[Study of microbial contamination of processed fresh vegetables and lettuce].

Investigations of microbial contamination and species composition of the Enterobacteriaceae family in fresh vegetables and lettuce has been conducted. The objects of study were new types of fresh ready-to-eat vegetable foods - salads, sliced vegetables and mixtures thereof, sampled at the main stages of production, including washing, antimicrobial treatment with sodium hypochlorite, and packaging in the film under vacuum. Quantitative analysis of Enterobacteriaceae levels in fresh and packaged vegetables and salads showed that their part in the total amount of microbial contaminants is large enough. Average Enterobacteriaceae content ranged from 2,14 to 3,34 lg cfu/g, reaching in some samples values 4,38-4,74 lg, comparable with the levels of total bacteria. Considerable species diversity of microflora contaminating ready-to-eat vegetable products has been found. Bacteria of the genera Enterobactel; Pantoea, Citrobacter, Serratia, Pseudomonas, Kluyvera, Klebsiella, Escherichia, Rahnella, Acinetobacter were found in the salads and sliced vegetables. In the tested samples most frequently detected Enterobacter spp. - 37% of identified strains and Pantoea spp - 25% of strains. The data on the composition and levels of microbial contaminants in vegetable and salad products highlight not only the need to monitor coliform bacteria - traditional indicators of faecal contamination of raw materials, but also the need to introduce criteria for the amount of Enterobacteriaceae.

[Direct detection of T-2- and HT-2-mycotoxins producers of fungi the genus Fusarium in food grain by PCR (report 2)].

The improvement of the Fusarium DNA extraction method has been undertaken in order to reduce the error of PCR analysis for detection of toxigenic Fusarium species, including those contained in the grain in the uncultureted state, directly in the grain. The efficiency of Fusarium DNA extraction methods (nucleotides sorption and CTAB method) has been compared. The efficiency of CTAB method combined with 10-fold weight increase of milled grain sample has been demonstrated. This approach revealed a greater number of Fusarium species, than PCR analysis of combined Fusarium mycelium from the same samples. The uncultureted F. langsethiae was detected in the DNA extract from a sample of barley, which was not identified in the combined sample of the mycelium. This sample of the grain has the highest levels of T-2/NT-2-toxins--0,075/0,345 mg/kg (determined by HPLC) among positive samples. F. sporotrichioides--a potential producer of T-2- and HT-2-toxins has been revealed by PCR method in other grain samples both containing and not containing these toxins. The biosynthesis of T-2- and HT-2-toxins on the PSA-medium in vitro has been studied for 10 single-spores F. sporotrichioides isolates, allocated from grain. Synthesized T-2-toxin content (measured by ELISA) ranged from 0.4 to 184.5 mg per l of medium. Three strains showed very high levels from 117.2 to 184.4 mg/l, two of which have been isolated from barley which don't contain these toxins. The absence of the toxin in grain samples does not guarantee the absence of high-level producers of mycotoxins. The direct detection of Fusarium spp. in grain by PCR analysis with extraction of fungal DNA by CTAB method along with increased sample weight has been shown to make possible the detection of a more number of species of Fusarium (including uncultureol strains) compared with mycological method with PCR analysis of the combined sample of the mycelium.

[Rapid determination of food grain infection by fungi Fusarium and their species detection (part 1)].

Currently contamination of food grains with Fusarium spp. is determined by conventional mycological methods and can last up to 30-40 days. The method specificity is highly dependent on the subjective evaluation of the researchers. The alternative to traditional mycological methods is detection by PCR. The purpose of the study was to improve the contamination analysis method of food grains infected by Fusarium for analysis time reducing and species detection specificity increasing. Investigations were carried out on food grains samples harvested in 2009-2011 from 5 federal regions of Russia. On the first stage, 100 grains were sowing on potato-sucrose medium and then incubated at 24 degrees C for 7-10 days. At the second stage, the Fusarium species composition grown from food grains was estimated by two methods. The first method was mycological for monospore isolates. The second one was PCR analysis of DNA extracts from the combined sample of Fusarium mycelium which grew on a foodgrains sample. Species-specific primers of such mycotoxins producers as F. graminearum, F. culmorum, F. sporotrchiodes, F. langsethiae, F. poae, F. avenaceum, F. tricinctum were used for PCR detection. The species composition of viable Fusarium spp. fungi revealed in foodgrains samples by mycological method completely concided with the results of PCR analysis. In result, the method that combines traditional mycological sowing for detection of viable species with PCR species detection of the mycelium in integrated sample has been developed. The method significantly reduces test duration (3-4 times) by excluding the sieving step in obtaining monospore fungi isolates for further species identification. The method also allows obtaining reliable data on the viable Fusarium species including producers of toxins in foodgrains. Thus, the idea of improving of the identification stage allowing reducing labor costs and increasing of the method specificity was realized.