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

Towards efficient production of highly optically pure d-lactic acid from lignocellulosic hydrolysates using newly isolated lactic acid bacteria

This 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

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

Background: 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

Schnelle Identifizierung von Mikroorganismen mittels MALDI-TOF MS

Sichere 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.The increasing demand on safe food with high quality poses a high challenge especially for perishable products. Microbiological sampling along the food processing chain is mainly focused on selected indicator microorganisms and unexpected potential human pathogenic bacteria may remain undetected. The detection of unexpected pathogenic bacteria is of great interest to avoid potential risks for consumers. The change microbial community of perishables exemplarily shown for mung bean sprouts was evaluated using plate count methods and MALDI-TOF MS. The total aerobic viable count of sprouts was 8–9 log CFU/g and among others bacteria from Bacillus cereus group, Yersinia sp., Enterobacter spp., Klebsiella spp., Pantoea spp., and Pseudomonas spp. were identified

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

Inhibition or Stimulation of Ochratoxin A Synthesis on Inoculated Barley Triggered by Diffuse Coplanar Surface Barrier Discharge Plasma

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins. Besides their high toxicity, mycotoxins are highly stable to physical, chemical or biological detoxification. Therefore, the treatment with cold atmospheric plasma could be one approach to reduce the amount of mycotoxins in different products. The aim of this study was to determine the influence of cold atmospheric plasma on the inactivation of Aspergillus niger and Penicillium verrucosum inoculated on barley and their production of OTA. Inoculated barley was treated with plasma generated by dry air, CO2 or CO2 + O2 for 1 or 3 min and stored for up to two weeks at 9, 25, or 37°C. Three minutes of air plasma treatment effectively significantly reduced the total mold count of both microorganisms by 2.5–3 log cycles. The production of OTA from A. niger was only low, therefore the treatment effect was indistinguishable. The treatment of P. verrucosum on barley after an incubation of five days using a CO2 + O2 plasma resulted in a reduction of the OTA content from 49.0 (untreated) to 27.5 (1 min) and 23.8 ng/g (3 min), respectively. In contrast, CO2 plasma caused an increase of the OTA amount from 49.0 (untreated) to 55.8 (1 min) and 72.9 ng/g (3 min). Finally, the use of air plasma resulted likewise in a decrease of the OTA concentration from 56.9 (untreated) to 25.7 (1 min) and 20.2 ng/g (3 min), respectively. Reducing the incubation time before the treatment to 24 h caused in contrast an increase of the OTA content from 3.1 (untreated) to 29.1 (1 min) and 20.7 ng/g (3 min). Due to the high standard deviation, these changes were not significant, but the tendencies were clearly visible, showing the strong impact of the plasma gas on the OTA production. The results show, that even if the total mold count was reduced, under certain conditions the OTA amount was yet enhanced, probably due to a stress reaction of the mold. Concluding, the plasma gas and incubation conditions have to be considered to allow a successful inactivation of molds and in particular their toxic metabolites

Impact of cold atmospheric pressure plasma processing on storage of blueberries

The 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

Sublethal Injury and Viable but Non-culturable (VBNC) State in Microorganisms During Preservation of Food and Biological Materials by Non-thermal Processes

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

Plasma applications for the treatment of bean sprouts : safety, quality and nutritional assessments under aqueous and gaseous set-ups

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Overall, it was found that the combined seed treatment with direct air CAP (350 W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for first time that aside from the stimulatory effect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can significantly enhance seedling growth. The positive outcome and further applications of different forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research.peer-reviewe

Aqueous and gaseous plasma applications for the treatment of mung bean seeds

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Germination assessments were performed in a test tube set-up flled with glass beads and the produced irrigation water. Overall, it was found that the combined seed treatment with direct air CAP (350W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for frst time that aside from the stimulatory efect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can signifcantly enhance seedling growth. The positive outcome and further applications of diferent forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research.peer-reviewe