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  • 1. Alriksson, B.
    et al.
    Rose, S. H.
    Van Zyl, W. H.
    Sjöde, A.
    Nilvebrant, N. -O
    RISE, STFI-Packforsk.
    Jönsson, L. J.
    Cellulase production from spent lignocellulose hydrolysates by recombinant aspergillus niger2009In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 75, no 8, p. 2366-2374Article in journal (Refereed)
    Abstract [en]

    A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard medium with a comparable monosaccharide content (e.g., 2,100 nkat/ml in spent bagasse hydrolysate compared to 480 nkat/ml in standard glucose-based medium). In addition, A. niger D15[egI] was also able to consume or convert other lignocellulose-derived compounds, such as acetic acid, furan aldehydes, and phenolic compounds, which are recognized as inhibitors of yeast during ethanolic fermentation. The results indicate that enzymes can be produced from the stillage stream as a high-value coproduct in secondgeneration bioethanol plants in a way that also facilitates recirculation of process water.

  • 2.
    Andersson, Rolf E.
    SIK – Svenska livmedelsinstitutet.
    Microbial lipolysis at low temperatures1980In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 39, no 1, p. 36-40Article in journal (Refereed)
  • 3. Artin, I.
    et al.
    Carter, A.T.
    Holst, Emma
    SIK – Institutet för livsmedel och bioteknik.
    Lövenklev, Maria
    SIK – Institutet för livsmedel och bioteknik.
    Mason, D.R.
    Peck, M.W.
    Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum type E2008In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 74, no 8, p. 2391-2397Article in journal (Refereed)
    Abstract [en]

    Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

  • 4. Borjesson, T.
    et al.
    Stollman, U.
    Schnurer, J.
    Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains1992In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 58, no 8, p. 2599-2605Article in journal (Refereed)
    Abstract [en]

    Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.

  • 5.
    Fachmann, M S R
    et al.
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience. DTU Technical University of Denmark, Denmark.
    Hoorfar, J
    DTU Technical University of Denmark, Denmark.
    Hansen, F
    DMRI Danish Technological Institute, Denmark.
    Christensen, J
    DTU Technical University of Denmark, Denmark.
    Mansdal, S
    DTI Danish Technological Institute, Denmark.
    Josefsen, M H
    DTU Technical University of Denmark, Denmark.
    Detection of Salmonella in meat in less than 5 hours by a low-cost and non-complex sample preparation method.2017In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 83, no 5, article id e03151-16Article in journal (Refereed)
    Abstract [en]

    Salmonella is recognised as one of the most important foodborne bacteria, and has a wide health and socioeconomical impact worldwide. Fresh pork meat is one of the main sources of Salmonella and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in few cases < 12 h, thus requiring at least two working shifts. Here, we report a rapid (< 5 h) and high throughput method, for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water, and a real-time PCR compatible sample preparation method, based on filtration, centrifugation, and enzymatic digestion, followed by fast cycling real-time PCR detection. The method was validated in an un-paired, comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n=140) were either artificially contaminated with Salmonella at levels: 0, 1-10 and 10-100 CFU/25 g, or naturally contaminated. Cohen's Kappa for degree of agreement between the rapid method and the reference was 0.64 and the relative accuracy, sensitivity and specificity for the rapid method were 81.4, 95.1 and 97.9 %, respectively. The limit of detection (LOD50) was 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low risk meat, providing savings for meat producers, and help contribute to improved food safety.

    IMPORTANCE: While the cost of analysis and hands-on time of the presented rapid method were comparable to reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage; consumers as well as retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in detection of other pathogenic bacteria in different sample types.

  • 6. Jakočiune, D.
    et al.
    Pasquali, F.
    Silva, C. S.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hoorfar, J.
    Klein, G.
    Manfreda, G.
    Olsen, J. E.
    Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 5, p. 1616-1622Article in journal (Refereed)
    Abstract [en]

    Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products. © 2014, American Society for Microbiology.

  • 7. Josefsen, M. H.
    et al.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hansen, T. B.
    Christensen, L. S.
    Olsen, J. E.
    Hoorfar, J.
    Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time pcr and propidium monoazide treatment, as a tool for quantitative risk assessment2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 15, p. 5097-5104Article in journal (Refereed)
    Abstract [en]

    A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT.) values (R2 = 0.993), with a quantification range of 1 × 102 to 1 × 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. Copyright © 2010, American Society tor Microbiology. All Rights Reserved.

  • 8.
    Löfström, Charlotta
    et al.
    Lund University, Sweden.
    Knutsson, R.
    Axelsson, C. E.
    Rådström, P.
    Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 1, p. 69-75Article in journal (Refereed)
    Abstract [en]

    A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.

  • 9.
    Lövenklev, Maria
    et al.
    SIK – Institutet för livsmedel och bioteknik.
    Artin, I.
    Hagberg, O.
    Borch, Elisabeth
    SIK – Institutet för livsmedel och bioteknik.
    Holst, Emma
    SIK – Institutet för livsmedel och bioteknik.
    Radstrom, P.
    Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 5, p. 2928-2934Article in journal (Refereed)
    Abstract [en]

    The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng·ml -1·unit of optical density-1 in the 10% CO 2 atmosphere and 126 ng·ml-1·unit of optical density-1 in the 70% CO2 atmosphere.

  • 10.
    Lövenklev, Maria
    et al.
    SIK – Institutet för livsmedel och bioteknik.
    Holst, E.
    Borch, Elisabeth
    SIK – Institutet för livsmedel och bioteknik.
    Radstrom, P.
    Relative neurotoxin gene expression in Clostridium botulinum type B, determined using quantitative reverse transcription-PCR2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 5, p. 2919-2927Article in journal (Refereed)
    Abstract [en]

    A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.

  • 11. Malorny, B.
    et al.
    Löfström, C.
    DTU Technical University of Denmark, Denmark.
    Wagner, M.
    Krämer, N.
    Hoorfar, J.
    Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment2008In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 74, no 5, p. 1299-1304Article in journal (Refereed)
  • 12. Quinto, E.J.
    et al.
    Arinder, Pernilla
    SIK – Institutet för livsmedel och bioteknik.
    Axelsson, L.
    Heir, E.
    Holck, A.
    Lindqvist, R.
    Predicting the concentration of verotoxin-producing Escherichia coli bacteria during processing and storage of fermented raw-meat sausages2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 9, p. 2715-2727Article in journal (Refereed)
    Abstract [en]

    A model to predict the population density of verotoxigenic Escherichia coli (VTEC) throughout the elaboration and storage of fermented raw-meat sausages (FRMS) was developed. Probabilistic and kinetic measurement data sets collected from publicly available resources were completed with new measurements when required and used to quantify the dependence of VTEC growth and inactivation on the temperature, pH, water activity (aw), and concentration of lactic acid. Predictions were compared with observations in VTEC-contaminated FRMS manufactured in a pilot plant. Slight differences in the reduction of VTEC were predicted according to the fermentation temperature, 24 or 34°C, with greater inactivation at the highest temperature. The greatest reduction was observed during storage at high temperatures. A population decrease greater than 6 decimal logarithmic units was observed after 66 days of storage at 25°C, while a reduction of only ca. 1 logarithmic unit was detected at 12°C. The performance of our model and other modeling approaches was evaluated throughout the processing of dry and semidry FRMS. The greatest inactivation of VTEC was predicted in dry FRMS with long drying periods, while the smallest reduction was predicted in semidry FMRS with short drying periods. The model is implemented in a computing tool, E. coli SafeFerment (EcSF), freely available from http://www.ifr.ac.uk/safety/EcoliSafeFerment. EcSF integrates growth, probability of growth, and thermal and nonthermal inactivation models to predict the VTEC concentration throughout FRMS manufacturing and storage under constant or fluctuating environmental conditions. © 2014, American Society for Microbiology.

  • 13.
    Snygg, Bengt Göran
    et al.
    SIK – Svenska livmedelsinstitutet.
    Andersson, J.E.
    Krall, C.A.
    Stollman, U.M.
    Akesson, C.A.
    Separation of botulinum-positive and -negative fish samples by means of a pattern recognition method applied to headspace gas chromatograms1979In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 38, no 6, p. 1081-1085Article in journal (Refereed)
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