Localising pectin in dairy products using direct immunostaining

The microstructure of fat and protein in foods is easily visualised using confocal laser scanning microscopy (CLSM). However, a method to visualise hydrocolloids in situ has not yet been published. The objective of this study was to develop a direct immunostaining technique for visualising the microstructure of pectin in dairy products. The cross- reactivity and stability of the monoclonal rat antibody JIM5, which binds to partially methyl-esterified pectin, was assessed by enzyme linked immuno sorbent assay (ELISA) at a variable pH (3.8, 4.2), salt (0, 1, 2%) and sugar (0, 10, 20, 40%) concentrations. The fluorophore Alexa 488 was conjugated to JIM5 and the conjugate was used for direct immunostaining of pectin in a yogurt and gelled dessert. JIM5 exhibited high specificity. No cross-reactivity was assessed with a range of hydrocolloids frequently present in dairy products, except a minor cross-reactivity with guar gum (29±2.2%) and locust bean gum (9.2±0.7%). The stability study of JIM5 showed that, in yogurt-like conditions 27% unimpaired antigen-binding activity was present relative to the activity at neutral pH. JIM5 with conjugated Alexa 488 was added to dairy products to observe the micro structure of pectin. Pectin was localised in yogurt and a gelled dessert, and it was confirmed that the microstructure of the products was unaffected by the direct immunostaining. In conclusion we have developed an in situ method able to localise pectin in dairy product microstructure.