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Human noroviruses (HuNoVs), a leading cause of food-borne gastroenteritis worldwide, are easily transferred via ready-to-eat (RTE) foods, often prepared by infected food handlers. In this study, the transmission of HuNoV and murine norovirus (MuNoV) from virus-contaminated hands to latex gloves during gloving, as well as from virus-contaminated donor surfaces to recipient surfaces after simulated preparation of cucumber sandwiches, was inspected. Virus transfer was investigated by swabbing with polyester swabs, followed by nucleic acid extraction from the swabs with a commercial kit and quantitative reverse transcription-PCR. During gloving, transfer of MuNoV dried on the hand was observed 10/12 times. HuNoV, dried on latex gloves, was disseminated to clean pairs of gloves 10/12 times, whereas HuNoV without drying was disseminated 11/12 times. In the sandwich-preparing simulation, both viruses were transferred repeatedly to the first recipient surface (left hand, cucumber, and knife) during the preparation. Both MuNoV and HuNoV were transferred more efficiently from latex gloves to cucumbers (1.2% ± 0.6% and 1.5% ± 1.9%) than vice versa (0.7% ± 0.5% and 0.5% ± 0.4%). We estimated that transfer of at least one infective HuNoV from contaminated hands to the sandwich prepared was likely to occur if the hands of the food handler contained 3 log10 or more HuNoVs before gloving. Virus-contaminated gloves were estimated to transfer HuNoV to the food servings more efficiently than a single contaminated cucumber during handling. Our results indicate that virus-free food ingredients and good hand hygiene are needed to prevent HuNoV contamination of RTE foods., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Human norovirus (HuNoV), which causes gastroenteritis, can be transmitted to food and food contact surfaces via viruscontaminated hands. To investigate this transmission in food processing environments, we developed a swabbing protocol for environmental samples, evaluated the stability of HuNoV in the swabs, and applied the method in the food industry. Swabs made of polyester, flocked nylon, cotton wool, and microfiber were moistened in either phosphate-buffered saline (PBS) or glycine buffer (pH 9.5) and used to swab four surfaces (latex, plastic, stainless steel, and cucumber) inoculated with HuNoV. HuNoV was eluted with either PBS or glycine buffer and detected with quantitative reverse transcription PCR. HuNoV recoveries were generally higher with an inoculation dose of 100 PCR units than 1,000 PCR units. The highest recoveries were obtained when surfaces were swabbed with microfiber cloth moistened in and eluted with glycine buffer after a HuNoV inoculation dose of 100 PCR units: 66% ± 18% on latex, 89% ±2% on plastic, and 79% ±10% on stainless steel. The highest recovery for cucumber, 45% ±5%, was obtained when swabbing the surface with microfiber cloth and PBS. The stability of HuNoV was tested in microfiber cloths moistened in PBS or glycine buffer. HuNoV RNA was detected from swabs after 3 days at 4 and 22°C, although the RNA levels decreased more rapidly in swabs moistened with glycine buffer than in those moistened with PBS at 22°C. In the field study, 172 microfiber and 45 cotton wool swab samples were taken from environmental surfaces at three food processing companies. Five (5.6%) of 90 swabs collected in 2010 and 7 (8.5%) of 82 swabs collected in 2012 were positive for HuNoV genogroup II; all positive samples were collected with microfiber swabs. Three positive results were obtained from the production line and nine were obtained from the food workers' break room and restroom areas. Swabbing is a powerful tool for HuNoV RNA detection from environmental surfaces and enables investigation of virus transmission during food processing.

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter(-l) purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C(2) to C(10)) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50 degrees C, but residual activity decreased sharply at 60 degrees C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

A novel colanic acid-degrading enzyme was isolated from a mixed culture filtrate obtained by enrichment culturing of a compost sample using colanic acid as carbon source. The enzyme was partially purified resulting in a 17-fold increase in specific activity. Further purification by Native PAGE revealed that the enzyme is part of a high-molecular weight multi protein complex of at least six individual proteins. The enzyme showed a temperature optimum at 50 degrees C while after 5h at 50 degrees C and pH7 still 70% of the total activity was left. The pH optimum was found to be pH7. Analysis of the degradation products showed that the enzyme is a novel 1,4-beta-fucoside hydrolase that liberates repeating units of colanic acid with varying degrees of acetylation. Km and Vmax of the enzyme were determined against the native substrate as well as its de-O-acetylated and depyruvated forms. Compared to the native substrate the affinity of the enzyme for the modified substrates was much lower. However, for the de-O-acetylated sample a dramatic increase in catalytic efficiency was observed. The native form of the substrate showed substrate inhibition at high concentrations, probably due to the formation of nonproductive substrate complexes. Removal of the acetyl groups probably prevents this effect resulting in a higher catalytic efficiency.

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.

Development of novel enzymatic methods for slime deposit control in paper mills requires knowledge of polysaccharide-producing organisms and the polysaccharide structures present in deposits. In this work, 27 polysaccharide-producing bacteria were isolated from slime samples collected from different parts of a paper machine. Most of the isolates produced polysaccharides in liquid culture and nine of them were selected for production of polysaccharides for characterisation. The selected isolates belonged to seven different genera: Bacillus, Brevundimonas, Cytophaga, Enterobacter, Klebsiella, Paenibacillus and Starkeya. Using ribotyping, partial 16S rDNA sequencing, physiological tests and fatty acid analysis, four of the nine isolates: Bacillus cereus, Brevundimonas vesicularis, K. pneumoniae and P. stellifer were identified to the species level. Production of polysaccharides by the selected isolates varied between 0.07 and 1.20 g L(-1), the highest amount being produced by B. vesicularis. The polysaccharides were heteropolysaccharides with varying proportions of galactose, glucose mannose, rhamnose fucose and uronic acids.

Aims: To discover novel laccases potential for industrial applications., Methods and Results: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C., Conclusions: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478., Significance and Impact of the Study: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases., (Copyright 2004 The Society for Applied Microbiology)

Bacterial strains were isolated from spruce wood chips and their ability to reduce the content of wood extractives was studied. Strains were screened by cultivation on liquid media containing wood extractives as the major nutrient. Some bacterial species could decrease remarkably the amount of extractives in the liquid media and reduced the amount of triglycerides, steryl esters and total extractives by 100, 20 and 39%, respectively. Spruce wood chips were treated in controlled conditions with selected bacteria to test their effects on the chips. All the bacteria grew well on wood chips. The effect of bacterial metabolism on wood extractives was significant. Bacterial treatments reduced the amount of lipophilic extractives by 16-38% in 1 week of treatment and up to 67% in 2 weeks. The most efficient strain removed 90, 66 and 50% of triglycerides, steryl esters and resin acids, respectively, in 2 weeks. These results indicate that bacteria may be promising agents for the removal of extractives for improved pulping and papermaking processes.

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.

Biofilm-degrading enzymes are potential agents for slime control in paper machines. In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes. Polysaccharide yields of 1.5-3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium. Two K. pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid. Fructose-containing polysaccharides were the main products of B. licheniformis and P. fluorescens. Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening. None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides.

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.