Avaliação da interação entre aflatoxina M1 e B1 com a fração proteica do leite

Abstract

Milk is one of the main sources of nutrients in the human diet and is a food that accompanies the human being throughout life, both as drinking milk as through its derivatives. However, there are countless forms and types of contamination that affect milk, especially among the contaminants of chemical order, aflatoxins. Among the methods of analysis of aflatoxins in milk and dairy products, the High Performance Liquid Chromatography (HPLC) stands out mainly due to its versatility, speed and accuracy of quantitative measurements. However, it is a complex system in which the properties of the constituents of the mobile phase are affected by changes in process conditions under which the experiments are performed. Typically the HPLC analysis conditions are determined empirically, using the "trial and error" in which numerous attempts are made without a more detailed study of the system. Therefore, the first step of this aimed to optimize multiresponses in HPLC, by selecting the optimal conditions as the mobile phase composition, its flow into the chromatographic system and the column temperature in order to identify, separate and quantify aflatoxin M1 (AFM1) and aflatoxin B1 (AFB1) simultaneously. Therefore, it was carried out experimental a mixture design for three components with restrictions combined with a 22 factorial design to the process variables (flow and column temperature). After defining the models for the dependent variables, a search of the optimum conditions was made using the sequential simplex method and the Derringer and Suich desirability functions. The variables evaluated were: mobile phase composition (acetonitrile, methanol and aqueous solution of acetic acid 1%), the mobile phase flow rate and column temperature. The chromatographic parameters obtained as responses were time and factor of retention for both aflatoxins, separation factor, column resolution and height of the peaks. After the design validation, analytical validation was performed through the analytical figures of merit: linearity, precision, accuracy and limits of detection and quantification. The experimental design carried out was able to produce reliable models that allowed better conditions estimation regarding multiple objectives. In the analytical validation of the chromatographic method, the analytical parameters evaluated were within the confidence interval, the method can be considered accurate and precise showing quantitation limits of 0.3 and 0.5 μg L-1 for AFB1 and AFM1, respectively, and linearity with R2> 0.99 for both aflatoxins. The second stage of the study aimed to evaluate the interaction between the AFB1 and AFM1 with dairy proteins, considering that studies show that those, especially AFM1, are located predominantly in the protein fractions. However, these studies did not evaluate the possibility of interaction between aflatoxins and dairy proteins, but only based on its quantification in the dairy protein fractions. Therefore, we sought through this study to evaluate a possible interaction between AFM1 and AFB1 with dairy protein fractions. Analyzes were performed by Fourier transformation infrared spectroscopy (FTIR) to assess possible changes in the secondary structure of dairy proteins when spiked with aflatoxins and Differential Scanning Calorimetry (DSC). For this purpose, preliminarily the spectra obtained were evaluated with standard casein and β-lactoglobulin in phosphate buffer saline solution (PBS) and a bovine milk model solution as well as in actual milk, whole and skim. In the second step, specific spectra bands were assessed through deconvolution and curve-fitting techniques. The results show that PBS was more suitable for the interaction study between aflatoxins B1 and M1 and the dairy proteins evaluated, β-lactoglobulin and casein. Changes in secondary structures suggest that although possibly occurring interactions with hydrophilic characters between β-lactoglobulin and aflatoxins were observed (especially with AFM1) also occur interactions with hydrophobic character (especially AFB1) with the hydrophobic β-lactoglobulin packages. Already with the casein, the changes introduced in protein secondary structure were more discreet but peak shifts were observed indicating structural changes of the protein, especially in the presence of AFB1, which suggests that chemical interactions occur between the components evaluated. The spectral changes, more evident with the β-lactoglobulin fraction than the casein fraction, suggests that although the quantification of aflatoxins is commonly higher in the casein fraction, it’s not possible to ensure that for this reason occur more tangible interactions between aflatoxins and casein than between aflatoxins and whey proteins (β-lactoglobulin). The greater quantify percentage of aflatoxins in the casein fraction, apparently, is attributed to the fact that this protein is found, in milk, in superior percentage to the whey proteins. Another hypothesis is the possibility of the aflatoxins evaluated are "masked" by being combined with β-lactoglobulin and not, therefore, being detected by conventional analytical methods leading to their underestimation in this fraction.