D. Lillehaug - Academia.edu (original) (raw)

Papers by D. Lillehaug

Research paper thumbnail of A high-throughput microcultivation protocol for FTIR spectroscopic characterization and identification of fungi

Journal of biophotonics, 2010

Characterization and identification of fungi in food industry is an important issue both for rout... more Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 ...

Research paper thumbnail of Characterization of food spoilage fungi by FTIR spectroscopy

Journal of Applied Microbiology, 2013

Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protoc... more Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. Methods and Results: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93Á9 and 94Á0% of the spectra were correctly identified at the species and genus level, respectively. Conclusions: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. Significance and Impact of the Study: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Research paper thumbnail of Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Applied and Environmental Microbiology, 2000

PCR techniques have significantly improved the detection and identification of bacterial pathogen... more PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene ( hlyA ) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies ...

Research paper thumbnail of A highly efficient and stable system for site-specific integration of genes and plasmids into the phage φLC3 attachment site (attB) of the Lactococcus lactis chromosome

Gene, 1997

An integration vector system based on the site-specific integration apparatus of the temperate la... more An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed. A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication. Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L. lactis. The above results, the observation that the phiLC3 attB site appear to be conserved in L. lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L. lactis.

Research paper thumbnail of The life cycles of the temperate lactococcal bacteriophage φLC3 monitored by a quantitative PCR method

FEMS Microbiology Letters, 2000

Research paper thumbnail of Use of Real-Time Quantitative PCR for the Analysis of  LC3 Prophage Stability in Lactococci

Applied and Environmental Microbiology, 2003

Bacteriophages are a common and constant threat to proper milk fermentation. It has become eviden... more Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage φLC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six φLC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the φLC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the φLC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencie...

Research paper thumbnail of Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea

Journal of Bacteriology, 1990

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at... more The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment r...

Research paper thumbnail of Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage φLC3

Molecular Genetics and Genomics, 2001

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris pha... more Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

Research paper thumbnail of Detection and quantification of Salmonella in pure cultures using 5′-nuclease polymerase chain reaction

Journal of food microbiology, Oct 15, 1999

Advances in detection and quantification assays based on nucleic acids conceivably will revolutio... more Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5'-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276-7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5'-nuclease-based kit for the quantification of Salmonella.

Research paper thumbnail of A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry

Letters in Applied Microbiology

The source tracking of fungal contamination in food industry is an important aspect of food safet... more The source tracking of fungal contamination in food industry is an important aspect of food safety. Currently all available methods are time consuming and require use of reference library that may limit the accuracy of the identification. In this study we report, for the first time, a library-independent FTIR spectroscopic approach for microbial source tracking of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on genus and species level. Therefore, such approach possesses great importance for food safety control in food industry.

Research paper thumbnail of Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends

Applied and environmental microbiology, 1991

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isom... more The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spont...

Research paper thumbnail of The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101

Applied and environmental microbiology, 1992

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular protei... more The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from pr...

Research paper thumbnail of An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages

Journal of Applied Microbiology, 1997

This report summarizes the results from an effort to optimize the double-agar plate assay for vis... more This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis. Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.

Research paper thumbnail of Characterization of food spoilage fungi by FTIR spectroscopy

Journal of Applied Microbiology, 2013

Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protoc... more Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. Methods and Results: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93Á9 and 94Á0% of the spectra were correctly identified at the species and genus level, respectively. Conclusions: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. Significance and Impact of the Study: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Research paper thumbnail of A high-throughput microcultivation protocol for FTIR spectroscopic characterization and identification of fungi

Journal of biophotonics, 2010

Characterization and identification of fungi in food industry is an important issue both for rout... more Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 ...

Research paper thumbnail of Characterization of food spoilage fungi by FTIR spectroscopy

Journal of Applied Microbiology, 2013

Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protoc... more Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. Methods and Results: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93Á9 and 94Á0% of the spectra were correctly identified at the species and genus level, respectively. Conclusions: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. Significance and Impact of the Study: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Research paper thumbnail of Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Applied and Environmental Microbiology, 2000

PCR techniques have significantly improved the detection and identification of bacterial pathogen... more PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene ( hlyA ) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies ...

Research paper thumbnail of A highly efficient and stable system for site-specific integration of genes and plasmids into the phage φLC3 attachment site (attB) of the Lactococcus lactis chromosome

Gene, 1997

An integration vector system based on the site-specific integration apparatus of the temperate la... more An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed. A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication. Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L. lactis. The above results, the observation that the phiLC3 attB site appear to be conserved in L. lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L. lactis.

Research paper thumbnail of The life cycles of the temperate lactococcal bacteriophage φLC3 monitored by a quantitative PCR method

FEMS Microbiology Letters, 2000

Research paper thumbnail of Use of Real-Time Quantitative PCR for the Analysis of  LC3 Prophage Stability in Lactococci

Applied and Environmental Microbiology, 2003

Bacteriophages are a common and constant threat to proper milk fermentation. It has become eviden... more Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage φLC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six φLC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the φLC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the φLC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencie...

Research paper thumbnail of Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea

Journal of Bacteriology, 1990

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at... more The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment r...

Research paper thumbnail of Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage φLC3

Molecular Genetics and Genomics, 2001

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris pha... more Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

Research paper thumbnail of Detection and quantification of Salmonella in pure cultures using 5′-nuclease polymerase chain reaction

Journal of food microbiology, Oct 15, 1999

Advances in detection and quantification assays based on nucleic acids conceivably will revolutio... more Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5'-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276-7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5'-nuclease-based kit for the quantification of Salmonella.

Research paper thumbnail of A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry

Letters in Applied Microbiology

The source tracking of fungal contamination in food industry is an important aspect of food safet... more The source tracking of fungal contamination in food industry is an important aspect of food safety. Currently all available methods are time consuming and require use of reference library that may limit the accuracy of the identification. In this study we report, for the first time, a library-independent FTIR spectroscopic approach for microbial source tracking of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on genus and species level. Therefore, such approach possesses great importance for food safety control in food industry.

Research paper thumbnail of Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends

Applied and environmental microbiology, 1991

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isom... more The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spont...

Research paper thumbnail of The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101

Applied and environmental microbiology, 1992

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular protei... more The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from pr...

Research paper thumbnail of An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages

Journal of Applied Microbiology, 1997

This report summarizes the results from an effort to optimize the double-agar plate assay for vis... more This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis. Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.

Research paper thumbnail of Characterization of food spoilage fungi by FTIR spectroscopy

Journal of Applied Microbiology, 2013

Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protoc... more Aims: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. Methods and Results: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93Á9 and 94Á0% of the spectra were correctly identified at the species and genus level, respectively. Conclusions: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. Significance and Impact of the Study: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.