Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement.

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement. We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.

Influence of octanoic acid addition to medium on some volatile compounds and PR-toxin biosynthesis by Penicillium roqueforti.

AIMS: The effect of wheat kernel medium supplementation with octanoic acid on the formation of PR toxin and some volatiles by Penicillium roqueforti was investigated. METHODS AND RESULTS: Octanoic acid was added to the medium once, prior to inoculation (4.55 mg g-1), or periodically (total 27.3 mg g-1) during the 10 day course of incubation. No octanoic acid was added to the reference sample. Levels of 2-heptanone, 2-heptanol and aristolochene, a volatile intermediate in PR toxin biosynthesis, were monitored using a solid phase microextraction (SPME) technique. The contents of PR toxin and ergosterol were determined after incubation. Aristolochene was observed in the reference sample, and 10.4 mg kg-1 of PR toxin was detected after 10 days. In cultures periodically supplemented with octanoic acid, no aristolochene or PR toxin were observed. However, in samples supplemented with octanoic acid only prior to incubation, the aristolochene level was 25% that in the reference sample, and PR toxin content was 3.4 mg kg-1. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that a high level of octanoic acid in the medium prevents PR toxin formation by P. roqueforti.

Antifungal activity of Bacillus sp. isolated from compost.

Four strains of Bacillus isolated from lupine compost exhibited an antifungal activity against six plant fungal pathogens (Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Trichothecium roseum, Fusarium solani, Fusarium oxysporum). It was significantly influenced by the composition of the cultivation media.