Testing the suitability of different high-performance liquid chromatographic methods to determine aflatoxin M1 in a soft fresh Italian cheese.

Aflatoxin M1 (AFM1) is a toxic undesirable compound in milk. AFM1 affinity for caseins causes a concentration effect during milk process for dairy transformation. In spite of this, no official method of analysis, nor maximum tolerance level for aflatoxin M1 in cheese have been established. Thus, the aim of this work was to test the suitability of different HPLC methods for the AFM1 quantification in soft cheese samples at three different contamination levels (low, medium and high, at respectively nearly 30, 100 and 250 ng/kg). Nine participants were selected among Italian laboratories accredited by the Italian accreditation body (ACCREDIA) for HPLC toxin analysis. They were asked to analyze samples applying the method routinely used. The different applied methods were compared, and precision and accuracy parameters were evaluated. The main differences among HPLC procedures were registered at the level of extraction step. The use of an enzymatic digestion for the extraction of the toxin from cheese seemed to be particularly advantageous and the use of immunoaffinity columns seemed to be determinant for the improvement of sensitivity at low contamination levels. In general, the applied methods well discriminated the 3 levels of contamination, even though they performed better at the medium and high concentration levels (100 and 250 ng/kg) than at the low one (30 ng/kg). In fact relative standard deviation for reproducibility at low level was higher (60.1%) than the same value at medium and high levels (22.8% and 28.9%, respectively).

Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk.

The affinity of aflatoxin M1 toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.

Influence of milk-clotting enzyme concentration on the alphas1-casein hydrolysis during soft cheeses ripening.

We studied the influence of the dose of milk-clotting enzyme on alphas1-CN degradation, soluble nitrogen production, and sensory profile for an Argentinean soft cheese: Cremoso Argentino. Five different types of cheeses were produced: 1) control cheeses with normal technology, 2) cheeses with inactivated milk-clotting enzyme, 3) cheeses with inactivated milk-clotting enzyme, without starter (acidified with glucono delta lactone), 4) cheeses with a half dose of milk-clotting enzyme, and 5) cheeses with a double dose of milk-clotting enzyme. Proteolysis was assessed by isoelectric focusing electrophoresis of the insoluble fraction at pH 4.6, followed by densitometric quantification. Soluble nitrogen at pH 4.6, expressed as a percentage of total nitrogen and defined as ripening index was also performed. A sensorial panel evaluated the cheeses at the end of ripening. The hydrolysis level of alphas1-CN depended on the milk-clotting enzyme dose used in cheese making. Cheeses without active coagulant did not show degradation at the end of ripening, while cheeses with half and whole doses showed proportional degradations to coagulant dose. Cheese with a double dose of coagulant did not show higher alphas1-CN hydrolysis than normal cheese. No difference was found between cheeses with and without microbiological starter, indicating that the selected culture, composed of thermophilic strains, was unable to attack the whole casein. A high linear correlation was found between ripening index and the relation Sensorial characteristics of cheeses agree with objective analysis. Cheeses without active coagulant were hard and crumbly, while cheeses with normal dose were soft and creamy.

Characterization of ewe's milk by capillary zone electrophoresis.

The purpose of the present work was to develop a procedure able to separate and identify the major protein components of ewe's milk by capillary zone electrophoresis (CZE). Thirty-five individual milk samples of Massese breed were analyzed using a coated capillary. The analyses were performed at pH 3.0 at a temperature of 40 degrees C in the presence of 6 M urea. The purification of casein fractions was carried out by preparative fast protein liquid chromatography and the CZE results were confirmed by polyacrylamide agarose gel electrophoresis (PAAGE). The identification of whey proteins was also carried out by comparison with high-performance liquid chromatography data. The present study permitted the identification of the major components of ewe's milk by high-resolution electropherograms and characteristic migration times (tM). it was also possible to detect the presence of genetic variants of beta-lactoglobulin. The tM of k-casein was determined after enzymatic action of chymosin by verifying the simultaneous formation of p-k-casein. In most of the samples a fast moving alpha-s2-casein variant was identified by comparison with PAAGE results. Minor genetic differences were found in other casein fractions for this pool of samples.

Lysozyme: just an additive or a technological aid as well?

The effects of lysozyme on coagulation of milk and cheese making were studied by means of the gelograph, tristimulus colorimetry, ANS-fluorescence (hydrophobicity) and SDS-PAGE. Lysozyme binding to caseins caused structural differences during coagulation and it is proposed that, if the products have similar qualitative properties, lysozyme might be used as a technological aid giving shorter clotting times and higher yields.