Biogenic amine-forming microbial communities in cheese.

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.

Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli.

This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.