on the influences that affect groundwater quality. The combination of high-throughput sequencing data together with flow cytometric measurements of total cell counts reveals absolute abundances among taxa, thus enhancing interpretation of bacterial dynamics. 16S rRNA high-throughput sequencing of 55 samples among six wells in a well field in Austria that is influenced by river bank filtrate within a time period of 3 months has revealed both, clear differences as well as strong similarity in microbiome compositions between wells and dates. A significant community shift from April to May occurred in four of six wells, suggesting that surface water flow regimes do affect these wells stronger than others. Triplicate sampling and subsequent sequencing of wells at different dates proved the method to be reproducible. Flow cytometric measurements of total cells indicate microbial shifts due to increased cell counts and emphasize the rise of allochthonous microorganisms. Typical freshwater bacterial lineages (Verrucomicrobia, Bacteroidetes, Actinobacteria, Cyanobacteria, Armatimonadetes) were identified as most increasing phyla during community shifts. The changes are most likely a result of increased water abstraction in the wells together with constant river water levels rather than rain events. The results provide important knowledge for future implementations of well utilization in dependency of the nearby Danube River water levels and can help drawing conclusions about the influence of surface water in the groundwater such that hygienically save and clean drinking water with a stable microbial community can be provided.

Im Rahmen eines Forschungsprojekts wurde bei einzelnen Quellfassungen eine kontinuierliche Überwachung zur Erfassung von Veränderungen der Trinkwassereigenschaften aufgrund hydrologischer Ereignisse durchgeführt. Die Quellfassungen wurden dabei mit einer kontinuierlichen Erfassung von Schüttung (in l/s mittels Messwehr und Wasserstand bzw. Wasserzähler), Wassertemperatur (in °C), elektrischer Leitfähigkeit (µS/cm), Trübung (FTU), spektralem Absorptionskoeffizient (SAK254) (Abs/m), gelöstem organischem Kohlenstoff (DOC mg/l) und Transmission (%) jeweils mittels 10-Minuten- bzw. Stundenwerten überwacht. Zusätzlich wurde zur Erfassung von unmittelbaren Niederschlagsereignissen im Fassungsgebiet eine Niederschlagsmessung (Zeit und Menge) eingesetzt und es wurden vergleichende Laboranalysen durchgeführt. Im Rahmen dieser Überwachung wurde zusätzlich die Anwendung der eingesetzten Messinstrumente an Extremstandorten (z. B. kein Stromanschluss) evaluiert.

As one of the emerging non-thermal technologies, pulsed light (PL) facilitates rapid, mild and residue-free microbial surface decontamination of food and food contact materials. While notable progress has been made in the characterization of the inactivation potential of PL, experimental data available on the tolerance development to the same (homologous) stress or to different (heterologous) stresses commonly applied in food manufacturing (e.g., acid, heat, salt) is rather controversial. The findings of the present study clearly indicate that both the homologous tolerance development against PL as well as the heterologous tolerance development from heat to PL can be triggered in Listeria monocytogenes. Further, conducted kinetic analysis confirmed that the conventionally applied log-linear model is not well suited to describe the inactivation of L. monocytogenes, when exposed to PL. Instead, the Weibull model as well as the log-linear + tail model were identified as suitable models. Transmission electron microscopic (TEM) approaches allow suggestions on the morphological alterations in L. monocytogenes cells after being subjected to PL.

The implementation and evaluation of biological nitrification as a possible treatment option for the small-scale drinking water supply of a rural Upper Austrian community was investigated. The drinking water supply of this community (average system input volume: 20 m(3)/d) is based on the use of deep anaerobic groundwater with a high ammonium content of geogenic origin (up to 5 mg/l) which must be treated to prevent the formation of nitrites in the drinking water supply system. This paper describes the implementation and operation of biological nitrification despite several constraints including space availability, location and financial and manpower resources. A pilot drinking water treatment plant, including biological nitrification implemented in sand filters, was designed and constructed for a maximum treatment capacity of 1.2 m(3)/h. Online monitoring of selected physicochemical parameters has provided continuous treatment performance data. Treatment performance of the plant was evaluated under standard operation as well as in the case of selected malfunction events.

Today, the increasing demand for minimally processed foods that are at the same moment nutritious, organoleptically satisfactory, and free from microbial hazards challenges the research and development to establish alternative methods to reduce the level of bacterial contamination. As one of the recent emerging nonthermal methods, pulsed light (PL) constitutes a technology for the fast, mild, and residue-free surface decontamination of food and food contact materials in the processing environment. Via high frequency, high intensity pulses of broad-spectrum light rich in the UV fraction, viable cells as well as spores are inactivated in a nonselective multi-target process that rapidly overwhelms cell functions and subsequently leads to cell death. This review provides specific information on the technology of pulsed light and its suitability for unpackaged and packaged meat and meat products as well as food contact materials like production surfaces, cutting tools, and packaging materials. The advantages, limitations, risks, and essential process criteria to work efficiently are illustrated and discussed with relation to implementation on industrial level and future aspects. Other issues addressed by this paper are the need to take care of the associated parameters such as alteration of the product and utilized packaging material to satisfy consumers and other stakeholders.

Modified atmosphere packaging (MAP) based on carbon dioxide (CO2) - nitrogen (N-2) gas mixtures has been applied to maintain the safety and quality of ready-to-eat (RTE) meat products. The use of argon (Ar) gas as a supplement to CO2-N-2 mixtures or as substitute for N-2 is a current approach to enhance the effectiveness of MAP. As there is limited information on the effect of Ar MAP on the growth behaviour or the survival of pathogenic bacteria in RTE foods, the aim of the present study was to assess the influence of Ar in MAP on the growth of Listeria monocytogenes and Escherichia coli strains under different conditions. For this purpose, a CO2-N-2 (20:80) atmosphere was compared with a CO2-N-2 Ar (30:30:40) and CO2 Ar (30:70) atmosphere based on the assessment of bacterial growth (8) on a gelatin-agar medium and ham. Additionally, a shelf life monitoring study was performed to evaluate the effect of these treatments on the background microflora of ham. The findings suggest that under the CO2-N-2 MAP the product matrices supported the growth (6 > 0.5 log CFU g(-1)) of L. monocytogenes throughout an observation period of 21 days at 4 -1- 2 degrees C. On the contrary, both MAP containing Ar were equally able to reduce the 8 below 0.5 log CFU g. In this regard it was irrelevant whether L monocytogenes was inoculated in depth (per slice) or at the surface (top slice) of the ham. Regarding the influence of the different gas atmospheres on E. coil all gas mixtures applied had the capacity to reduce the S of E. coli below 0.5 log CFU g(-1). Further, shelf-life extension could not be managed with the gas atmospheres considered. (C) 2015 Elsevier Ltd. All rights reserved.