The role of copper in the manufacture of Finnish Emmental cheese.

The effects of added copper in the manufacture of Finnish Emmental cheese were studied. Consequently, cheeses were produced with or without the copper supplement and a facultative heterofermentative strain, Lactobacillus rhamnosus Lc705, which is currently utilized as a protective culture in large-scale manufacture in Finland. Cheeses were examined at 1, 7, 30, 60, and 90 d from the microbiological, chemical, and sensory points of view. Organic acid production was affected by the presence of copper in the cheeses. The addition of copper to cheesemilk increased the level of primary proteolysis and slowed secondary proteolysis as measured by nitrogen content in different extracts after citrate fractionation of cheeses, in pH 4.4-soluble nitrogen and 5% phosphotungstic acid-soluble nitrogen, respectively. The presence of copper appears to positively regulate the sensory characteristics of the cheese produced in our conditions; in particular, consistency was affected significantly. The role of the Lb. rhamnosus Lc705 protective strain has not been shown to have important effects on most of the parameters that influence the final quality of the cheeses. Although the traditional plating systems for revealing bacterial populations during cheese manufacture did not reveal any drastic differences caused by the presence of copper, the results from chemical and sensory analyses suggest that its use plays a significant role in the regulation of bacterial physiological and biochemical activities, which in turn affect the sensory quality of Emmental cheese.

Effects of copper on germination, growth and sporulation of Clostridium tyrobutyricum.

The effects of copper (Cu(2+)) on spore germination, vegetative growth and sporulation of Clostridium tyrobutyricum, which is capable to causing texture and flavour defects in Emmental cheese, were studied. Spore suspensions of three different strains were used as starting material for two experimental set-ups. The first studied the effects of supplemented (0-30 ppm) copper in RCM medium during spore germination and vegetative growth of C. tyrobutyricum measured by plating. The second set-up studied the effects of copper (0-30 ppm) in RCM medium during growth and sporulation of C. tyrobutyricum as measured by optical density at 550 nm and by platings after heat treatment of the samples respectively. Inhibition of germination, vegetative growth and sporulation processes by copper was strain-dependent. Both sporulation and germination were more sensitive than vegetative growth of C. tyrobutyricum to the inhibitory effects of copper. Copper, at the concentrations investigated in this study, inhibits spore germination of C. tyrobutyricum strains. Consequently copper may reduce the risk of late blowing spoilage from in the germination of C. tyrobutyricum spores during the ripening period of Emmental cheese.

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture.

AIMS: To determine the effects of supplemented copper (Cu2+) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. METHODS AND RESULTS: Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. CONCLUSIONS: Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.

Integration of the group c phage JCL1032 of Lactobacillus delbrueckii subsp. lactis and complex phage resistance of the host.

AIMS: Sequences related to Lactobacillus delbrueckii phage JCL1032 genome integration, the maintenance of lysogeny and putative immunity genes were characterized. Phenotypic changes of the JCL1032 lysogens were investigated. METHODS AND RESULTS: Integration of JCL1032 DNA into the host genome and the location of phage and bacterial attachment sites were studied by standard molecular methods. The frequency of lysogenization was 10(-7), and stable lysogeny was an even rarer phenomenon. JCL1032 integrates its genome into two distinct host genes of unknown functions. According to EOP (efficiency of plating) and adsorption tests JCL1032 lysogens showed resistance against several virulent and temperate Lactobacillus phages at different steps of phage infection. CONCLUSIONS: Temperate JCL1032 integrates its genome into bacterial DNA with exceptionally low frequency. JCL1032 lysogens express a complex phage resistance against several Lact. delbrueckii phages. An antagonistic arms race between the temperate phage and its host is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the genome integration of a group c Lact. delbrueckii phage has been described. The characterized lysogens could facilitate studies on Lact. delbrueckii phage receptors and phage resistance mechanisms. The genetic information gained from this study benefits the development of integration vectors and phage resistant starters.

Preliminary study of ultrasonic structural quality control of Swiss-type cheese.

There is demand for a new nondestructive cheese-structure analysis method for Swiss-type cheese. Such a method would provide the cheese-making industry the means to enhance process control and quality assurance. This paper presents a feasibility study on ultrasonic monitoring of the structural quality of Swiss cheese by using a single-transducer 2-MHz longitudinal mode pulse-echo setup. A volumetric ultrasonic image of a cheese sample featuring gas holes (cheese-eyes) and defects (cracks) in the scan area is presented. The image is compared with an optical reference image constructed from dissection images of the same sample. The results show that the ultrasonic method is capable of monitoring the gas-solid structure of the cheese during the ripening process. Moreover, the method can be used to detect and to characterize cheese-eyes and cracks in ripened cheese. Industrial application demands were taken into account when conducting the measurements.

Antibiotic resistance of raw-milk-associated psychrotrophic bacteria.

The present study proposed to evaluate the breadth of antibiotic resistance among psychrotrophic bacteria spoiling raw milks in Finland. A core of 60 isolates, retrieved from farms, trucks, and silos, phenotypically characterized, were analysed with ATB PSE strips, composed of a panel of 17 beta-lactams and non-beta-lactams antibiotics, representatives of five classes. Surprisingly, 60% of the psychrotrophs harboured multiresistant traits. We observed an increase of this feature along the cold chain of raw milk transportation.

Phenotypic characterization of raw milk-associated psychrotrophic bacteria.

Among the 68 isolates, selected from 13 raw-milk samples in Finland (that originate from farm, truck or silo tanks), 60 (88%) were psychrotrophs. All the isolates were characterized by the determination of their spoilage and phenotypic features: proteolytic and lipolytic activities, the production of lecithinases and hemolytic factors were considered. Phenotypic characterization of the isolates was mainly performed with API 20NE and BIOLOG GN2 identification systems; the results were system-dependent, although the presence of representatives of the Pseudomonas genus (for the majority of the isolates) was suggested by both systems. The results of the numerical profile analyses by API 20NE proposed that some strains might be members of Stenotrophomonas, Burkholderia and Acinetobacter genera; however, the identity of many isolates remained doubtful or controversial.

The genome of the virulent phage Lc-Nu of probiotic Lactobacillus rhamnosus, and comparative genomics with Lactobacillus casei phages.

The complete 36,466-bp genome sequence of the virulent phage Lc-Nu of probiotic Lactobacillus rhamnosus was determined. The linear dsDNA with a GC-content of 44.2% contained 3' single-stranded cohesive ends of 12 nucleotides. A total of 51 putative open reading frames (orfs) were predicted. Lc-Nu showed to be evolutionary closely related to the temperate Lactobacillus casei phages phi AT3 and A2. High DNA homology with phi AT3 was shared over the late transcribed genes, and the highest homology with A2 was within the genetic switch region. The truncated cI-like repressor was the only lysogeny related gene left, which strongly suggested Lc-Nu to be recently evolved from a temperate origin. Three putative methylases and endonucleases were detected from the region of early-transcribed genes. The putative origin of replication within the putative gene orf34 homologous to replisome organizers resembled to that of lambdoid phages. The present study suggested Lc-Nu to be a new candidate for the proposed Sfi21-like species.

Two self-splicing group I introns interrupt two late transcribed genes of prolate-headed Lactobacillus delbrueckii phage JCL1032.

Two group I introns were detected from the late gene region of the prolate-headed phage JCL1032 of Lactobacillus delbrueckii. Introns JCL-I1 and JCL-I2 interrupt orf602 and orf1868 encoding a phage terminase large subunit (TerL, 69.7 kDa) and a putative tape measure protein (TMP, 202 kDa), respectively. The introns JCL-I1 (226 bp) and JCL-I2 (322 bp) were efficiently self-spliced in vivo. Both introns were classified to the subgroup IA1 having all the conserved structures necessary for splicing, but lacking the ability to encode endonucleases or other gene products. The introns JCL-I1 and JCL-I2 shared restricted nucleotide sequence similarity with each other and with the group I terL intron of Lb. delbrueckii phage LL-H. No match was found for JCL-I1 in the homology searches. Instead, the primary sequence from the joining region of P8 and P7 to P9 of the intron JCL-I2 was homologous to the group I intron of Bacillus mojavensis; the orf142 introns I1, I2 and I3 of Staphylococcus aureus phage Twort; the group I intron of phage Bastille (Bacillus thuringiensis); and to the group IA3 intron of Monomastix species.

Characterization of the 16S-23S and 23S-5S rRNA intergenic spacer regions of dairy propionibacteria and their identification with species-specific primers by PCR.

In this study, the 16S-23S and 23S-5S rRNA intergenic spacer region sequences of Propionibacterium acidipropionici, P. freudenreichii ssp. freudenreichii and ssp. shermanii, P. jensenii and P. thoenii were determined. The sequences were shown to vary greatly between the species. Specific primer pairs were derived from the 16S-23S rRNA spacer sequences and used for the identification of the species by PCR.

Phage-related DNA polymorphism in dairy and probiotic Lactobacillus.

Various DNA-based methods are presently being applied for identification of industrial bacterial cultures including dairy starter and probiotic strains of Lactobacillus. The success of strain-specific identification depends on the power of the DNA-based methods to reveal intraspecies DNA polymorphism. This study reveals that all eleven arbitrarily chosen Lactobacillus rhamnosus starter, laboratory and probiotic strains contain Lb. rhamnosus phage Lc-Nu related nucleotide sequences. One of these highly homologous regions in the genome of phage Lc-Nu was the 2.4kb HindIII fragment, which has been sequenced. Nucleotide sequence analysis suggested that one side of the 2.4kb HindIII fragment encodes a phage Lc-Nu helicase and accordingly represents an early gene region of phage Lc-Nu genome. Five forward and five reverse primers were derived from the nucleotide sequence of the 2.4kb HindIII fragment of phage Lc-Nu DNA for PCR-based identification of the eleven Lb. rhamnosus strains included in this study. Six different types of PCR product patterns were obtained. Among the patterns three were unique to particular Lb. rhamnosus strains. The results suggest that phage-related DNA sequences are, surprisingly, distributed widely among the Lb. rhamnosus strains, and that these sequences could also be a source of DNA polymorphism to apply for DNA-based identification of bacterial strains. Phage Lc-Nu related DNA homology was also found in the chromosome of Lb. casei, the species closely related to Lb. rhamnosus.

Electrophoretic methods for fractionation of native and heat-denatured bovine beta-lactoglobulin.

We have developed three electrophoretic methods for analytical and preparative separation of native and heat-denatured beta-lactoglobulin. The methods can be applied, e.g., to optimize dairy milk processing, especially in laboratories which are not provided with expensive column chromatographic instruments. The methods consist of native PAGE followed by electroelution. Consequently, the eluted proteins are in solution in a biologically active and native form, and can therefore be used immediately for further analyses.

Fats and fatty acids as growth factors for Lactobacillus delbrueckii.

The effects of various fats and fatty acids on the growth of Lactobacillus delbrueckii strains have been studied using modified MRS broth without Tween 80 as a basic growth medium. Among the six L. delbrueckii strains studied all except one strain required Tween 80 or Tween 20 as a fatty acid supplement for the growth. Tween 40 and Tween 60, which contain solely medium and long chain saturated fatty acids, inhibited the growth of all L. delbrueckii strains when present as a sole fat supplement in MRS broth. Free oleic acid but not free lauric acid could substitute Tween 80 or Tween 20 supplement suggesting that unsaturated fatty acids are essential growth factors for most L. delbrueckii strains. Among the natural food oils tested, the oils containing the lowest amounts of saturated long chain fatty acids promoted the growth of L. delbrueckii most effectively. Especially cellular C18:1 and C19 cyclopropane fatty acid contents of L. delbrueckii were strongly affected by exogenous fatty acid composition and by strain suggesting genetic diversity and polymorphism among the genes encoding and/or regulating cyclopropane synthase. In addition obviously most if not all L. delbrueckii strains lack particular synthase, desaturase and/or dehydrase activities required for de novo synthesis of long chain unsaturated fatty acids. These biochemical features could be used as informative chemotaxonomic characteristics for L. delbrueckii starter strain identification and selection.

Application of siderotyping for characterization of Pseudomonas tolaasii and "Pseudomonas reactans" isolates associated with brown blotch disease of cultivated mushrooms.

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and "P. reactans." The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401-407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 "P. reactans" isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some "P. reactans" isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one "P. reactans" siderovar were found.

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.

Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons.

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.

Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539.

A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages.

Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers.

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.

Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR.

Spacer regions between the 16S and 23S rRNA genes of different dairy and probiotic lactic acid bacteria were amplified by the polymerase chain reaction (PCR) with conserved primers and the nucleotide sequences of these spacer regions were determined. Amplification/oligonucleotide primer pairs were designed for Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus and Streptococcus thermophilus based on the differences in the nucleotide sequences of the 16S-23S rRNA spacer regions. Also a primer pair identifying both Lb. paracasei and Lb. rhamnosus was designed. In addition to conventional PCR in a heat block a rapid PCR method in an Air Thermocycler (ATC) with glass capillaries was used.

The early gene region completes the nucleotide sequence of Lactobacillus delbrueckii subsp. lactis phage LL-H.

Transcription of genes from phage LL-H can be divided into an early phase and a late phase. The early gene region was located in a 5.9-kb segment of the phage LL-H genome and it was part of the sequence that completed the phage LL-H genome sequence, 34 659 bp in size. Phage LL-H is the first completely sequenced Lactobacillus phage. In the main coding strand of phage LL-H genome 48 putative ORFs could be detected, but only four small putative ORFs could be found in the opposite strand. The ORFs covered 85.6% of the main coding strand. Function could be assigned to eleven of the phage LL-H ORFs either by biochemical analyses or by database homologies. A single-strand-binding protein, SSB, was detected in addition to the previously determined functions (small and large subunits of terminase, intron-encoded endonuclease, six structural proteins, phage lysin). For 15 additional ORFs of phage LL-H homology was detected in databases, but no function could be inferred for them.