Preparation of bacteria Lactobacillus and Bifidobacterium, standard aflatoxin ,Aspergillus toxinogenic and Chemicals and instrumental analyses
Strains of Lactobacillus and Bifidobacterium fermentum PTCC 1744 Bifidobacterium bifidum PTCC 1644 and Aspergillus parasiticus toxinogenic PTCC 5286 from Scientific and Industrial Research Organization of Iran (IROST) were received. The mixture standard solutions of AFB1, AFB2, AFG1, AFG2, purchased from Sigma (St. Louis, MO, USA). Chloroform, acetonitrile, methanol, Sabouraud glucose agar (SGA) obtained from Merck (Darmstadt, Germany). Other chemicals and solvents were of analytical grade or HPLC grade. Deionized water was purified by the Milli-Q-Plus ultra pure water system LCTech (GmbH, Germany) supplied Immunoaffinity column (Afla clean). HPLC apparatus consisted of a Smartline HPLC pump 1000, a PDA detector 2800 and a degasser 5000, all from Knauer(Berlin, Germany). The data were acquired and processed by Chrom Gate software (version 3.3.1) from Knauer (Berlin, Germany). Detection performed by fluorescence with excitation and emission wavelengths of 365 and 418 nm (10). Chromato-graphic separation was achieved on a Lichrospher 100 RP & EC C8 reverse phase column (C8, 25×0.46 cm i.d. 5 µm particle size) from Teknokroma (Barcelona, Spain).
Cultured standard strain of Aspergillus parasiticus in yeast extract sucrose broth (YESB) and determination of dry weight and aflatoxin.
In a 100 ml Erlenmeyer flask containing 25 ml of liquid medium YESB 5×104 spres/ml of strain Aspergillus parasiticus is cultured. After storage for seven days on a shaker incubator at 30 °, the resulting mycelial mass medium by Tiffany fourfold isolated and dried in an oven at 70 °. The mycelial dry weight was calculated, and the amount of aflatoxins in liquid medium is determined by HPLC.effect of Bifidiobacterium bifidum PTCC 1644 and Lactobacillus fermentum PTCC 1744 in preventing fungal growth and aflatoxin.
1 ml of 18-h cultures of lactic acid bacteria in the nutrient broth added to 100 ml Erlenmeyer flasks containing 25 ml of liquid medium YESB , and simultaneously amount of 5×104 spres/ml spores of Aspergillus parasiticus toxinogenic added to each the flasks. After 7 days the Shaker incubator maintained at 30 °, resulting mycelial mass by Tiffany fourfold separated and dried at 70 ° it is. Afterwards , Mycelium dry weight was calculated and the amount of aflatoxin in liquid medium are determined by HPLC.
Effect of extracellular metabolites of lactic acid bacteria on the reduction of standard aflatoxin
72-hour cultures of bacteria in nutrient broth bacterial cells using centrifugation in 4000 g for 15 min were precipitated. The supernatant from the membrane filter 0.22 micrometer was filtered. The 0.25 ml of basic solution standard aflatoxin B1 (1000 ppb) was mixed with 1.75 ml of filtrate to a final concentration of 125 ppb reach it.Aflatoxin extraction.
Aflatoxin production in YESB medium was extracted three...