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Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.


Key message

Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity.


Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.

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Aspartic proteases


Bovine serum albumin


Hairy roots


Phosphate-buffered saline


Phenylmethylsulfonyl fluoride




Schenk and Hildebrandt medium


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This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through project “Proteases from Cynara cardunculus L.: study of gene expression and establishment of alternative production platforms” (ref. PTDC/BAA-AGR/30447/2017), Research Unit Bioresources 4 Sustainability (ref. UID/Multi/04551/2013), and PhD Fellowship to AF (ref. PD/BD/114488/2016, Plants for Life PhD Program). MS data were obtained by the UniMS – Mass Spectrometry Unit, ITQB/IBET, Oeiras, Portugal. The authors wish to thank Isaura Simões and Rita B. Santos for helpful discussions throughout the course of this work, Bruno Alexandre for help with the MS analysis, and Andreas Schiermeyer for critical reading of the manuscript.

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AF and RA conceived and designed the research. AF and ASP conducted experiments. ACF and CP contributed reagents and tools for hairy root cultures and gene expression analysis, respectively. AF and RA wrote the manuscript. All authors analyzed the data and approved the manuscript.

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Correspondence to Rita Abranches.

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The authors declare that they have no conflict of interest.

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Communicated by Maike Petersen.

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Folgado, A., Pires, A.S., Figueiredo, A.C. et al. Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.. Plant Cell Rep 39, 89–100 (2020).

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  • Cardoon
  • Hairy roots
  • Proteolytic activity
  • Enzyme
  • Protease
  • Cardosin