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초록·키워드
Stable carbon isotope ratio (δ<sup>13</sup> C) analysis of isolated honey protein serves as a robust internal standard method for authenticity assessment. However, conventional protein precipitation protocols rely on sulfuric acid(H₂ SO₄), which poses severe safety hazards and risks acid catalyzed degradation of abundant sugars. This study evaluated a milder and safer alternative protocol by substituting sulfuric acid with phosphoric acid(H₃PO₄) and systematically optimized key pretreatment parameters including acid concentration, reaction temperature, reaction time, washing frequency, and drying methods using six distinct honey samples. The experimental results indicated that while theδ<sup>13</sup> C values remained highly stable and consistent across most tested variables, the physical yield and procedural stability of protein recovery were significantly influenced by each parameter. Based on empirical findings, the optimal pretreatment configuration was established as 0.5 M phosphoric acid concentration, a reaction temperature of 60°C, a reaction duration of 30 min, a three-cycle washing procedure, and freeze-drying. Specifically, the 0.5M concentration minimized the variation in protein recovery yields across samples to ensure maximum procedural stability. A temperature of 60°C provided the most favorable and robust protein yields, while a 30 min reaction duration maximized time efficiency without sacrificing recovery mass. For post precipitation refinement, three washing cycles effectively eliminated interfering matrix components while preventing excessive protein loss. Furthermore, compared to hot-air oven drying which induced minor thermal degradation and a scorched appearance, freeze-drying successfully preserved the physical quality and texture of the isolated proteins into a fine powder. Taken together, this optimized framework offers an efficient, reliable, and standardized pretreatment protocol that ensures both stable protein recovery and isotopic accuracy, making it highly suitable for routine honey authenticity testing via isotope ratio mass spectrometry.
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