[Alternaria toxins in tomato products marketed in the Russian Federation].

Tomatoes and tomato products are widely produced and consumed throughout the world. Alternaria spp. are the main cause of alternariosis (black mold disease) on fresh tomatoes, both in the field and after harvesting. Alternaria toxins are widespread contaminants of tomato products. The aim of the present study was to evaluate the contamination of tomato processing products from the domestic market with Alternaria toxins, as well as to assess their intake by humans through the consumption of tomato juices. Material and methods. The content of Alternaria toxins (alternatiol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid) was determined in 64 samples of tomato products (paste, ketchup, juice) by high-performance liquid chromatography coupled to tandem mass-spectrometric detection (HPLC-MS/MS). Results. The priority Alternaria toxins for tomato paste, ketchup and juice were tenuazonic acid (61% of 64 samples, in amounts from 20.0 to 1065.5 µg/kg), altenuene (52%, 8.9-200.1 µg/kg) and alternariol (27%, 12.2-561.6 µg/kg). Samples of tomato paste turned out to be the most contaminated with Alternaria toxins while tomato juice samples were the least contaminated. At the same time, several toxins were found in 91% of tomato paste samples, 35% of ketchups, and 23% of tomato juices. Conclusion. To the best of our knowledge, the present study is the first survey devoted to Alternaria toxins contamination of tomato paste, ketchup and tomato juice sold on the Russian market. The high frequency of their contamination with tenuazonic acid, altenuene and, to a lesser extent, alternariol has been established, which indicates a potential risk to human health when tomato processing products are consumed. This indicates the need for a hygienic assessment of contamination the above products with tenuazonic acid, altenuene and alternariol. When calculating the potential intake of Alternaria toxins for different age population groups, it was shown that high levels of alternariol (up to 56.77 ng/kg body weight per day) could be obtained under daily consumption of tomato juice by adults and children under three years of age, as well as tenuazonic acid when consuming tomato juice contaminated at the 95th percentile level as part of the diet in organized groups for orphans and children without parental care.

[Mycotoxins in spices consumed in Russia].

Spices and herbs have been used since ancient times as flavor and aroma enhancers, colorants, preservatives and traditional medicines. As many other plant products, they can be exposed to contaminants, ones of which are mycotoxins, secondary metabolites of fungi. Such contamination can occur during harvesting, processing and storage, distribution, retailing and consumer use. Although they are used and consumed in small quantities, but added to a wide variety of products, especially ready-to-eat products. So the assessment of their contamination with mycotoxins is very important. The aim of the study was to investigate the contamination of spices and herbs with mycotoxins of fungi of the genera Aspergillus, Penicillium, Fusarium and Alternaria, as well as to assess the mycotoxins intake per person when consuming these food groups. Material and methods. Concentration of mycotoxins in 155 samples of spices and herbs was determined by ultra high-performance liquid chromatography coupled to tandem mass-spectrometric detection (UHPLC-MS/MS). The list of mycotoxins included deoxynivalenol, aflatoxins, ochratoxin A, zearalenone, T-2 toxin, fumonisins, sterigmatocistin, HT-2 toxin, diacetoxyscirpenol, enniatins, beauvericin, neosolaniol, citreoviridin, mycophenolic acid, citrinin, tentoxin, altenuene, alternariol and its monomethyl ether. Results. Among the regulated in plant products mycotoxins in the studied samples there were found aflatoxins (B1 - in 19% of samples, from 0.4 to 48.2 µg/kg, B2 - 8%, from < limit of quantitation (LOQ) to 3.2 µg/kg, G1 - 2%, 0.75-21 µg/kg, G2 - 5%, 0.5- 12.5 µg/kg), ochratoxin A (15% samples, 0.8-14 µg/kg), fumonisin B1 (8%, 16.1-722.6 µg/kg), and fumonisin B2 (14%, < LOQ - 79.6 µg/kg). T-2 toxin and deoxynivalenol were found in 10% of samples (< LOQ - 6.5 µg/kg and < LOQ - 65.5 µg/kg respectively), zearalenone - in 4 samples (1.7-106.2 µg/kg), HT-2 toxin - in 8 samples (5.4-19.8 µg/kg). Among little-studied (emergent) mycotoxins in the spices and herbs samples there were found tentoxin (in 36% of samples, in an amount from 0.7 to 10.9 µg/kg), altenuene (in 8%, 14.5-161.5 µg/kg). 10% of the samples were contaminated with alternariol and its methyl ether (from less than LOQ to 12.8 and < LOQ to 55.7 µg/kg, respectively), 4% - with sterigmatocystin (0.4-7.8 µg/kg), 5% - mycophenolic acid (13.1-297 µg/kg), 2% of the samples were contaminated with citrinin and enniatin B (< LOQ - 27.7 and 0.1-1 µg/kg), in 9 samples (6%) beauvericin was detected (< LOQ - 1.7 µg/kg). Over 60% of samples were contaminated with more than one mycotoxin. The content of aflatoxin B1 exceeded the maximum permissible level set in the EU (5 µg/kg) in nine samples. Conclusion. To the best of our knowledge, the present study is the first in the Russian Federation to report results indicating to the contamination of spices and herbs with mycotoxins. High occurrence of aflatoxins, tentoxin, ochratoxin A and fumonisin B2 has been observed. In calculating the potential exposure of mycotoxins, the possibility of high levels of aflatoxin B1 intake have been shown to be possible, which could lead to a public health risk when consuming contaminated spices, herbs and foods containing them.

[Mycotoxins contamination of cocoa products and carob marketed in the Russian Federation].

Cocoa is a daily basic food for many people all over the world. Also, people engaged in healthy lifestyle often prefer carob. The aim of the present study was to evaluate occurrence of Aspergillus, Penicillium, Fusarium and Alternaria secondary metabolites in cocoa and carob available in the Russian Federation and assess mycotoxin's intake with these products. Material and methods. Concentration of 27 mycotoxins in 63 samples of cocoa and carob products was determined by ultra high-performance liquid chromatography coupled to tandem mass-spectrometric detection (UPLC-MS/MS). The list of mycotoxins included regulated ones (aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, T-2 toxin, zearalenone), their derivatives and structural analogs (A and B trichothecenes), and emergent mycotoxins (alternariol, its monomethyl ether, altenuene, tentoxin, citrinin, sterigmatocistin, cyclopiazonic and mycophenolic acids, enniatins, beauvericin). Results. 29 of 41 cocoa samples were positive for beauvericin, contamination level was from 1.6 to 2184.8 µg/kg. Tentoxin, sterigmatocystin and alternatiol monomethyl ether were detected in 3 samples; their concentration varied in the ranges 0.7-1.2, 1.5-3.3 and 4.0-7.8 µg/kg correspondingly. Carob samples were positive for alternariol (14 of 22 samples, 1.5-43.1 µg/kg); tentoxin (13 samples, 0.5-8.7 µg/kg), mycophenolic acid (6 samples, 6.9-8.2 µg/kg) and for alternatiol monomethyl ether (3 samples, 1.0-1.2 µg/kg). Several samples of cocoa were contaminated with regulated mycotoxins: zearalenone (18 of 41 samples, 2.1-24.6 µg/kg), ochratoxin A (14 samples, 0.75-12.0 µg/kg) and aflatoxin B1 (2 samples, 0.59 and 0.86 µg/ kg). Several carob samples were contaminated with fumonisin B2 (7 of 22 samples, 4.2-5.2 µg/kg), ochratoxin A (5 samples, 0.5-1.4 µg/kg) and aflatoxin B1 (3 samples, 0.15-0.18 µg/kg). The ochratoxin A content in two instant cocoa beverages exceeded its maximum level (5 µg/kg) set for some plant products. Conclusion. To the best of our knowledge, the present study is the first survey devoted to emergent mycotoxin contamination of carob and cocoa alternative products marketed in the Russian Federation. The high occurrence of such mycotoxins as aflatoxins, beauvericin and ochratoxin A in these products indicates a potential health risk and the need for a hygienic assessment of cocoa and carob products' contamination not only with regulated in cocoa products aflatoxin B1, but with other mycotoxin including emergent ones. The risk of dietary intake of aflatoxins, beauvericin and ochratoxin A with cocoa products (especially with instant cocoa beverages) has been revealed for children over 7 years old in organized groups.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

[Dried fruits marketed in Russia: multi-mycotoxin contamination].

Mycotoxins (MT) - secondary metabolites of micromycetes - are natural contaminants of plant products. Fruits are particularly susceptible to fungal contamination and MT accumulation due to high sugar content. It can occur at any production stage: during vegetation, drying and storage. The most hazardous MT - aflatoxins (AFLs) and ochratoxin A (OTA) - are regulated in dried fruits in some countries. However, their maximum levels (ML) were not set in Russia yet. The present research was aimed at the evaluation of MT contamination of dried fruits marketed in Russia. Material and methods. 54 samples of dried dates (n=11), apricots (n=9), raisins (n=9), prunes (n=7), figs (n=6), apples (n=3) and mixtures for compote (n=9) were analyzed for 32 MTs by HPLC-MS/MS with positive/negative ESI in the MRM mode. Results. OTA and fumonisins (FBs) were the major regulated contaminants, their occurrence proved to be 10 and 17% correspondingly. Emergent metabolites of Fusarium spp. enniatin A (ENN A, 22%) and beauvericin (BEA, 15%); Penicillium spp. mycophenolic and cyclopiazonic acids (MPA and CPA, about 19%); Alternaria spp. tentoxin (TE, 17%) were detected also. Two-thirds of positive samples were contaminated with two and more MTs. All studied samples could be referred as safe within the EU regulations. Conclusion. Some kinds of dry fruits proved to be susceptible to contamination with particular MTs. Characteristic pattern for raisin was the OTA and MPA combination, for figs - FBs and CPA. According to literature data concerning occurrence and safety of MTs and the results of our survey, the long-term monitoring of AFLs and OTA in dry fruits and AFLs, OTA, FBs and CPA in figs from different regions of Russia is necessary to assess the need for setting of MLs of these MTs.

[Сontamination of baby foods with ochratoxin A].

Mycotoxin ochratoxin A (OTA) is a widespread contaminant of raw cereal grains with nephrotoxic activity. Сereal-based baby foods (BF) are an important component of the infant diet. In Russia, the presence of OTA in grainbased BF is not allowed (<0.5 ng/kg), in the EU maximum limit of toxin in BF is 0.5 ng/kg. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) set for OTA a provisional tolerable weekly intake of 100 ng/kg bw; the European Food Safety Authority (EFSA) ­ 120 ng/kg bw. The purpose of this study was to investigate the OTA content in BF (infant cereals and canned food) and assess the relevant risk to the health of children first year of life. The analysis of OTA was performed by immunoaffinity column clean-up and high performance liquid chromatography with fluorescence detection. The limit of detection and limit of quantification for OTA were 0.10 and 0.50 ng/kg, respectively. The content of OTA in BF was represented as maximum (Max), mean (M), median (Мe) and 90 percentile (90%) of all samples. The 554 BF samples based on corn, rice, buckwheat, millet, wheat, oats, barley, and mixtures of cereals were studied. OTA was detected in 32 samples of BF: in 30 of the 312 samples of infant cereals (Max ­ 4.95 ng/kg; M ­ 0.09 ng/kg, Me and 90% ­ 0 ng/kg) and in 2 of the 242 samples of canned food (0.34 and 0.37 ng/kg). 20 samples of BF were contaminated with OTA above the maximum limit (≥0.50 ng/kg). BF, exhibited the highest incidence of OTA, were buckwheat-based (13 of 41 samples, Max ­ 2.52 ng/kg, M ­ 0.36 ng/kg, Me ­ 0 ng/kg, 90% ­ 1.57 ng/kg) and mixed-grain (12 of 115 samples, Max ­ 4.95 ng/kg, M ­ 0.10 ng/kg, Me ­ 0 ng/kg, 90% ­ 0.14 ng/kg) infant cereals. OTA was also detected in 2 of 40 samples of oat-based infant cereals (0.19 and 0.60 ng/kg), in 2 of 72 samples of rice-based infant cereals (0.18 and 0.48 ng/kg) and in 1 of 37 samples of wheat-based infant cereals (0.13 ng/kg). None of the 4 samples of corn-based and of the 3 samples of millet-based infant cereals contained OTA. Calculations showed that for infants the daily ochratoxin A dietary intake did not exceed 6.8 ng/kg bw. This value of OTA intake is below the levels, proposed as tolerable intake. Thus, one can conclude that cereal-based BF (infant cereals and canned food) are moderately contaminated with OTA and there is not a significant toxicological risk to the health of children of first year of life. However, identification of BF samples containing OTA above the maximum limit, demonstrates the need for thorough monitoring of the quality of the products from the manufacturers and regulatory authorities.

[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.

Fusariotoxins in Russian Federation 2005-2010 grain harvests.

Monitoring results of food grain contamination with fusariotoxins-deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FB1&FB2), T-2 and HT-2 toxins-are presented. Harvests of 2005-2010 in different regions of Russia were investigated. The occurrence of DON in wheat was 8%, barley 9%, oats 4%, rye 2% and maize 2%. The highest frequency of ZEN contamination was found in oats, the lowest in wheat. Calculated average daily intake of DON varied from 0.066 to 0.096 µg/kg body weight, the highest being found in the Southern region, but substantially lower than the provisional maximum tolerable daily intake. The results of enzyme-linked immunosorbent assay and high-performance liquid chromatography-mass spectrometry analysis demonstrated the presence of T-2 toxin in 14% and HT-2 toxin in 17% of all samples. The maximum level of T-2 toxin was exceeded only in one sample of barley. Relatively high frequency and levels of FB1&FB2 contamination were found in maize.

[Application of immunochemical and chromatographic techniques for monitoring of trichothecene mycotoxins type A in grains].

A sensitive HPLC-MS technique after immunoaffinity clean-up has been developed for the simultaneous determination of T-2 and HT-2 toxins of in the food grains. The limit of detection of method was 0,002 mg/kg for T-2 toxin and 0,005 mg/kg for HT-2 toxin. Comparison of existing immunochemical and chromatographic techniques of trichothecenes type A determination in grains was made. New combined scheme of monitoring of grain contamination by trichothecenes type A was proposed. It was based on screening by ELISAs followed by confirmation of positive samples by developed HPLC-MS technique with preliminary immune affinity purification of extracts. Proposed modification of immunochemical and chromatographic techniques allowed to lower the influence of grain matrix on the analytical results and to reduce of the probability obtaining of false-positive determination. Developed approach was used for determination of trichothecenes type A in natural contaminated grain samples.

[Fusariotoxins in the food production in the Russian Federation: situation 2006-2008 years].

The results of monitoring of the fusarium mycotoxins (deoxynivalenole (DON), zearalenone (ZL), T-2 and HT-2 toxins, fumonisins B1 and B2) contents in the food wheat, barley, rye, oats and corn (2006-2008 crops) in different regions of Russia were presented. DON occurrence in the food wheat, rye and barley were 9, 4 and 0,8% accordingly. DON wasn't detected in the food oats and maize in these years. ZL occurrence was relatively high and varied between 4 and 40%. The estimated daily intake of DON per 1 kg of body weight in Russia on average varied from 0,066 to 0,096 mg. The daily intake of DON in the North-Caucasian region was twice higher than in Russia on average and it didn't exceed tolerable daily intake (TDI) of DON - 1 mg/kg of body. HPLC-MC analyses results indicated that 14 and 16% of all investigated grain samples were contaminated by T-2 and HT-2 toxins accordingly. T-2 toxin content in grains was lower then MTL (0,1 mg/kg). High frequency and levels of fumonisins contamination were detected in the food corn.

[Study of fumonisins B1 and B2 contamination of baby food].

Fumonisins content were studied in the baby foods, grits and flour by HPLC. FB1 +FB2 were found in 95% of 129 samples of grits and flour in quantities from 30 to 4350 microg/kg (mean: 580 microg/kg). Fumonisins were found in baby food in 52% of snacks in the range from 10 to 720 microg/kg (mean: 35 microg/kg). Processed cereal-based and cereal and dairy-based food contained FB1+FB2 in 39% of cases in the amount from 10 to 9200 microg/kg (mean: 244 microg/kg). Fumonisins were not found in canned baby food on the cereal-based with meat, fish or fruit products.

[Production of fumonisins by Fusarium moniliforme strains isolated from corn grain]. / Obrazovanie fumonizinov shtammami Fusarium moniliforme, vydelennymi iz zerna kukuruzy.

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).