Development of bacteria identification array to detect lactobacilli in Thai fermented sausage.

To improve the quality and safety of food products, there is a need in the food industry for a reliable method for simultaneously monitoring multiple bacterial strains. Microarray technology is a high-throughput screening approach that can provide an alternative for bacteria detection. A total of 164 bacteria-specific probes were designed from 16S rRNA gene sequences to target 12 bacteria species, including lactic acid bacteria and selected food pathogens. After fabrication onto aminosilane-coated slides, hybridization conditions of the array were optimized for high specificity and signal intensities. The array was applied to detect 12 bacteria individually and was specific to all (Lactobacillus plantarum group, L. fermentum, L. brevis, L. delbrueckii, L. casei, L. sakei, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes) except L. animalis. Multiplex detection using mixed bacteria populations was evaluated and accurate detection was obtained. The feasibility of using the array to detect the target bacteria in food was evaluated with Thai fermented sausages (Nham). Meat samples were collected on days 2, 3 and 7 after natural fermentation, L. plantarum-inoculated fermentation and L. brevis-inoculated fermentation before applying to the array. The naturally-fermented Nham contained L. sakei, L. delbrueckii, L. plantarum and L. fermentum. The L. plantarum-inoculated Nham showed a similar lactic acid bacteria population but the positive signal level for L. plantarum was higher than with natural fermentation. The L. brevis-inoculated Nham contained L. brevis, L. plantarum, L. delbrueckii and L. fermentum. The array was used to monitor bacteria population dynamics during the fermentation process. The naturally-fermented and L. brevis-inoculated samples showed lower positive signal levels of L. plantarum on day 2, but signals gradually increased on days 3 and 7 of the fermentation. In contrast, the L. plantarum-started fermentation showed a higher positive signal level on day 2 than the natural and L. brevis-inoculated samples, and the positive signal level remained high on days 3 and 7. The bacteria identification array was proven to be useful as an alternative method to detect and monitor target bacteria populations during food fermentation.

Garvieacin Q, a novel class II bacteriocin from Lactococcus garvieae BCC 43578.

Lactococcus garvieae BCC 43578 produces a novel class II bacteriocin, garvieacin Q (GarQ), 70 amino acids in length and containing a 20-amino-acid N-terminal leader peptide. It is cleaved at the Gly-Gly site to generate the mature GarQ (5,339 Da), which is especially inhibitory against Listeria monocytogenes ATCC 19115 and other L. garvieae strains.

Biogenic amine formation in Nham, a Thai fermented sausage, and the reduction by commercial starter culture, Lactobacillus plantarum BCC 9546.

Biogenic amines are of concern for sausage due to their toxicological effects on nervous, blood pressure, gastric and intestinal systems. In this study, the influence of raw pork meat quality and starter culture inoculation on biogenic amines accumulation in Nham, a Thai traditional fermented pork, were studied. Before Nham processing, pork meat was stored at 30°C for 6h, and at 4 and -20°C for 2days. Formation of biogenic amines (cadaverine, putrescine, histamine and tyramine) was significantly higher in Nham processed from stored meat. Accumulation of these biogenic amines in Nham reduced significantly by the addition of Lactobacillus plantarum BCC 9546, a commercial Nham starter culture. The results highlight the importance of using fresh meat products and the inclusion of an appropriate starter culture to minimise the formation of biogenic amines during the process of Nham fermentation.

Reconstitution and function of Tetragenococcus halophila chaperonin 60 tetradecamer.

Tetragenococcus halophila originally isolated from soy sauce is a halophilic lactic acid bacterium which can grow under 4 M sodium chloride. T. halophila chaperonin composed of a core moiety of chaperonin 60 (cpn60) and a lid moiety of chaperonin 10 (cpn10), is thought to contribute to host halotolerant capability. In this report, we reconstituted and characterized the core complex of T. halophila chaperonin by using a recombinant T. halophila cpn60 (Tcpn60) overexpressed in Escherichia coli. The reconstitution of Tcpn60 was performed in the presence of 10 mM MgCl2, 2 mM ATP and 0.8 M (NH4)2SO4 and the resultant oligomer was purified by gel filtration chromatography. Electron microscopy of the reconstituted Tcpn60 revealed a double toroidal tetradecameric structure that is characteristic of bacterial cpn60. The T. halophila tetradecamer cpn60 exhibited an ATPase activity and a refolding activity of both chemically and thermally denatured enolases under wide range of salt concentrations. Furthermore, we demonstrated that heterologous expression of Tcpn60 allowed the normal growth of host Escherichia coli cells under salt stress conditions and this effect was further enhanced by co-expression with Tcpn10. These results suggested that Tcpn60 contributes to the halotolerance property of T. halophila cell as a tetradecamer complex, probably associated with the Tcpn10 complex.

Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli.

Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.