Tapani Alatossava

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.

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.

Specific identification of certain probiotic Lactobacillus rhamnosus strains with PCR primers based on phage-related sequences

PCR primers derived from Lactobacillus rhamnosus phage Lc-Nu genome were used to screen the presence of phage-related sequences in Lb. rhamnosus strains. Several primer pairs derived from structural and replication gene regions of phage Lc-Nu amplified PCR products of expected sizes from bacterial strains revealing phage-related sequences in 10 of 11 Lb. rhamnosus strains. Strain-specific PCR primers for three probiotic Lb. rhamnosus strains were derived from these phage-related sequences for identification and detection purposes. Specificity of these primers was tested against 11 Lb. rhamnosus strains and over 40 other bacterial strains.

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.

Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

BackgroundUse of lactose-rich concentrates from dairy processes for the induction of recombinant genes expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments.ResultsShake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction.ConclusionWhey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

Effect of heat denaturation on beta-lactoglobulin-induced gastrointestinal sensitization in rats: denatured betaLG induces a more intensive local immunologic response than native betaLG.

Beta‐lactoglobulin (βLG) is one of the first foreign antigens encountered by a newborn child, and it is the major allergen causing cows milk allergy. Heat denaturation causes changes to the protein structure, but the significance of heat‐induced changes for immunogenicity or allergenicity is not known. To clarify how heat denaturation affects allergenicity and immunogenicity, we immunized Hooded‐Lister rat pups with intra‐peritoneal injections of native or heat‐denatured βLG at days 43 and 62 after birth. The animals were then fed native and denatured milk products twice weekly from 73 to 101 days of age with a feeding tube, after which they were allowed cheese and milk ad libitum, until they were killed on day 131. Total immunoglobulin (Ig)E and βLG‐specific IgG1 and IgG2a levels were determined from serum samples. Spontaneous interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) production was measured from duodenal specimens, and specimens of gastrointestinal mucosae were studied for the presence of inflammatory cells. The rats immunized with native βLG had higher levels of total serum IgE than the unimmunized controls or the rats immunized with heat‐denatured βLG, while heat‐denatured βLG induced a significantly more intensive mononuclear inflammatory cell and eosinophil infiltration in the gastroduodenal mucosa. The βLG‐specific IgG antibody and IL‐4 and IFN‐γ responses were similar in the two groups of immunized animals. Hence, denaturation modifies the immunogenic and allergenic properties of βLG. Heat‐denatured βLG induces a more intensive local reaction in the gastrointestinal mucosa, while there is some evidence for enhanced systemic allergic sensitization by native βLG.

Characterization of the genome region encoding structural proteins of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H

Two regions from the genome of the virulent Lactobacillus delbrueckii subsp. lactic bacteriophage LL-H were sequenced (2330 and 12939 bp; 44% of the 34.6-kb genome). Together with the previously sequenced region containing the major capsid protein-encoding gene (2498 bp), the sequence had 21 open reading frames (ORFs) on the main coding strand. Only two putative ORFs were detected on the complementary strand. The ORFs covered 93.2% of the sequence. All but four of the ORFs were preceded by a ribosome-binding site. Only four longer non-coding stretches of sequences (175-278 nucleotides (nt) in size) were present. The longest of the non-coding regions contained an A + T-rich sequence that is surrounded by eight perfect copies of an 8-nt sequence that is present both as direct and inverted repeats. This region could represent the origin of replication. All the previously mapped structural protein-encoding genes of phage LL-H were included in the sequence. Genes were identified for the following five proteins: gp19 (encoded by gene g17), gp58 (g71), gp61 (g57), gp75 (g70) and gp89 (g88). N-terminal amino-acid sequencing was performed on gp19 and gp75, and it was found that the N-terminal Met had been post-translationally removed from both proteins.

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.

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.