In Portugal several traditional cheeses are made using cardoon flower extracts (Cynara cardunculus) as milk coagulant. The enzymes present in these extracts, responsible for milk clotting, have been identified as aspartic proteases. Cardosins A and B are the best characterized, and exhibit proteolytic activity similar to chymosin and pepsin respectively. Additionally these plant enzymes have higher proteolytic activity, than the animal counterpart, which results in a slightly bitter taste and low curd firmness. These unique characteristics render to this kind of cheeses a smooth texture and a unique intense flavor. However the use of dried flower extracts does not offer a consistent product and significantly hinders the industrial upscaling of cheese production and larger commercial applications. Therefore, cheese production processes must be improved in order to minimize the element of uncertainty and maximize the output of the desired product.
This project aims to explore the potential of plant cell and hairy root cultures as an alternative platform to produce these plant proteases. Plant tissue cultures offer lower production costs, large-scale production in industrial bioreactors, product consistency, containment, compliance with regulatory requirements and the possibility of genetic engineering.
To accomplish this, the project will follow a multi-scale approach, where initially factors that control of the APs gene expression will be assessed. After that, and taking into account the insights obtained from these initial studies, enzyme productivity using plant cell and hairy root cultures will be improved at the laboratory scale with a sequential scale up and proof of concept towards the end of the project.
For production we will use cells and hairy roots from cardoon. Expression level and activity of the target enzymes will be assessed. Optimum culture conditions for higher production yields will be determined. Concurrently BY2 tobacco cells will also be used as a heterologous expression system for production. Given that all the production systems selected are of plant origin we expect that these cells will perform properly post translational modifications retaining full milk clotting activity. Proteases produced in this way will be purified and tested for casein hydrolysis and milk clotting activity. The project also envisages a production scale up and cheese manufacturing trials to assess the quality of the final product.
Besides a substantial input on the knowledge of the regulatory mechanisms of APs expression, the expected impacts of this project include the development of a new added-value product. This will contribute for the improvement of a traditional Portuguese product, making it a safe standardized good in order to provide new markets.