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

Folgado, André; Pires, Ana Sofia; Figueiredo, Ana Cristina; Pimentel, Catarina; Abranches, Rita

doi:10.1007/s00299-019-02475-1

Original Article
Published: 03 October 2019

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

André Folgado¹,
Ana Sofia Pires¹,
Ana Cristina Figueiredo²,
Catarina Pimentel³ &
Rita Abranches ORCID: orcid.org/0000-0003-3173-3701¹

Plant Cell Reports volume 39, pages 89–100 (2020)Cite this article

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Abstract

Key message

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

Abstract

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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Abbreviations

APs:: Aspartic proteases
BSA:: Bovine serum albumin
HR:: Hairy roots
PBS:: Phosphate-buffered saline
PMSF:: Phenylmethylsulfonyl fluoride
PVPP:: Polyvinylpolypyrrolidone
SH:: Schenk and Hildebrandt medium

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Acknowledgements

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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Authors and Affiliations

Plant Cell Biology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA), Universidade Nova de Lisboa, 2780-157, Oeiras, Portugal
André Folgado, Ana Sofia Pires & Rita Abranches
Faculdade de Ciências da Universidade de Lisboa, Centro de Estudos do Ambiente e do Mar (CESAM Lisboa), Centro de Biotecnologia Vegetal (CBV), 1749-016, Lisbon, Portugal
Ana Cristina Figueiredo
Genomics and Stress Laboratory, Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA), Universidade Nova de Lisboa, 2780-157, Oeiras, Portugal
Catarina Pimentel

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André Folgado
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Ana Sofia Pires
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Ana Cristina Figueiredo
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Catarina Pimentel
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Rita Abranches
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Contributions

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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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). https://doi.org/10.1007/s00299-019-02475-1

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Received: 24 May 2019
Accepted: 23 September 2019
Published: 03 October 2019
Issue Date: January 2020
DOI: https://doi.org/10.1007/s00299-019-02475-1

Keywords

Cardoon
Hairy roots
Proteolytic activity
Enzyme
Protease
Cardosin

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