Proline in Honey: A Complementary Biochemical Indicator in Quality, Authenticity, and Adulteration Assessment

Proline is a major free amino acid in honey and has long been used as a biochemical indicator of honey quality, maturity, and authenticity. Its association with the biochemical contribution of bees during nectar transformation gives it a distinctive role among conventional quality parameters. However, proline concentration is influenced by botanical origin, geographical conditions, pollen content, bee-feeding practices, harvest time, maturation, storage, and heat treatment. Therefore, proline should not be used as a standalone criterion for honey quality or adulteration. This review evaluates the biological origin of proline in honey, its role in quality and maturity assessment, its limitations, its relevance to botanical and geographical origin determination, and its variation in honeys from Türkiye. Analytical approaches for proline determination, including spectrophotometric, chromatographic, mass spectrometric, kit-based, sensor-based, and multi-analytical systems, are also discussed. The literature indicates that low proline levels may indicate counterfeit honey, intensive sugar feeding, or insufficient maturation. However, reliable interpretation requires combined evaluation with HMF, diastase activity, electrical conductivity, sugar profile, C4 sugar ratio, amino acid profile, phenolic/mineral content, isotopic analyses, and chemometric methods. Proline should therefore be considered a complementary indicator within multi-analytical honey authentication systems.