In our study the presence of bacteria, yeast, and microscopic fungi was evaluated. Three forms of corn silage were made including silage without additive, silage with microbial additive (lactic acid bacteria), and silage with nutritional additive (urea). Silage additives were applied to the matter within the recommended dosage, then the matter was ensiled into plastic bags and stored at a constant temperature. After 5.5 months of storage, average samples for microbial and mycotoxins analysis were taken. From microbiological points, the plate count agar method for enumeration of total count of bacteria, lactic acid bacteria, enterococci, yeasts, and microscopic fungi and mass spectrometry for microbiota identification were used. In total, 43 species of bacteria and yeasts and 6 genera of microscopic fungi were identified from all samples of corn silages. The most isolated species were Lentilactobacillus buchneri and Kazachstania exigua from bacteria resp. yeasts and Aspergillus and Penicillium from microscopic fungi. Mycotoxins were determined by HPLC-MS/MS and divided into two groups as regulated and emerging. In the corn silages only Fusarium mycotoxins were observed. All corn silages, regardless of the addition of the additive, were the highest in nivalenol content. Deoxynivalenol and beauvericin with the highest concentrations were present in silage with urea. Although the mycotoxins content of the variants changed, these changes were not statistically significant. In general, addition of lactic acid bacteria Lentilactobacillus buchneri and Lacticaseibacillus casei and urea as silage additives affect the microbial diversity; however, the hygienic quality of whole crop corn silage was not negatively changed.
Addition of silage additives is commonly concentrated on the improvement of nutritive and fermentative features of silages, but in recent years, mycotoxic contamination is also a feature monitored after the treatment. This study aimed to evaluate the effect of silage additives, in the concrete inoculant consisting of a mixture of Lactobacillus buchneri LN40177, Lactobacillus casei LC329090 and urea, on the concentration of major mycotoxins in maize silages. The ensilage mass was made in three variants, which consisted of maize silage (control variant C), maize silage with the addition of a commercial inoculant (variant A) and maize silage with the addition of urea (variant U). The commercial inoculant was added to the cut mass at a dose of 1 g/t of mass and urea at a dose of 5 kg/t of mass. After application of silage additives, the mass was stored and sealed in silage units. Mycotoxin analysis was performed using Veratox tests with ELISA reader, whereas average samples were prepared according to established protocols. The immuno-enzymatic method revealed that all samples of maize silage showed 100% contamination. The maize silages were characterized by the highest concentration of deoxynivalenol regardless of the treatment. The results confirmed the effect of silage additives on mycotoxic contamination of maize silages. Compared to the control variant, the commercial inoculant had negative increasing effect (P<0.05) on deoxynivalenol, ochratoxins and zearalenone, and a positive decreasing effect (P<0.05) on T-2 toxin and fumonisins. The urea addition resulted in significant reduction of T-2 toxin and increase of zearalenone.
In the rye silages (control and with additive) microbiota isolation and identification (total count of bacteria, coliform bacteria, enterococci, lactic acid bacteria and microscopic filamentous fungi) and occurrence of mycotoxins were evaluated. Total count of bacteria on Plate count agar at 30 °C for 48-72 hours, coliform bacteria on McConkey agar at 37 °C for 24-48 hours, enterococci on Enterococcus selective agar at 37 °C for 48-72 hours, lactic acid bacteria on De Man, Rogosa and Sharpe agar, Mayeux, Sandine and Elliker and All Purpose TWEEN® agar 37 °C for 48-72 hours in microaerophilic condition and microscopic filamentous fungi on Malt extract agar at 25 °C for 7 days were evaluated and identified with mass spectrometry. The most isolated family from both types of silage was Lactobacillaceae. The most isolated species from the control group was Lentilactobacillus buchneri (22%) and lowest number of isolated species was Mucor spp. The most isolated species from silage with additive was Lactobacillus gasseri (16%). The ELISA method (enzyme-linked immunosorbent assay) with using reader at wavelength 650 nm for detection and quantification of mycotoxins (total aflatoxins, total ochratoxins, total fumonisins, deoxynivalenol, zearalenone and T-2/HT-2 toxin) was used. The rye silages of control and with additive were characterized by occurrence of all analyzed mycotoxins, whereas deoxynivalenol was mycotoxin with the highest concentration. The rye silages with additive had significantly lower content of total aflatoxins compared to the control, while significantly higher concentration of total ochratoxins, deoxynivalenol, zearalenone and T-2/HT-2 toxin was determined.
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