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.

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 filled with glass beads and the produced irrigation water. 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.

Thermal Impact on the Culturable Microbial Diversity Along the Processing Chain of Flour From Crickets (Acheta domesticus).

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.

Safety Control of Whole Berries by Cold Atmospheric Pressure Plasma Processing: A Review.

HIGHLIGHTS: CAPP technology has high application potential for decontamination of berries. Impacts of CAPP in aspects of food safety and security still need to be addressed. Optimized treatment parameters need to be investigated for each berry type.

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.

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.

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 microbiome's 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.

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.

Impact of different water activities (a w) 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 a w-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 a w-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 (a w), 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 (a w = 1) to 75 (a w = 0.9) min at 105°C to 3 (a w = 1) to 15 (a w = 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 a w 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.

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 plasma's 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.

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.

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.

Analysis of the bacterial community within carrot wash water.

Vegetables are washed after harvest to remove unwanted organic and inorganic particles, but wash water contaminated with certain pathogenic microorganisms can potentially contaminate produce. In this study, the microbial diversity of wash water was analyzed in samples taken from a carrot-processing facility. A 16S rRNA gene library with 427 clones was constructed and analyzed by amplified rDNA restriction analysis. For taxonomic classification, the 16S rRNA gene nucleotide sequences of 94 amplified rDNA restriction analysis fingerprints were determined. Each fingerprint indicates a distinct group of microorganisms. The nucleotide sequences were assigned to corresponding reference species. The most prevalent genus was Tolumonas , with 26% of the clones, followed by Acinetobacter and Flacobacterium , with 11% each. The latter two genera contain species that are known to cause nosocomial infections. The fourth most common genus was Arcobacter , comprising 9% of all clones. Some species of Arcobacter are considered to be emerging food pathogens, mainly associated with the contamination of meat products. So far, they have not been considered as contaminants of fresh produce. Based on the sequence data, an Arcobacter-specific PCR assay was developed to facilitate the detection of vegetable-associated Arcobacter strains.