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中国精品科技期刊2020
龙欢,郭锐,王佳宏. 耐乙醇酶制备及一步法高效提取文冠果叶皂苷J. 食品工业科技,2026,47(8):273−284. doi: 10.13386/j.issn1002-0306.2025050306.
引用本文: 龙欢,郭锐,王佳宏. 耐乙醇酶制备及一步法高效提取文冠果叶皂苷J. 食品工业科技,2026,47(8):273−284. doi: 10.13386/j.issn1002-0306.2025050306.
LONG Huan, GUO Rui, WANG Jiahong. Ethanol-tolerant Enzyme Preparation and One-step Efficient Extraction of Saponins from Xanthoceras sorbifolium LeavesJ. Science and Technology of Food Industry, 2026, 47(8): 273−284. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2025050306.
Citation: LONG Huan, GUO Rui, WANG Jiahong. Ethanol-tolerant Enzyme Preparation and One-step Efficient Extraction of Saponins from Xanthoceras sorbifolium LeavesJ. Science and Technology of Food Industry, 2026, 47(8): 273−284. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2025050306.

耐乙醇酶制备及一步法高效提取文冠果叶皂苷

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

  • 摘要: 为解决目前使用酶和超声两步法提取存在有机试剂消耗量高和提取时间长的问题,实现文冠果叶皂苷高效提取,本研究从腐殖土壤中定向筛选耐乙醇的产酶菌株,根据其发酵后获得混合酶的耐乙醇特性,设计一步超声-酶-乙醇提取文冠果叶皂苷工艺并与超声辅助酶解提取(两步法)进行比较。通过单因素及响应面试验优化条件,采用超高效液相色谱-串联质谱法(Ultra-performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)、环境扫描电子显微镜(Scanning electron microscopy,SEM)、傅立叶变换红外光谱(Fourier transform infrared spectroscopy,FT-IR)和X射线衍射(X-ray diffraction,XRD)表征和解析了文冠果叶残渣的结构特征。结果表明,黑曲霉Aspergillus niger 42发酵的混合酶耐乙醇效果最佳,其分泌的耐乙醇纤维素混合酶(内切葡聚糖酶酶活254±0.05 U/mL)在40%乙醇中稳定性显著(P<0.001)(β-葡萄糖苷酶和木聚糖酶相对酶活分别保留78.67%±0.25%和55.34%±4.00%),且不产生赭曲霉毒素,安全性高。响应面试验显示,以混合酶作为溶液,无水乙醇作为溶质,在最优条件下(超声温度55 ℃、功率300 W、乙醇浓度40%、时间30 min),皂苷得率达14.98%±0.08%,较超声乙醇提取(7.42%±0.23%)提升101.89%,且乙醇用量较两步法减少30%,皂苷得率提高59%(9.42%±0.04%)。UPLC-Q-TOF MS证实一步法提取物的皂苷组成未变且较两步法含量显著增加。SEM、FT-IR和XRD结果分析表明,一步法较两步法处理对原料表观结构的破坏更大,对结构中的O-H、C=C和C-O-C等化学键被破坏更明显且无定形区被充分降解,该一步法兼具皂苷得率高、溶剂消耗低、操作条件温和及环境友好等优势,为文冠果叶皂苷的绿色高效提取提供了新解决方案,对其他天然活性物质的提取提供借鉴。

     

    Abstract: 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.

     

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