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<p>Milk-clotting enzymes are essential in cheesemaking, traditionally sourced from animal-derived rennet. However, growing demand for plant-based and microbial alternatives has driven interest in new enzyme sources. <i>Onopordum macracanthum</i>, a species from the <i>Asteraceae</i> family, remains unexplored as a potential source of milk-clotting proteases. This study aims to purify and characterize these enzymes from <i>O. macracanthum</i> flowers and evaluate their potential as rennet substitutes. Proteases were extracted using acidic extraction and purified through size-exclusion and anion-exchange chromatography. The purified enzymes were analyzed via SDS-PAGE and assessed for milk-clotting and proteolytic activities using casein hydrolysis assays. Response surface methodology (RSM) was applied to optimize key parameters influencing milk-clotting activity (MCA), including temperature, pH, and calcium chloride concentration. Two distinct active fractions, identified as dimers with varying subunit compositions, were obtained. Fraction 2 exhibited significantly higher milk-clotting and proteolytic activity than Fraction 1. Both fractions effectively degraded bovine caseins, with Fraction 2 demonstrating superior activity. RSM analysis revealed that temperature, pH, and calcium chloride concentration significantly influenced MCA, with interactions between these factors playing a critical role. The optimal activity was achieved at 39.19&#xa0;°C, pH 5.5, and 18.55 mM CaCl<sub>2</sub>. To the best of our knowledge, this is the first study to report the purification and characterization of milk-clotting proteases from <i>O. macracanthum</i>. The enzymes demonstrated good technological properties, positioning the plant as a valuable alternative coagulant source for the dairy industry.</p>

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Milk-clotting proteases from Onopordum macracanthum flowers: purification, characterization, and potential applications in cheesemaking

  • 期刊论文

  • 发表日期：

    2025年5月8日

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  • Abdellah Zikiou,
  • Fairouz Saci,
  • Samah Fiala,
  • Kamal Dahdah

摘要

Milk-clotting enzymes are essential in cheesemaking, traditionally sourced from animal-derived rennet. However, growing demand for plant-based and microbial alternatives has driven interest in new enzyme sources. Onopordum macracanthum, a species from the Asteraceae family, remains unexplored as a potential source of milk-clotting proteases. This study aims to purify and characterize these enzymes from O. macracanthum flowers and evaluate their potential as rennet substitutes. Proteases were extracted using acidic extraction and purified through size-exclusion and anion-exchange chromatography. The purified enzymes were analyzed via SDS-PAGE and assessed for milk-clotting and proteolytic activities using casein hydrolysis assays. Response surface methodology (RSM) was applied to optimize key parameters influencing milk-clotting activity (MCA), including temperature, pH, and calcium chloride concentration. Two distinct active fractions, identified as dimers with varying subunit compositions, were obtained. Fraction 2 exhibited significantly higher milk-clotting and proteolytic activity than Fraction 1. Both fractions effectively degraded bovine caseins, with Fraction 2 demonstrating superior activity. RSM analysis revealed that temperature, pH, and calcium chloride concentration significantly influenced MCA, with interactions between these factors playing a critical role. The optimal activity was achieved at 39.19 °C, pH 5.5, and 18.55 mM CaCl2. To the best of our knowledge, this is the first study to report the purification and characterization of milk-clotting proteases from O. macracanthum. The enzymes demonstrated good technological properties, positioning the plant as a valuable alternative coagulant source for the dairy industry.