Abstract:To explore the potential combination of enzymatic hydrolysis and treatments with ultrasonication or alkaline hydrolysis with the purpose of achieving the effective wall-disruption and extraction of nutrients in pine pollen. Methods Scanning electron microscopy (SEM) was used to observe the microstructure of pine pollen, and high-performance liquid chromatography was used to achieve the qualitative and quantitative analysis of phenols. Results The antioxidant values such as total phenols, total flavonoids, and ABTS of the soluble part of pine pollen turned to be the highest after the treatment with 12.5% alkaline protease for 1 h, with the specific values reaching 5.97 ± 0.09 mg GAE/g dw, 1.65 ± 0.04 mg RE/g dw, and 95.12 ± 1.84 mg μM TE/g dw, respectively. Total phenols, total flavonoids, ABTS free radical scavenging ability and iron ion reduction ability increased by 107%, 113%, 73% and 81%, respectively compared with untreated pollen after enzymatic hydrolysis combined with ultrasound and 0.3 mol/L NaOH treatment. The results of scanning electron microscopy revealed that the wall-disruption of pine pollen was further intensified after the treatment with enzymatic hydrolysis-ultrasonication-alkaline hydrolysis, and the pollen contents were spilled outward from the clefts, which was consistent with the trends of physicochemical indexes. The optimal process conditions were 0.3 M NaOH ultrasonic-alkali treatment for 1 h, adjusting the pH to 7, and enzymatic hydrolysis with 12.5% alkaline protease at 50°C for 1 hour. Conclusion Ultrasonic alkali treatment can assist enzymatic hydrolysis to break the wall of pine pollen, and nutrients such as small peptides, polyphenols and flavonoids were further dissolved, which promotes the high-value utilization of pine pollen.