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Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture

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Abstract

Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy—consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step—we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

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Acknowledgments

This work was funded by Fundo Europeu de Desenvolvimento Regional (FEDER) Funds through the Operational Competitiveness Programme (COMPETE) by National Funds through the Fundação para a Ciência e a Tecnologia (FCT) under the project PTDC/AGR-ALI/102540/. Authors would like to acknowledge grant PEst-C/SAU/LA0001/2013-2014.

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Authors and Affiliations

  1. Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, 3060-197, Cantanhede, Portugal

    Carla Malaquias Almeida, Carlos Faro & Isaura Simões

  2. Instituto Politécnico de Coimbra—Escola Agrária, Bencanta, 3045-601, Coimbra, Portugal

    David Gomes

  3. CNC—Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal

    Carlos Faro & Isaura Simões

  4. Edifício UC-Biotech, Biocant-Parque Tecnologico de Cantanhede, Nucleo 04, Lote 8, 3060-197, Cantanhede, Portugal

    Isaura Simões

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  1. Carla Malaquias Almeida
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  4. Isaura Simões
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Correspondence to Isaura Simões.

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Almeida, C.M., Gomes, D., Faro, C. et al. Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture. Appl Microbiol Biotechnol 99, 269–281 (2015). https://doi.org/10.1007/s00253-014-5902-5

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  • DOI: https://doi.org/10.1007/s00253-014-5902-5

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