Algerian cardoon flowers express a large spectrum of coagulant enzymes with potential applications in cheesemaking

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Abstract

Proteases from flowers of Algerian cultivated and wild cardoons were purified, characterised and compared with those purified from flowers of a Portuguese variety. Three cardosins (A0, A and B) were obtained from each variety. All of them were dimeric and comprised heavy and light chain. Cardosins from Algerian varieties presented higher molecular masses and were less glycosylated than their Portuguese counterparts. Milk coagulation and curd yield parameters revealed a large difference between cardosins A0, A and B and among the same cardosin from different origins. The enzymatic specificity of cardosins, studied against β-chain of oxidised insulin and κ-casein, showed no prevalent effect of varieties. However, compared with cardosins A0 and A, cardosin B was more proteolytic and led to more complex digestion profiles. The present study reports the first description of the diversity of cardosins in Algerian cardoon flowers and sustains their potential use as milk coagulants for cheesemaking.

Introduction

Several enzymes from various origins (animals, plants and microorganisms) can coagulate milk and are used in cheese making (Hyslop, 2003, Jacob et al., 2011, Yegin and Dekker, 2013). Plant coagulants have a strong coagulant activity and are accessible for farmers. Their use in cheese manufacturing is mainly driven by the chronic shortages in rennet, an enzyme extracted from the stomach of young calves (Hafid et al., 2019, Llorente et al., 2014, Shah et al., 2014, Tito et al., 2020).

Nonetheless, some studies report limitations to the use of plant coagulants, mainly traduced by some defects in texture and in sensorial quality of the produced cheeses (Shah et al., 2014). However, proteases from the cardoon Cynara cardunculus L. seem to be an exception (Roseiro et al., 2003, Sousa and Malcata, 2002). In fact, it was shown that the aqueous extract of the cardoon flowers is a successful substitute of animal rennet used for centuries to produce traditional cheeses along the Mediterranean basin (Roseiro et al., 2003). These cheeses usually have a regional character and are representative of small-scale production in rural areas. They are an important source of income for local communities and have a high impact on the preservation of specific regional breeds of cows, sheep and goat (Folgado & Abranches, 2020). Portugal, Spain and Italy have a large variety of PDO traditional cheeses produced using cardoon flowers as coagulant: Serra da Estrela, Serpa and Azeitão in Portugal; La Serena, Manchego and Torta del Casar in Spain; Fiore sardo and Cacio Fiore in Italy (Aquilanti et al., 2011, Francisco-José et al., 2010, Llorente et al., 2014, Macedo and Malcata, 1997, Ordiales et al., 2016, Prados et al., 2007, Roseiro et al., 2003).

The milk-clotting activity of C. cardunculus flowers extract is due to several aspartic proteases (Aps): cardosins (A, B, E, F, G and H) as well as cyprosins 1, 2 and 3 (Cordeiro et al., 1994, Sarmento et al., 2009). Cardosins represent around 70% of total proteins content in cardoon flowers (Oliveira et al., 2010). Cardosins A and B are the most studied and characterised. These two proteases are able to cleave the Phe105–Met106 bond of k-casein: cardosin A acting like chymosin while cardosin B has a pepsin-like activity what gives to the extract of cardoon flowers a good aptitude to coagulate milk (Veríssimo, Esteves, Faro, & Pires, 1995). Recent studies reported a considerable variability in cardosin contents of cardoon flowers (Amira et al., 2017, Barracosa et al., 2018). This variability in enzymes composition, has an influence on the characteristics of the yielded curds and therefore, could have an impact on cheese properties (Gomes et al., 2019).

In Algeria, two varieties of cardoon are known and widely distributed: cultivated cardoon (C. cardunculus. L. var. altilis) and the wild or spontaneous cardoon (C. cardunculus L. var. sylvestris). Rural population uses cardoon flowers to prepare numerous traditional fresh cheeses, namely Tiklilt or Djben, usually manufactured from cow, goat or sheep milk (Aquilanti et al., 2011, Benheddi and Hellal, 2019). However, in Algeria, cardoon is still somehow an underutilised source. Thereby, the valorisation of milk coagulant enzymes from C. cardunculus may be economically beneficial to stimulate the local cheese-making industry: knowing that the Algerian cheese factories import most of their needs (e.g., coagulants), cardoon flowers could be an interesting alternative to the imported rennet. The present study aims to characterise the proteases occurring in the flowers of the two Algerian cardoon varieties and evaluate their milk coagulant properties. Furthermore, a comparison with the enzymes extracted from the Portuguese cardoon variety is made.

Section snippets

Plant material

Cardoon flowers used in the present study were collected from two locations (Akbou, 36°29′08.9″N 4°36′35.6″E and Tizi Ouzou, 36°36′17.9″N 3°59′06.6″E) in the region of Kabylia, middle north of Algeria. Flowers collected in Akbou were identified as the wild cardoon C. cardunculus L. var. sylvestris (labelled as ALG1) while flowers collected at Tizi Ouzou were from the cultivated cardoon C. cardunculus L. var. altilis (labelled as ALG2). ALG1 and ALG2 pistils were collected from well-developed

Chromatographic profiles protein recovery and electrophoretic patterns

Cardosins A0, A and B were purified from three varieties of C. cardunculus (ALG1, ALG2 and POR). The purification of cardosins was based the protocol of Sarmento et al. (2009) that includes two successive chromatographic steps. First, crude extracts were fractioned by size exclusion chromatography. For the three varieties, the proteolytic activity was recovered as a single peak (Fig. 1a), but with different elution times; ALG1 active fraction was eluted after 43 min, while those from ALG2 and

Discussion

To reach self-sufficiency, Algeria made the promotion of the dairy sector among its main priorities. Finding local sources of coagulant enzymes to support the needs of cheese factories could reduce the heavy reliance to imports. In our previous work (Zikiou & Zidoune, 2019), the aqueous extract of wild cardoon flowers (C. cardunculus var. sylvestris) was partially characterised and used in cheesemaking as a substitute of the imported rennet to manufacture a Camembert type cheese. The present

Conclusion

Overall, our findings confirm the possibility of using flowers from wild or cultivated cardoon as a coagulant in cheese production. The use of crude extracts or of pure cardosins could yield cheeses with different proprieties; Algerian cardoon flowers offer a spectrum of coagulant enzymes with potential applications in cheesemaking. The valorisation of this crop by the production of coagulants may be economically beneficial and could be a good alternative to support the needs of the national

Acknowledgements

Abdellah Zikiou was supported by the Algerian DGRSDT “Projet à intérêt socio-économique contrat N° 240”. The authors wish to thank Programa de Desenvolvimento Rural 2014-2020 (PDR2020) under Portugal 2020 and through Fundo Europeu Agrícola de Desenvolvimento Rural (FEADER) for the financial support of iCheese Project (PDR2020-101-031002).

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