Purpose: The aim of this study is to investigate and understand the behavior of two probiotic bacterial strains; Lactobacillus acidophilus LA5, Bifidobacterium animalis/lactisBB12 in corporation with yogurt starters to eliminate aflatoxin M1 in yogurt.
Methods: High Performance Liquid Chromatography equipped with fluorescence detector was used to determine Aflatoxin M1 in yogurt. The method was optimized and validated according to Commission Decision BS EN ISO 14501:2007 by using the conventional validation approach, according to the standard, selectivity, recovery, and precision of the method.
Standard samples of known aflatoxin M1 concentrations (0.1 to 10 µg/l) were injected and a linear calibration curve was obtained. In order to validate the method, the mean recoveries were calculated at three different levels of aflatoxin M1 additions (0.5, 1.0, and 1.5-fold the MRL).
Later reconstituted milk with added aflatoxin M1 at concentrations of 0.050 and 0.100 µg/l was fermented with two probiotic bacteria (Lactobacillus acidophilus LA5, Bifidobacterium animalisBB12) in corporation with yogurt starter cultures to reach pH value of 4.6. The prepared yogurt samples were stored at 4 °C for 4 weeks. Qualitative and quantitative tests concerned with aflatoxin M1 were performed by the application of HPLC using validated method.
Results: The results show that the calibration curve was linear for the concentrations of 0.1–10 µg/l when injected. The average recoveries were determined on three different days for various concentrations (0.025, 0.050, 0.075 µg/kg) in septimal orders (72.57 – 86.66%) with RSD in the range of 2.56 – 8.41 %. The limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 µg/kg and 0.015 µg/kg, respectively. The method was not affected by slight variations of some critical factors as pre-treatments and different storage conditions.
The results of the validation and the method used are in agreement with method described by Commission Regulation 401:2006: EC which might be regarded rapid, reproducible and simple to apply.
AFM1 levels in yogurt samples showed a significant decrease (P < 0.01) as compared to those initially added to milk. The growth of lactic acid bacteria was not affected by the presence of aflatoxin M1 except Streptococcus thermophilus that showed a significant (P < 0.01) decrease in the yogurt containing the toxin at high concentration. During the course of fermentation, AFM1 was significantly decreased (P < 0.01) in yogurts at both contamination levels; however, storage at refrigerated conditions did not affected the levels of aflatoxin significantly (P > 0.01). Final loses of 33 to 39% were observed at the end of both fermentation and refrigeration practice.
Significance: It therefore might be suggested that the strains of probiotic bacteria which were studied in this research work and are currently used in the food industries, due to their cost-effective and detoxification potentials employed at great demand for decreasing of aflatoxin M1 level in milk products.