No abstract

Milk proteins are commonly used as ingredients in the food industry because of their functional properties, but they can cause severe reactions in milk-allergic individuals. In this work, two enzyme-linked immunosorbent assay (ELISA) formats were developed to detect bovine beta-lactoglobulin. The indirect competitive ELISA involved the use of anti-beta-lactoglobulin antisera, and the sandwich ELISA involved the use of specific antibodies isolated using a beta-lactoglobulin immunosorbent material. The effect of heat treatment on immunoreactivity of the protein in buffer and in milk was determined with both assays. The amount of immunoreactive protein in buffer and in milk decreased as determined by the sandwich ELISA, whereas the amount increased when measuring with the competitive ELISA. Several food products, including meat, bakery products, sauces, and snacks, were analyzed. With both assays, 10 of 11 products in which the ingredient list included the terms "powdered milk" or "milk proteins" contained beta-lactoglobulin. However, the beta-lactoglobulin concentration in these products obtained with the competitive ELISA were much higher than those obtained with the sandwich ELISA. These differences could be explained by the fact that the determination of beta-lactoglobulin concentration by immunoassay is influenced by differences in antibody recognition of the protein present in highly processed foods. Therefore, the antigen-binding properties of antibodies used in a particular immunoassay are important for a correct interpretation of results obtained in food processed at high temperature.

The effect of heat treatment on the denaturation of Cry1A(b) protein expressed in transgenic maize was studied over a temperature range of 69 to 77 degrees C. Denaturation of Cry1A(b) protein was measured by the loss of reactivity with its specific antibodies using a sandwich ELISA. The process of denaturation was studied by analyzing the values of inmunoreactive protein after each heat treatment by kinetic analysis. Denaturation of Cry1A(b) protein was best described assuming a reaction order of 1.5. D-values calculated were 4338, 2350, 1272, 734, and 601 s at 69, 71, 73, 75, and 77 degrees C, respectively. Z-value was estimated to be 9.0 degrees C and the activation energy value was 266.15 kJ/mol. Thermodynamic parameters for the process of denaturation of Cry1A(b) protein were also calculated. The high values of the enthalpy of activation and the positive values of the entropy of activation obtained for Cry1A(b) protein are typical of a reaction in which the denaturation of the protein is the rate-determining process that predominates over an aggregation process during heating.

Aims:  The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results:  Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 103 spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions:  FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study:  This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.

Enzyme-linked immunosorbent assay (ELISA) kits (indirect competitive and sandwich formats) to determine either b-lactoglobulin or ovomucoid were evaluated in model foods. A cut-off value was established for each kit to consider food samples as positive for milk or egg addition. Sausage and bread were positive at lower percentages of added milk using the sandwich format (0.005 and 0.05%) than the indirect competitive format (0.05 and 0.25%) and pâté was positive at 0.25% milk addition for both formats. Sausage was positive at 0.005%, and bread at 0.05% added egg for indirect competitive and sandwich formats, whereas pâté was positive at 0.25% egg only by the indirect competitive assay. The concentration of added milk and egg to give a positive result depends on heat treatment, being higher for pâté (sterilized), followed by bread (baked) and sausage (pasteurised). The particularities of each format and the heat processing applied influenced the determination by ELISA of allergenic proteins in foods.