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.
| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |