目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过...目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过分子对接与生物传感技术揭示其与苦味受体的相互作用机制,实现苦味属性从传统经验判定到分子水平表征的跨越,为完善白术质量控制体系提供支撑。方法采用D101大孔吸附树脂结合紫外分光光度法,制备并筛选富含内酯类成分的白术组分;通过UPLC-MS/MS与分子对接技术,在分子水平解析白术组分与味觉受体的相互作用特征;采用生物传感器与UPLCMS/MS联用技术,实现白术组分苦味关键质量属性的精准辨识。结果成功制得白术不同极性组分11个,其中白术醇提物70%组分的总内酯含量最高,为后续性味研究的理想载体;分子对接实验显示白术组分与Ⅱ型味觉受体家族14号成员(taste receptor type 2 member 14,TAS2R14)的结合展现出明显优势,在分子层面揭示了白术组分具有强烈趋于苦味属性的作用特质;进一步构建苦味生物传感器,对白术组分的苦味属性进行表征,结果显示白术组分与TAS2R14的相互结合强度为49.0ng/L,属于强相互作用,表明以内酯类成分为主的白术组分具有显著的苦味属性,该结果与分子对接结果相互印证,增强了实验结论的可靠性;最后,联合UPLC-MS/MS技术,共辨识得到包括白术内酯Ⅱ在内的苦味关键质量属性13个,在物质基础层面表明白术组分与苦味受体TAS2R14的结合受关键质量属性驱动,为理解白术内酯类成分苦味特性提供直接证据。结论通过多技术融合的系统性研究,从分子相互作用到物质基础层面揭示了白术内酯类成分苦味属性的本质,所发现的苦味关键质量属性为建立基于苦味特征的白术质量控制方法奠定了理论基础,此外,提出的技术路线为中药苦味物质基础研究提供了新的方法学参考。展开更多
This study aimed to clarify the molecular basis of bitter peptides in lager beer and their impacts on multidimensional sensory attributes of beer body.A systematic workflow was established that included peptide identi...This study aimed to clarify the molecular basis of bitter peptides in lager beer and their impacts on multidimensional sensory attributes of beer body.A systematic workflow was established that included peptide identification by reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry(RPLC-QTOFMS),machine learning screening,receptor docking with the bitter taste receptor TAS2R14,molecular mechanics/generalized Born surface area(MM/GBSA)analysis and single-peptide addition tests.Under stringent criteria(-10logP≥15 for database hits and de novo ALC≥90%),2056 peptides were identified;their sequences were enriched in aromatic and branched hydrophobic residues,and most peptides contained three to five amino acids.Machine learning and docking showed that the overall mean docking score was about-6.77,with tripeptides and tetrapeptides performing best(about-7.37 and-6.90,respectively).The ten top candidates displayed docking scores dominated by aromatic and Pro-containing motifs,and their MM/GBSAΔGbind values ranged from about-134 to-82 kcal/mol.The best-scoring peptide HCLPY gave a docking score of about-10.14,and the stability of HCLPY,SFIPLI and AWIQP was mainly supported by van der Waals and lipophilic terms,consistent with binding in a hydrophobic cavity.By combining interaction patterns with binding-pocket geometry,four peptides(SFIPLI,AWIQP,YVGW and HCLPY)were selected as representative bitter peptides.Sensory evaluation showed that,at a constant bitterness level of the control beer(4.8 on a 0-10 scale),singlepeptide addition led to bitterness intensities of 4.95,4.75,4.45 and 4.25 for HCLPY,SFIPLI,YVGW and AWIQP,respectively(p>0.05),but markedly redistributed other flavor dimensions.Relative to the control,hop aroma increased by+33.3%to+74.1%and aftertaste was extended by up to+12.7%.These results indicated flavor redistribution through cross-modal integration and modulation at the oral interface rather than simple additive bitterness.Overall,the work shifted the simple assumption that stronger binding necessarily produced stronger bitterness toward a framework in which molecular binding led to perceptual reweighting and redistribution of overall flavor,and it proposed short,sequence-designed peptides as controllable inputs for flavor engineering.展开更多
文摘目的旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱(ultra-performance liquid chromatography with electrospray tandem mass spectrometry,UPLC-MS/MS)的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象,通过分子对接与生物传感技术揭示其与苦味受体的相互作用机制,实现苦味属性从传统经验判定到分子水平表征的跨越,为完善白术质量控制体系提供支撑。方法采用D101大孔吸附树脂结合紫外分光光度法,制备并筛选富含内酯类成分的白术组分;通过UPLC-MS/MS与分子对接技术,在分子水平解析白术组分与味觉受体的相互作用特征;采用生物传感器与UPLCMS/MS联用技术,实现白术组分苦味关键质量属性的精准辨识。结果成功制得白术不同极性组分11个,其中白术醇提物70%组分的总内酯含量最高,为后续性味研究的理想载体;分子对接实验显示白术组分与Ⅱ型味觉受体家族14号成员(taste receptor type 2 member 14,TAS2R14)的结合展现出明显优势,在分子层面揭示了白术组分具有强烈趋于苦味属性的作用特质;进一步构建苦味生物传感器,对白术组分的苦味属性进行表征,结果显示白术组分与TAS2R14的相互结合强度为49.0ng/L,属于强相互作用,表明以内酯类成分为主的白术组分具有显著的苦味属性,该结果与分子对接结果相互印证,增强了实验结论的可靠性;最后,联合UPLC-MS/MS技术,共辨识得到包括白术内酯Ⅱ在内的苦味关键质量属性13个,在物质基础层面表明白术组分与苦味受体TAS2R14的结合受关键质量属性驱动,为理解白术内酯类成分苦味特性提供直接证据。结论通过多技术融合的系统性研究,从分子相互作用到物质基础层面揭示了白术内酯类成分苦味属性的本质,所发现的苦味关键质量属性为建立基于苦味特征的白术质量控制方法奠定了理论基础,此外,提出的技术路线为中药苦味物质基础研究提供了新的方法学参考。
基金supported by the Beijing Elite Scientist Sponsor-ship Program of BAST(No.BYESA.2023055)the National Key Research and Development Program of China(No.2022YFD2101205)+1 种基金Research Foundation for Youth Scholars of Beiiing Technology and Business University(JRFYS2025)the National Natural Science Foundation of China(No.32322068).
文摘This study aimed to clarify the molecular basis of bitter peptides in lager beer and their impacts on multidimensional sensory attributes of beer body.A systematic workflow was established that included peptide identification by reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry(RPLC-QTOFMS),machine learning screening,receptor docking with the bitter taste receptor TAS2R14,molecular mechanics/generalized Born surface area(MM/GBSA)analysis and single-peptide addition tests.Under stringent criteria(-10logP≥15 for database hits and de novo ALC≥90%),2056 peptides were identified;their sequences were enriched in aromatic and branched hydrophobic residues,and most peptides contained three to five amino acids.Machine learning and docking showed that the overall mean docking score was about-6.77,with tripeptides and tetrapeptides performing best(about-7.37 and-6.90,respectively).The ten top candidates displayed docking scores dominated by aromatic and Pro-containing motifs,and their MM/GBSAΔGbind values ranged from about-134 to-82 kcal/mol.The best-scoring peptide HCLPY gave a docking score of about-10.14,and the stability of HCLPY,SFIPLI and AWIQP was mainly supported by van der Waals and lipophilic terms,consistent with binding in a hydrophobic cavity.By combining interaction patterns with binding-pocket geometry,four peptides(SFIPLI,AWIQP,YVGW and HCLPY)were selected as representative bitter peptides.Sensory evaluation showed that,at a constant bitterness level of the control beer(4.8 on a 0-10 scale),singlepeptide addition led to bitterness intensities of 4.95,4.75,4.45 and 4.25 for HCLPY,SFIPLI,YVGW and AWIQP,respectively(p>0.05),but markedly redistributed other flavor dimensions.Relative to the control,hop aroma increased by+33.3%to+74.1%and aftertaste was extended by up to+12.7%.These results indicated flavor redistribution through cross-modal integration and modulation at the oral interface rather than simple additive bitterness.Overall,the work shifted the simple assumption that stronger binding necessarily produced stronger bitterness toward a framework in which molecular binding led to perceptual reweighting and redistribution of overall flavor,and it proposed short,sequence-designed peptides as controllable inputs for flavor engineering.
