Interactions between macro-molecules and starter cultures in the cheese curd using antibody phage display technique and bioimaging
Special Symposium - Innovations in Food Technology (LMC Congress)
Meals - Convenience, Gastronomy & Quality (Food-1a)
Keywords: Cheese ripening, casein, proteolysis, phage display, antibody
Objectives
To investigate the ripening of cheese in a microscopic level, we generated the antibody fragments against specific casein sequences and a dipeptidase (pepD) from LAB (Lactic Acid Bacteria) by using antibody phage display technique.
The selected casein sequences traverse the cleavage sites of enzymes responsible for proteolysis in cheese during ripening. The antibody fragments against these casein sequences will be used to detect the proteolysis of casein. So long as the cleavage site is intact, the antibody will bind to the casein. However, after cleavage of the peptide bond by the enzymes, the antibody will no longer be able to recognize the substrate.
The antibody fragments against dipeptidase can be used to trace the distribution and location of dipeptidases in cheese matrix, and also be possible for monitoring the lysis of LAB.
Methodology
Antibody Phage display is a molecular technique by which antibody fragments (scFv, single chain variable antibody fragment) can be expressed at the surface of bacteriophage particles (usually M13 bacteriophage). Such phages thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed scFv, but also have the capacity to replicate. Populations of variant scFvs can be displayed on phage, by which means to generate an in vitro library of scFvs. Using this technique, immunization is by passed and functional antibodies against target antigen can be generated by panning.
The antibody fragments will be labeled by fusion with different fluorescent proteins. Bioimaging techniques (Confocal Laser Scanning Microscopy) can then be utilized to follow the selected macro-molecules of cheese during ripening.
Results
An efficient method for producing scFvs specifically against small peptides by antibody phage display technique has been developed.
The monoclonal scFvs against three small peptides from αs1-casein sequence and dipeptidase of Lactococcus lactis were generated.
The specificity and affinity of these scFvs against their target antigens were identified by ELISA and Western Blotting.
Conclusions
We have generated antibody fragments specifically against the interesting macro-molecules in cheese matrix. And now we are investigating the methods to use these antibody fragments as probes on cheese. It is expected that this project will provide an unprecedented knowledge about the micro-scale changes in the cheese matrix during ripening, which may be developed to evaluate and predict the influence of processing parameters and starter cultures on the final food cheese quality.
Presented Wednesday 19, 16:50 to 17:05, in session Meals - Convenience, Gastronomy & Quality (Food-1a).
