Mikrowellen‐PECVD für kontinuierliche Großflächenbeschichtung bei Atmosphärendruck

Microwave PECVD for continuous wide area coating at atmospheric pressure Plasma processes are applied for a variety of surface modifications. Examples are coatings to achieve an improved corrosion and scratch protection, or surface cleaning. Normally, these processes are vacuum based and therefore suitable to only a limited extend for large area industrial applications. By use of atmospheric pressure plasma technology integration in continuously working manufacturing lines is advantageously combined with lower costs and higher throughput. Microwave plasma sources present powerful modules for plasma enhanced chemical vapour deposition at atmospheric pressure. At Fraunhofer IWS processes and equipment as well as application specific materials are developed. The coatings are suitable for scratch resistant surfaces, barrier and corrosion protective layers or anti‐reflex layers on solar cells. The film properties achieved are comparable with those produced by low pressure processes.     

Comparison of sodium hypochlorite-based foam and peroxyacetic acid-based fog sanitizing procedures in a salmon smokehouse: survival of the general microflora and Listeria monocytogenes

The effects of fog sanitization with peroxyacetic acid (hydrogen peroxide, peracetic acid, and acetic acid in combination) on general hygiene (aerobic plate count) and on Listeria monocytogenes were assessed in a slicing area at a salmon smokehouse and compared with the effects of foam sanitization with sodium hypochlorite (routinely performed at the smokehouse). Two hundred twenty-three environmental samples were collected with sponges and swabs after each of the sanitization procedures, and 68 samples were collected during production. The total culturable aerobic plate count was determined for each sample, and a total of 288 bacterial strains were randomly isolated and tentatively identified to genus level by physiological and biochemical tests. The microflora was dominated by Neisseriaceae, Enterobacteriaceae, and lactic acid bacteria during production. Foam sanitization caused a change in the composition of the flora, with Pseudomonas spp. and Alcaligenes spp. being the dominant gram-negative bacteria and Kurthia spp. and Bacillus spp. being the surviving gram-positive bacteria. Bacteria were very sensitive to fog sanitization, and yeasts accounted for almost half of the surviving flora. By a selective isolation method, strains of L. monocytogenes were isolated and subsequently characterized by random amplified polymorphic DNA (RAPD) typing. Following foam sanitization, 14 to 42% of the samples contained <10 CFU per site, whereas 29 to 78% of the samples collected after fog sanitization contained this level of bacteria. The prevalence of L. monocytogenes was unchanged, but L. monocytogenes was found only in poorly cleaned areas such as drains. The RAPD types for all positive samples were identical to the type that had persisted in the smokehouse since 1995, indicating the importance of drains as a niche.     

A new expression system for protein crystallization using trimeric coiled-coil adaptors

We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils, using the corresponding GCN4 variants.     

Prevalence and survival of Listeria monocytogenes in Danish aquatic and fish-processing environments

Listeria monocytogenes contamination of ready-to-eat food products such as cold-smoked fish is often caused by pathogen subtypes persisting in food-processing environments. The purpose of the present study was to determine whether these L. monocytogenes subtypes can be found in the outside environment, i.e., outside food processing plants, and whether they survive better in the aquatic environment than do other strains. A total of 400 samples were collected from the outside environment, fish slaughterhouses, fish farms, and a smokehouse. L. monocytogenes was not detected in a freshwater stream, but prevalence increased with the degree of human activity: 2% in seawater fish farms, 10% in freshwater fish farms, 16% in fish slaughterhouses, and 68% in a fish smokehouse. The fish farms and slaughterhouses processed Danish rainbow trout, whereas the smokehouse was used for farm-raised Norwegian salmon. No variation with season was observed. Inside the processing plants, the pattern of randomly amplified polymorphic DNA (RAPD) types was homogeneous, but greater diversity existed among isolates from the outside environments. The RAPD type dominating the inside of the fish smokehouse was found only sporadically in outside environments. To examine survival in different environments, L. monocytogenes or Listeria innocua strains were inoculated into freshwater and saltwater microcosms. Pathogen counts decreased over time in Instant Ocean and remained constant in phosphate-buffered saline. In contrast, counts decreased rapidly in natural seawater and fresh water. The count reduction was much slower when the natural waters were autoclaved or filtered (0.2-microm pore size), indicating that the pathogen reduction in natural waters was attributable to a biological mechanism, e.g., protozoan grazing. A low prevalence of L. monocytogenes was found in the outside environment, and the bacteria did not survive well in natural environments. Therefore, L. monocytogenes in the outer environment associated with Danish fish processing is probably of minor importance to the environment inside a fish production plant.     

Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [Pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim

Data on the prevalence and growth of Listeria monocytogenes in lightly preserved fish products from subtropical and tropical regions are very scarce. Our research describes L. monocytogenes that was detected in 5% of the packages of cold-smoked surubim, a native Brazilian freshwater fish that we analyzed, and shows that the strains isolated were of the same random amplified polymorphic DNA subtype as the strains that were isolated from the same factory 4 years earlier. A bacteriocinogenic strain of Carnobacterium piscicola (strain C2), isolated from vacuum-packed cold-smoked surubim, and two C. piscicola strains, isolated from vacuum-packed, cold-smoked salmon, were capable of limiting or completely inhibiting the growth of an L. monocytogenes (strain V2) isolated from surubim in fish peptone model systems incubated at 10 degrees C. Monocultures of L. monocytogenes reached 108 CFU/ml (g), whereas the growth of L. monocytogenes was completely inhibited by C. piscicola C2. The bacteriocinogenic C. piscicola A9b+ and its nonbacteriocinogenic mutant A9b- reduced maximum Listeria levels by 2 to 3 log units. Both bacteriocinogenic C. piscicola strains prevented listerial growth in cold-smoked fish juices (surubim and salmon). Although the carnobacteria grew poorly on cold-smoked surubim at 10 degrees C, the strains were able to reduce maximum Listeria counts by 1 to 3 log units in an artificially inoculated product (surubim). We conclude that Brazilian smoked fish products harbor L. monocytogenes and should be stabilized against the growth of the organism. C. piscicola C2 has the potential for use as a bioprotective culture in surubim and other lightly preserved fish, but further studies are required to optimize its effect.     

Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P < 0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P < 0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P < 0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.     

AFM Images of Complexes between Amylose and Aspergillus niger Glucoamylase Mutants,Native and Mutant Starch Binding Domains: A Model for the Action of Glucoamylase

Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild‐type A. niger starch binding domains (SBDs), and mutant SBDs (W563K and W590K) lacking either of the two starch binding sites. The images are interpreted in terms of a favourable binding between the amylose chains and the wild‐type SBDs, leading to the formation of ring‐like structures, in which parallel strands of the amylose molecule bind to both binding sites on the SBDs. The SBDs are seen to form a template for the assembly of an expanded amylosic double helix. This model for amylose‐SBD binding has been used to propose a molecular mechanism for the role of the SBD in the hydrolytic action of glucoamylase on starch granules. The SBDs are considered to recognise the ends of amylosic double helices formed by the short amylosic chains present as branches on the amylopectin molecules, and displayed on the face of crystalline lamellae. This allows the binding and immobilisation of chain ends by the SBD, facilitating binding and cleavage by the exo‐acting catalytic domain.     

(Un)expected Similarity of the Temporary Adhesive Systems of Marine,Brackish, and Freshwater Flatworms

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.     

Tungsten and Molybdenum 2,4,6‐Trimethylbenzylidyne Complexes as Robust Pre‐Catalysts for Alkyne Metathesis

A series of 2,4,6‐trimethylbenzylidyne tungsten and molybdenum complexes was prepared from the tribromides mer‐[MesCMBr₃(dme)] ( 9a , M=W; 9b , M=Mo, dme=1,2‐dimethoxyethane). Successive reaction of complexes 9 with lithium or potassium hexafluoro‐tert‐butoxide and lithium 1,3‐di‐tert‐butylimidazolin‐2‐imide, (Im^t‐BuN)Li, afforded the imidazolin‐2‐iminato complexes [MesCM{OC(CF₃)₂Me}₂(Im^t‐BuN)] ( 10a , M=W; 10b , M=Mo), whereas the reactions of 9 with three equivalents of LiOSi(O‐t‐Bu)₃ or KOC(CF₃)₃ gave [MesCM{OSi(O‐t‐Bu)₃}₃] ( 11a , M=W; 11b , M=Mo) and [MesCM{OC(CF₃)₃}₃] ( 12a , M=W; 12b , M=Mo), respectively. For comparison, the benzylidyne complex [PhCMo{OSi(O‐t‐Bu)₃}₃] ( 7b ) was also prepared, and its molecular structure together with those of 10a , 10b , 11b , 12a and 12b were established by X‐ray diffraction analysis. Complexes 10 and 11 were employed as pre‐catalysts for the alkyne metathesis of the test substrate 3‐pentynyl benzyl ether ( 13 ) at low catalyst loadings (1 mol%) in the presence of molecular sieve (5 Å). Comparative studies of these 2,4,6‐trimethylbenzylidyne species (MesCM) with their benzylidyne analogues (PhCM) revealed that the increased steric bulk renders the former more stable and manageable in air in solid form for shorter periods of time, but at the expense of a slower initiation, which requires higher temperatures or longer reaction times.