Ethanol-tolerant Enzyme Preparation and One-step Efficient Extraction of Saponins from Xanthoceras sorbifolium Leaves

To address the current issues of high consumption of organic solvent and prolonged extraction time inherent in the two-step method involving enzymes and ultrasound for efficient extraction of saponins from Xanthoceras sorbifolium leaves. A specific strain of black aspergillus fungus from humus soil was selectively isolated in this study. Based on the ethanol tolerance characteristics of the mixed enzymes obtained after fermentation, a one-step method using ultrasonication-enzyme-ethanol extraction process for saponins from Xanthoceras sorbifolium leaves was designed and compared with the ultrasonication-assisted enzymatic extraction (two-step method). The optimized conditions through single factor and response surface experiments were employed to analyze samples using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) to characterize and analyze the structural features of the Xanthoceras sorbifolium leaf residue. As a results, the ethanol-tolerant cellulase mixed enzyme secreted by this strain, exhibiting an endoglucanase activity of 254±0.05 U/mL, which was demonstrated remarkable stability in 40% ethanol, with relative enzyme activities of β-glucosidase and xylanase retained at 78.67%±0.25% and 55.34%±4.00%, respectively. Importantly, the mixed enzyme does not contain ochratoxin, ensuring high safety. Under optimal conditions (ultrasonic temperature 55 ℃, power 300 W, ethanol concentration 40%, time 30 min) with mixed enzyme as a solution and anhydrous ethanol as a solvent, the response surface experiments revealed that saponin yield reached 14.98%±0.08%, representing a 101.89% increase compared to ultrasonic ethanol extraction (7.42%±0.23%). Furthermore, ethanol consumption was reduced by 30% relative to the two-step method, while saponin yield improved by 59% (9.42%±0.04%). UPLC-Q-TOF MS analysis confirmed that the saponin composition unchanged remained from one-step method and showing a significant increase in content compared to the two step method. Besides, SEM, FT-IR and XRD further confirmed the effectiveness of one-step method, inducing more substantial material degradation than the two-step method, more pronounced disruption of O-H, C=C and C-O-C bonds and complete degradation of amorphous regions. This one-step method combined the advantages of high saponin yield, low solvent consumption, mild operating conditions, and environmental friendliness, providing a novel solution for the green and efficient extraction of saponins from Xanthoceras sorbifolium leaves and offering valuable insight for the extraction of other natural active compounds.