Effects of dietary n-3 polyunsaturated fatty acids and ... - FINS

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1 st WorkshopXIII International Feed Technology Symposiummycotoxins. Mycotoxins are naturally occurring secondary metabolites of severaltoxigenic microfungi that contaminate the whole food chain, from the agriculturalcultures to the plate of consumers. Mycotoxins occur sporadically both seasonally andgeographically. In farm animals, mycotoxins exert their effects through four primarymechanisms: (1) intake reduction or feed refusal, (2) alteration in nutrient content of feedin nutrient absorption and metabolism, (3) effects on the endocrine and exocrine systemsand (4) suppression of the immune system. In routine animal feed screening, mycotoxinsare usually found at relatively low levels. Limited information exists regarding theeffects of low levels of multiple mycotoxins in livestock. It has been suggested thatcombinations of mycotoxins at low concentrations may have negative effects onlivestock, even though the concentrations of individual mycotoxins are well belowconcentrations reported to cause negative effects [26].The main mycotoxins classes of concern produced by fungi in the genera Aspergillus,Penicillium and Fusarium include the aflatoxins, ochratoxin A, trichothecenes andfumonisins. Moisture and/or temperatures is the single most important factor indetermining if and how rapidly molds will grow in feeds. Improper storage accompaniedby too high a temperature and elevated moisture content in the grain favours furthermycotoxin production and leads to reduction in grain quality [16, 22].The aims of this work were: 1) to determine the mycobiota in pig feed samples, 2) toevaluate the feedstuffs' mycotoxins contamination, especially aflatoxins, ochratoxin,deoxinivlenol and zearalenone, 3) to correlate the mycobiota presence versus specificmycotoxins contamination in respect of the effects of the moisture content and wateractivity (aw), on the growth and mycotoxins production.MATERIAL AND METHODSSamplesThe research materials consisted of 18 representative pig feed samples which werecollected directly at animal farms from different provinces of Serbia during six monthperiod. The samples (each about 1 kg) were stored at 4 °C and analysed the day aftercollection. On the same day, the moisture content of the feeds samples was determinedby drying.ReagentsStandards of AFT, OTA, ZEA and DON were purchased from Sigma-Aldrich ChemieGmbH. All other solvents and reagents were analytical grade.Isolation and identification of fungalCulturable fungal spore concentrations are presented in terms of colony-forming units(CFU)/g of samples. Isolation and quantitative enumeration of fungal propagules weredone on solid media using the surface- spread method by blending a 10 g portion of eachsample with 90ml of 0.1% peptone water solution. Serial dilutions, 10 -1 to 10 -6concentration, were made from each material and 0.1ml aliquots were inoculated intriplicate on two media potato dextrose agar (PDA) and Czapek yeast extract agar(CYA) of fungal enumeration. After 3-7 days, growing fungal colonies were transferredto Czapek yeast extract agar (CYA) and incubated at 25 C in the dark for 7 days.Macrofungi and moulds were identified to genera/species by their macro- andmicromorphology features using appropriate identification keys [14, 17, 18, 24, 28].103
1 st WorkshopXIII International Feed Technology SymposiumChemical analysisAll pig feed samples were analysed with validated methods and under quality assuranceconditions.Moisture contentThe moisture content of the pig feed samples was determined by drying at 105°C untilthe weight did not change further [6]. The a w value of the grain was measured in ahygroscope (GBX Scientific Instruments FA-St/1, tastatura model MX 3700/ML 4700) ata temperature of 20°C.Analysis for aflatoxins (AFT), ochratoxin A (OTA) deoxynivalenol (DON) andzearalenone (ZEN)The detection of these mycotoxins in all samples was performed by TLC usingappropriate methodology [7, 8, 15, 19].Statistical analysisDifferences in the mean levels of mycotoxins contamination across the three groups ofpositive samples was calculated by analysis of variance and then by a Student’s t-test.Additional posttests were applied to evaluate differences between groups withstatistically significant variation among means. The differences with p values smallerthan 0.05 were considered statistically significant.RESULTS AND DISCUSSIONMycoflora analysisThe results obtained from the mycoflora analysis in the samples are presented in Figure1 and 2. Occurrence of mycotoxins contamination and mean contamination levels ofmycotoxins (mg/kg) in samples originating from region where samples were collectedare presented in Figure 3 and 4.Total fungal counts ranged from 10 5 to 40x10 5 cfu/g. In our study, the highest fungalcount (40x10 5 cfu/g) and average total fungal counts was detected in pig feed samplescollected from region Bogatić. From the 18 samples analysed, seven samples had cfu/ghigher than the limit established by Serbian regulations (3x10 5 ) [20]. These samplesincluded 3 samples originating from Vladimirci and 4 samples from Bogatić locality.Fungal count is an indicator of the quality of feeds and should not exceed 1x10 5 cfu/g[2]. It is worth mentioning that in such a samples only Penicillium species mostlybelonging to F. equiseti were isolated.Species determination revealed great heterogeneity. A total of six genera and 14 speciesof moulds were identified. With the exception of Alternaria alternata, and Mucorracemosus which occurred only in one to two samples, the rest of the species were foundin more than one sample in all locations surveyed. The most frequently isolated funguswas Penicillium species (Fig. 2), a mould present on 17 (94,4%) of the 18 samplesexamined, and comprised 30,6% ofthe total fungal population. Other frequently isolatedmoulds included Fusarium (55,5%) and Paecilomyces (44,4%) genera. Fusariumisolates were identified as F. equseti, F. moniliforme, F. nivale F. semitectitum, F.tricinctum and F. avanaceum, which are proven toxogenic moulds. Other fungi from thegenera Aspergillus (22%), Mycor (11,1%) and Alternaria (5,5%) were represented in aless amount. Among fourteen species were identified, only Paecilomyces was notpotentially mycotoxins produced fungi. The incidences of Penicillium species increased104
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Effects of dietary n-3 polyunsaturated fatty acids and ... - FINS

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