Identification of a species-specific aminotransferase in Pediococcus acidilactici capable of forming α-aminobutyrate.

During cheese ripening, the bacterial strain Pediococcus acidilactici FAM18098 produces the non-proteinogenic amino acid, α-aminobutyrate (AABA). The metabolic processes that lead to the biosynthesis of this compound are unknown. In this study, 10 P. acidilactici, including FAM18098 and nine Pediococcus pentosaceus strains, were screened for their ability to produce AABA. All P. acidilactici strains produced AABA, whereas the P. pentosaceus strains did not. The genomes of the pediococcal strains were sequenced and searched for genes encoding aminotransferases to test the hypothesis that AABA could result from the transamination of α-ketobutyrate. A GenBank and KEGG database search revealed the presence of a species-specific aminotransferase in P. acidilactici. The gene was cloned and its gene product was produced as a His-tagged fusion protein in Escherichia coli to determine the substrate specificity of this enzyme. The purified recombinant protein showed aminotransferase activity at pH 5.5. It catalyzed the transfer of the amino group from leucine, methionine, AABA, alanine, cysteine, and phenylalanine to the amino group acceptor α-ketoglutarate. Αlpha-ketobutyrate could replace α-ketoglutarate as an amino group acceptor. In this case, AABA was produced at significantly higher levels than glutamate. The results of this study show that P. acidilactici possesses a novel aminotransferase that might play a role in cheese biochemistry and has the potential to be used in biotechnological processes for the production of AABA.

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

Evaluation of baker's yeast in honey using a real-time PCR assay.

Occasionally, melissopalynological analysis reveals the presence of baker's yeast (Saccharomyces cerevisiae) in honey sediments. A field experiment reproducing a common spring bee feeding practice, using sugar paste containing baker's yeast, was performed to understand how S. cerevisiae are introduced into honey. Apart from classical microscopy, a real-time quantitative PCR (qPCR) system specific for S. cerevisiae was established for quantification of S. cerevisiae in honeys. Results showed that S. cerevisiae cells are stored in the honey of the brood combs and are also transferred into honey in the supers. The concentrations of S. cerevisiae were highest in honey of the brood frames immediately after the feeding and decreased over time to low concentrations at the end of the year. A high content of S. cerevisiae cells were also found in the honey from supers of the spring harvest. Observed S. cerevisiae cells were not able to multiply in a high-sugar environment, such as honey, and their viability decreased rapidly after addition to the honey. The screening of 200 Swiss honeys revealed the presence of S. cerevisiae in 4.5% of the samples, as determined by microscopy and qPCR. Finally, the method described here may indicate an unwanted sucrose addition to honey through bee-feeding.

Lactic Acid Bacteria as Markers for the Authentication of Swiss Cheeses.

The manufacture of traditional Swiss-type cheeses adheres to strict rules, so as to guarantee quality and purity of the end product. This raises production costs and means consumers pay more. It also opens the door to cut-rate forgeries claiming to be made to the stringent standards and causing considerable economic losses to the entire dairy sector. In order to combat product counterfeiting, Agroscope has developed proof-of-origin cultures that allow the identification of copycats. Carefully selected lactic acid bacteria, having uniquely located insertion sequence elements, are proliferated by fermentation and subsequently dried by lyophilization. The proof-of-origin culture is added during the cheese production process and sustains maturation. These so-called 'biological markers' can be traced using polymerase chain reaction (PCR) methods, which allow authentication even if the cheese is cut into pieces or grated. They do not lead to any alteration of the cheese's taste or texture, and are compatible with the strict 'protected designation of origin' (PDO) specifications. The proof-of-origin cultures are used for the protection of several traditional Swiss-cheese varieties, such as Emmental PDO, Tête de Moine PDO, and Appenzeller(®). A market survey of Emmental PDO showed that the system is effective in revealing fraud and has the power to enforce corrective measures.

Catabolism of serine by Pediococcus acidilactici and Pediococcus pentosaceus.

The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L- and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.