【作者】 武雯；

【导师】 陈忠秀； 邓少平；

【作者基本信息】 浙江工商大学 ， 食品科学与工程， 2011， 硕士

【摘要】 甜味是食品科学的一个基本问题,甜味感官特征的评价一直依赖于人的感官品评。由于品评人员个体、性别及阅历等生理和心理的差异,很大程度上制约了对甜味感受真实、客观的表达。而且,基于实验条件的限制和甜味感觉的特殊性,人们至今没有完整理解甜味分子的感受机理。而在甜味抑制剂研究方面,虽然人们对甜味抑制剂作用位点达成了共识,但有关甜味抑制物质起作用的机制是竞争性的还是非竞争性的,仍然没有统一的结论。本课题组前期以富勒醇C60(OH)18作为人工甜味化学受体模型,运用等温滴定量热(ITC)方法探索甜味识别的热力学,研究结果表明富勒醇对人工甜味剂、天然甜味剂都具有较好的识别效果,其作为模型化合物研究甜味机理具有可行性。本论文进一步运用感官品评、ITC、核磁共振(NMR)和分子模拟(MS)等多种实验手段共同研究人工甜味受体模型与甜味抑制剂,甜味异构体、甜味增强剂的识别作用,以期获得甜味机理方面更多的信息。本课题研究的内容有：(1)人工甜味化学受体模型与甜味抑制剂作用的研究采用甜味抑制剂(HPMP)和十四种常用甜味剂,通过前期建立的人工甜味受体模型ITC滴定模式,获得抑制过程中热力学参数,实验结果表明富勒醇优先自发识别HPMP,疏水作用力在识别过程中起着重要作用,HPMP和甜味剂同富勒醇的结合具有竞争性。采用“快速作用-有无法”的品评模式进行HPMP感官实验,通过比较加入甜味抑制剂前后甜味剂察觉阈值,发现HPMP对不同结构甜味剂的抑制效果不同,离子对其作用无明显影响,抑制效果可以用Ⅰk(甜味抑制度)量化表示。比较富勒醇识别过程中热力学参数与Ⅰk,发现热力学平衡常数的比值(K1/K2)与Ⅰk有一定的相关性,K1/K2比值越大,甜味抑制度Ⅰk越高,由此佐证了甜味抑制机理可能是甜味抑制物质竞争地与味蕾受体结合产生的。实验结果进一步说明利用人工受体模型研究甜味机理的有效性。(2)人工甜味受体模型与甜味剂异构体作用的研究通过ITC、NMR和MS等技术共同研究富勒醇与单糖异构体的作用,实验结果表明在溶液中多种单糖异构体共存的情况下,富勒醇与p型异构体结合更稳定,优先与之形成氢键,此过程中释放出的能量可以提供α型异构分子转化成p型异构分子所需的构象转化能,打破溶液中原有的变旋平衡,向生成p型异构体的方向移动,导致溶液中p型异构体比例明显增加。因此可以推测,单糖分子接近味蕾受体时,可能也引起甜味剂异构体的平衡移动,导致异构体的比例发生变化,具体表现为各种糖类异构体的甜度差异。(3)人工甜味化学受体模型与甜味增强剂作用的研究通过MS手段进行富勒醇分子与甜味增强剂的相互作用的研究,结果表明甜味增强剂与富勒醇形成氢键后,还与甜味分子形成较强氢键,这样就拉近了甜味分子与富勒醇的距离,增强了甜味分子与富勒醇的氢键强度,使其更好地与富勒醇进行识别。因此可以推测,甜味增强剂在与味蕾受体结合的同时,还能与甜味分子形成较强氢键,从而增强了甜味分子与味蕾受体的识别作用,实际表现为增强剂单独使用不产生甜味,但和甜味剂共同使用能有效增强甜感。(4)不同结构的人工甜味受体模型与甜味剂相互作用的研究通过分子动力学模拟,对现有人工甜味受体模型C60(OH)18进行结构优化,实验结果表明富勒醇随碳数增加碳球笼体积增大,在理想稀溶液中,分子表面疏水作用力增加,无法更好与甜味分子识别；富勒醇羟基数的增加会使其与单糖分子的结合能增加,识别作用更加明显,但是羟基数过多会减小分子表面疏水作用力,分子表面的羟基因为距离过近会相互吸引形成分子内,增大识别过程中噪音能量,不利于富勒醇分子与单糖分子的识别作用。因此,结构优化结果表明C60(OH)20更适合作为人工甜味受体模型。总之,本文在前期研究的基础上,采用多种实验手段,进一步利用富勒醇作为人工甜味受体的模型化合物研究了它与甜味剂异构体、甜味抑制剂和甜味增强剂的相互作用,在目前人们未能获得甜味蛋白精细结构之前,研究结果对于理解甜味识别、甜味抑制机理和甜味增强机理具有一定的意义,同时也丰富了甜味机理研究的仿生化学。更多还原

【Abstract】 Sweet taste is a basic issue of food science while evaluation of sweetness organoleptic property always relies on panelist sensory evaluation. Nobody has completely understood sense mechanism of sweet molecules due to limited experimental condition and specialty of sweet taste. Although researchers have made some agreement on acting spot of sweetness inhibitor in relating researches, there’s still no uniformed conclusion regarding whether sweetness inhabiting mechanism is competitiveness or not.Recently, our team used C60(OH)18 as a synthetic sweetness receptor model, and isothermal titration calorimetry (ITC) as a tool to discover the thermodynamic mechanism of the molecular recognition of sweetness. The result showed a fullerenols-based synthetic model can effectively recognize both artificial and natural sweeteners. Our model truly helps to understand the mechanism of the recognition of sweetness.This thesis further applies multiple experimental approaches such as sensory evaluation, ITC, NMR(Nuclear Magnetic Resonance) and MS(molecular simulate) to research on recognition of artificial receptor model, sweetness inhibitor, sweetness isomer and sweetness enhancer in order to obtain more information about mechanism of sweetness. The main research work is as follows:(1) Research on Artificial Receptor Model and Sweetness InhibitorThe thermodynamics of the mimetic interaction of HPMP and 14 sweeteners with fullerenols as a synthetic sweet receptor model was elucidated by ITC technique. Thermodynamic parameters revealed that fullerenols recognize HPMP firstly on account of hydrophobic force, and competitive binding with fullerenols do exist when the sweeteners and HPMP are presented in the solution concurrently."rapid action-yes or no way" sensory evaluated method was conducted to get the threshold value of each sweetener. The efficiency of HPMP inhibiting each sweetener was expressed by the change of sweetness intensity. Test result implied that the inhibitor showed discriminating inhibition effect on each sweet compound, and ion has no significant effect on suppressive effectivity (Ik)The combined results of sensory evaluation and ITC thermodynamics revealed that the larger value of the ratio of two equilibriums constant K1/K2, the more effectively HPMP inhibit the sweetness of the sweetener. Thus the results proof that maybe the HPMP suppressive effect on human sweet taste by a competitive mechanism. Further certification of the artificial sweet receptor is a useful model that helps to provide chemical information of sweetness recognized mechanism.(2) Research on Artificial Receptor Model and Sweetness IsomersReaction of fullerenols and monosaccharides isomer is jointly researched by application of ITC, NMR and MS technology. Experimental result shows that fullerenols andβ-isomer has more stable coalition and can form hydrogen bond preferentially while multiple monosaccharides isomers existing in solution at meantime. Such process can release necessary conformation conversion energy for a-isomers transfer intoβ-isomers thus to break original mutarotation balance of solution and shift toward generating direction ofβ-isomers, causing significant increase ofβ-isomers in solution. Therefore it can be attributed that monosaccharides molecular may cause equilibrium shifting of sweet isomer when approaching taste buds receptors, then proportion change of isomer may incur and specifically expressed as sweetness difference of various sweet isomers.(3) Research on Artificial Sweetness Chemical Receptor Model and Sweetness EnhancerResearches on interactions of fullerenols module and sweetness reinforcing agent has been conducted by MS approach. The result shows that sweetness reinforcing agent can form relatively strong hydrogen bond after forming hydrogen bond with fullerenols,hence, distance between sweet module and fullerenols is shortened while hydrogen bond intensity of sweet module and fullerenols is improved for better recognition with fullerenols. It can be estimated that sweet reinforcing agent can form relatively strong hydrogen bond while combing with taste buds receptor to improve recognition of sweet module and taste buds receptor.(4) Research on Interactions Between Artificial Sweet Receptor Model with Different Structures and EdulcoratorStructural optimization of artificial sweetness receptor model C60(OH)18 by molecular dynamics simulation shows that fullerenols grows along with enlarging size of carbon spheres. Hydrophobic interaction increases on module surface in idea diluted solution; therefore we’re unable to make better identification with sweetness module. Increasing fullerenols hydroxyl groups will improve binding energy of monosaccharides molecular and more obvious recognition, but excessive hydroxide numbers will decrease hydrophobic interaction increases on module surface. Hydroxid on module surface will make interaction to form intramolecular and increase noise energy during recognition process, therefore make against to recognition reaction of polyhydroxy fullerenols module and monosaccharides molecular. Hence, structural optimization result shows that C60(OH)20 is more suitable to be used as artificial sweetness receptor model.In conclusion, the thesis applies multiple experimental approaches to further study interactions among fullerenols which is used as model compound of artificial sweetness receptor, edulcorator isomer, sweetness inhibitor and sweetness reinforcing agent. The research result has certain significance for people to understand sweetness inhibitor, sweetness inhibiting mechanism and sweetness strengthening mechanism before as certaining fine structure of sweet protein while also enriches biomimetic chemistry of sweetness mechanism study.更多还原