苦味传递机制与苦味肽研究进展

毕继才，崔震昆，张令文，林泽源，江海洋，莫海珍

(河南科技学院食品学院,河南新乡 453003)

味觉是哺乳动物尤其是人类的重要生理感觉之一[1]。哺乳动物在选择自己的食物源时,食物的味道是一个关键筛选条件[2]。典型哺乳动物的味蕾是由50～100个紧凑排列的味觉受体细胞(taste-receptor cells,TRCs)组成,每个成熟味觉受体细胞的寿命只有5～20 d,新的味觉受体细胞是通过味觉干细胞的不断分化更新产生[3]。成熟味觉受体细胞可以感受到五种基本味型:苦味、咸味、鲜味、甜味和酸味[4]。有学者认为最新研究发现一些新的味道应该考虑进去,如醇厚(kokumi)味[5]、脂肪味[6]和金属味[7]。简言之,味觉的传递过程就是食品中的风味化学物质激活了存在于味蕾中的一系列特异化味觉受体细胞,味觉受体细胞将这些刺激信号通过神经传导的方式呈递到神经中枢系统[8-9]。

食品中的风味物质种类很多,其中由蛋白质水解产生的呈味肽(taste peptide)是由Juerg Solms在1969年第一次正式系统报道的[10]。呈味肽是一些对食品风味有贡献的蛋白质序列片段,如多肽、二肽等。呈味肽不仅具有多种味型,如酸、甜、苦、咸等,还具有诸多生物活性功能,如降低血压、抗氧化、抗菌、免疫调节、降低脂肪等[11]。其中,苦味肽往往是消费者厌恶的一类呈味肽,它们通常在发酵、陈化和酶水解的食品产物中产生[12]。

本文系统地梳理了苦味的信号递呈途径,并对苦味肽的结构特征和脱苦的方法等研究成果的研究进展进行了综述。

1 味觉的生理学基础和苦味传递机制

1.1 味蕾

舌头是第一个与食品接触并感知其味道的器官。味蕾(taste bud)是簇状柱形感觉细胞,不均匀地分布在舌背部表面的乳突(papillae)结构表面[13]。味觉细胞按照功能不同可以分为四种,分别是暗细胞(I型),亮细胞(Ⅱ型),中间细胞(Ⅲ型)和基细胞(Ⅳ型)[9,14](如表1)。其中Ⅱ型细胞是最为主要的味觉识别和传导细胞。

表1 不同类型味蕾细胞及其功能Table 1 Different types of taste buds cell and their functions

大多数Ⅱ型细胞均含有细胞信号传导中重要的拥有七次跨膜结构域的G蛋白偶联受体(G protein-coupled receptor,GPCR),包含两种味觉受体,即1型味觉受体(T1R)和2型味觉受体(T2R),每种味觉受体相应地仅响应于一种味道(例如,甜味或苦味,但不能两者兼有)。其中T2R特异性地结合苦味[15],而T1R可以分为三个亚基:T1R1,T1R2和T1R3,通常可以在味蕾细胞中共表达[16]。人受体T1R2和T1R3识别天然或合成的甜味剂,而T1R1和T1R3对L-谷氨酸等鲜味物质进行识别[17]。另外,唾液在味觉呈递过程中的作用不容忽视。唾液可以为促味剂提供溶剂,将促味剂扩散到受体细胞的功能位点。此外,唾液可以刺激味觉感受细胞(TRCs)从而影响味觉。Matsuo曾设想用人造唾液来帮助治疗味觉功能障碍的疗法[18]。

1.2 苦味受体

苦味、甜味、鲜味受体同属于G蛋白偶联受体大家族[19],分别被T2Rs受体、T1R2/T1R3异源二聚体和T1R1/T1R3异源二聚体所识别[20]。与T1Rs不同,苦味受体T2Rs一般认为是单聚体,但最新研究表明，他们同样也可形成同源二聚体和异源二聚体[21]。啮齿动物中有40多个T2R家族成员,人类有25条功能基因编码T2Rs,但编码T1R的基因只有3条[22]。人类和啮齿动物单独的T2R仅仅可以辨别一种或几种苦味化合物,如T2R3仅能感知一种化合物(94种不同的天然和合成化合物进行测试),而T2R14至少可以感知33种化合物[23]。而单一的苦味物质却经常能够活化多种不同的T2Rs,如奎宁能够活化九种不同的人类T2Rs受体[22]。苦味物质与受体之间相互作用产生第二信号,活化下游的一系列生理反应。

1.3 苦味传递途径

甜味、鲜味和苦味受体信号传递途径尽管具有多样性,但T1R和T2Rs却拥有共同的细胞内信号通路。不同的味觉受体GPCR均能与异源三聚体G蛋白偶联,包括Gβ3和Gγ13、Gαgus(也称为味蛋白)、Gα14和Gαi[24-26]。早期学者认为Gα亚基可以激活cAMP信号,但最近研究表明，它主要是调节Gβγ亚基的作用。通过蛋白激酶A激活并维持信号蛋白处于响应状态,cAMP也具有长期调节信号蛋白的功能[27]。当T1Rs和T2Rs被呈味物质激活时,Gβγ二聚体被释放,激发磷脂酶Cβ2以调节细胞内Ca2+含量[24]。胞质Ca2+水平升高导致瞬时受体电位阳离子通道TRPM5开放,TRPM5开放可有效地使味细胞的阳离子通道去极化[28-30]。目前,关于味道传递途径中的许多细节问题还未有明确研究结论。

2 苦味肽来源和结构特征

2.1 苦味肽来源

一些食品中的苦味被认为是食品的典型味觉特征。苦味一般很难被人们接受,但在特定食品如啤酒、咖啡或干酪等的感官标准中却是非常重要的呈味特性。苦味肽仅是众多呈苦味物质中的一类,苦味肽除了能够呈现出苦味之外,有些苦味肽还具有一定的生理功能,起到预防慢性疾病的作用[31-33]。Gordon[12]和Speck[34]等在1965年第一次报道了在乳清培养发酵过程中产生具有苦味的肽。苦味肽包括有二肽、三肽、六肽、八肽乃至几十个氨基酸组成的肽,广泛存在于天然食品和发酵食品中,尤其在蛋白质含量丰富的食品发酵过程中会产生大量的苦味肽[35]。蛋白质水解产物的味道可以因为蛋白质种类,水解条件和蛋白水解酶应用的不同而发生变化[12]。

蛋白来源的苦味肽产生的途径主要是用酶法水解的方式,实际中常用的蛋白质水解的酶有三类:植物蛋白酶如木瓜蛋白酶[36]、菠萝蛋白酶[37]等，动物蛋白酶如胃蛋白酶[38-39]、胰蛋白酶[40]、胰凝乳蛋白酶[41]等和微生物蛋白酶如碱性蛋白酶[42]、风味蛋白酶[43]、中性蛋白酶[42]、复合蛋白酶[43]、枯草杆菌蛋白酶[44]。目前用的较多的蛋白酶有胰蛋白酶、木瓜蛋白酶、中性蛋白酶和碱性蛋白酶。如用胃蛋白酶水解大豆蛋白可以获得10种苦味肽,分别是Phe-Leu,Leu-Phe,Leu-Lys,Arg-Leu,Gly-Leu,Arg-Leu-Leu,Tyr-Phe-Leu,Glu-Tyr-Phe-Leu,Ser-Lys-Gly-Leu,Phe-Ile-Glu-Gly-Val[45-46]。利用胰蛋白酶水解酪蛋白获得三种苦味肽,分别是Gly-Pro-Phe-Pro-Ile-Val,Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys,Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Ahe-Gly-Lys[47-48]。利用枯草杆菌蛋白酶水解酪蛋白获得的苦味肽也是三种,分别是Leu-Val-Pro-Arh-Tyr-Phe-Gly,Arg-Gly-Pro-Pro-Phe-Ile-Val,Val-Tyr-Pro-Phe-Pro-Pro-Gly-Ile-Asn-His[41]。另外,随着呈味肽发掘数量的猛增,研究单独肽与其生物功能的关联性十分耗时。因此,生物信息学数据库对于发掘更多的呈味肽十分有用。如BIOPEP数据库含有大量感官肽和氨基酸的信息,可为研究者提供一些感官肽的生物活性最新信息[49]。

2.2 苦味肽结构特征

苦味肽是目前最容易描述的一类味觉感官肽,对苦味肽的科学评价和测量主要利用傅里叶转换拉曼光谱技术进行分析,研究确定苦味肽的相对苦度(Rcaf)。相对苦度Rcaf指是以1 mmol/L咖啡因溶液的阈值作为标准,此时的相对苦度Rcaf为1.0[50]。

呈味肽的味型特征与肽的疏水性关系密切[51],而疏水性又与氨基酸组成、氨基酸序列、肽链长度、空间结构等相关[52]。首先,肽的疏水性能由肽中氨基酸的组分和序列决定,其中脯氨酸(Pro)是主要的苦味肽的贡献者[53]。多肽结构包含Pro时,可更容易地结合到T2Rs受体上[54]。此外,当肽中出现甘氨酸(Gly),丙氨酸(Ala),缬氨酸(Val),亮氨酸(Leu),苏氨酸(Tyr)和苯丙氨酸(Phe)时也可以增强肽的疏水性,从而影响与苦味受体的结合[55]。此外,肽的分子量、立体参数和空间结构等同样是苦味的影响因素[56]。如Wang等报道显示，中度大小的肽通常比较大或者较小的肽段表现出的苦味更为突出。分子质量(MW)在1.9～3.3 kDa之间的肽被称为是高苦肽段,而较大或者较小分子质量的肽段表现出较温和的苦度[57]。多肽苦味还与肽中是否存在亲水性基团和碱性氨基酸残基有关。有学者认为，多肽中亲水性基团与疏水性基团在空间相距0.3 nm时便会产生苦味[47]。

在探究苦味肽的结构与苦味化学机理的研究中,学者们引用了Q值做为评估苦味的指标。Q值表示肽的平均疏水性。Q=∑ΔGi/n,其中ΔGi指将氨基酸链(肽)从乙醇溶液中转移到水溶液中所需平均自由能量(以cal/mol表示),n为肽链的氨基酸残基数[58]。当Q值大于1400 cal/mol时呈现苦味,Q值小于1300 cal/mol时不呈现苦味,Q值介于1300～1400 cal/mol之间则不能判断肽是否具有苦味[58]。Q参数与作为苦味肽组分的氨基酸的疏水性有相关性,这种相关性称为Q定则[12,59]。但Q定则只是经验规律而并不是绝对的,有些肽如Glu-Val-Leu-Asn序列的Q值为1162.5 cal/mol,它同样显示出苦味[60]。Kim等用胰蛋白酶水解大豆11S抗蛋白原获得的水解产物,水解产物中被描述为明显苦味的苦味肽序列如下:Leu-Ala-Gly-Asn-Gln-Glu-Glu-Glu,Asn-Leu-Gln-Gly,Gly-Ile,Glu-Gln-Pro-Gln-Gln-Asn-Glu,Ala-Gly-Asn-Pro-Asp-Ile-Glu-His-Pro-Glu,Gly-Asn-Pro-Asp-Ile-Glu-His-Pro,Asn-Ala-Leu-Pro-Glu和Asn-Asn-Glu-Asp-Thr。他们的Q值都在80～1474 cal/mol之间。被描述为适度苦味的苦味肽序列如下:Arg-Pro,Gly-Tyr,Ser-Ala-Glu-Phe-Gly,Glu-Gln-Gly-Gly-Glu-Gln-Gly,Ala-Leu-Glu-Pro-Asp-His-Arg,Asn-Ala-Leu-Glu-Pro-Asp-His-Arg-Val-Glu,Gly-Lys-His-Gln-Gln-Glu-Glu-Glu-Asn-Glu-Gly-Gly,Lys-Leu-His-Glu-Asn-Ile-Ala-Arg,Gly-Met-Ile-Tyr-Pro-Gly,Tyr-Glu-Gly-Asn-Ser,Ile-Gly-Thr-Leu-Ala-Gly-Ala,Asn-Phe-Asn-Asn-Gln-Leu-Asp-Gln-Gln-Thr-Pro-Arg。他们的Q值是从0(如Glu-Gln-Gly-Gly-Glu-Gln-Gly)到1670 cal/mol(Arg-Pro肽)[61]。这些事实表明,肽的Q值尽管能提供一些参考信息,但苦味判断并不能简单地基于Q值来预测。值得注意是,血管紧张素转换酶(Angiotensin Converting Enzyme,ACE)活性抑制所需要的结构域与苦味肽的结构相似,因此,很多苦味的二肽表现出ACE活性的抑制作用,如苦味三肽Phe-Phe-Phe[35,62-63]。

3 苦味肽脱苦技术研究

食品中的苦味往往是人们厌恶的味道,发酵食品中的苦味影响消费者的接受度,制约着发酵食品的市场化。因此,对于苦味食品我们通过脱苦技术改良可以提高它的感官评定。食品工业中的脱苦技术需要考虑以下几个因素:是否影响食品组分的活性(如苦味肽的保健功能);是否影响食品的消化率、吸收率和生物利用度;是否能高效地分离纯化;是否是最经济的除去/掩蔽嫌忌味道的技术等。这里介绍几种常见的苦味肽脱苦方法:

3.1 选择分离法

选择性分离脱苦主要是基于肽理化性质的差异性,采用物理吸附、等电点沉淀、溶剂萃取和色谱分离等方法将蛋白水解液中的苦味肽分离出来的方式。如苦味的酪蛋白和明胶蛋白水解液可以用活性炭吸附达到降低苦味的目的[12]。疏水肽在其等电点(pI)附近时具有非常低的溶解度,通过将溶液pH调整到苦味肽的等电点附近,便可将其沉淀去除[64]。另有研究者用大孔树脂分离制备出酱油中的多肽,结果表明XAD-16树脂是吸附多肽最有效的树脂[65]。另外,通过用仲丁醇、含水乙醇或异丙醇水溶液共沸萃取,使得酶促蛋白质水解产物中的苦味肽在醇相中浓缩[66]。随着现代分离技术的发展,用色谱的方法除去和制备呈味肽逐渐普及,如在发酵香肠中,肌原纤维蛋白发生强烈的蛋白水解,产生小的肽和游离氨基酸,用质谱联用的液相色谱法(Liquid Chromatograph Mass Spectrometer,简称LC-MS)进行多肽的分离和鉴定,同样可以进行苦味肽的分离[67]。

3.2 掩盖法

掩盖法能够有效地掩盖食品感官味道,如在苦味食品中添加脱脂牛奶,大豆酪蛋白和酪蛋白水解产物可以掩盖其原本的感官味道[68-69]。谷氨酸盐和腺苷酸或者5′-核苷酸钠盐以及具有鲜味的多肽,如αGlu-Asp,αGlu-Glu,αGlu-Ser和αGlu-Glu-Glu同样可以掩盖苦味肽的苦味[70-71]。另外,味觉物质的浓度和不同的味道刺激可以受到味道增强子和/或抑制子的作用,如低浓度时,咸-酸和酸-苦可以互相增强,但是在咸味环境下则可抑制苦味[17]。Kim是第一个发现并报道苦味和鲜味肽在受体水平相互作用的人。他们用五种大豆来源的鲜味肽Glu-Asp,Glu-Glu,Glu-Ser,Asp-Glu-Ser,Glu-Gly-Ser分别与具有明显苦味的苦味剂水杨苷一起进行实验。实验结果显示，五种具有鲜味的大豆鲜味肽(Glu-Asp,Glu-Glu,Glu-Ser,Asp-Glu-Ser和Glu-Gly-Ser)以非竞争性的方式抑制水杨苷诱导细胞内钙流,细胞内钙离子水平升高是苦味物质结合苦味受体引起的一个重要指标,但对照组的无味肽Gly-Gly却未见此结果[17]。

3.3 酶法

酶法脱苦与其他的脱苦方式相比具有突出的优点:更高的水解效率、反应条件温和、不破坏食品营养成分、水解过程便于控制等。因此用酶法脱苦是目前国内外研究的热点。Tamura等[69]用羧肽酶对苦味肽进行水解或结构修饰,最终能将苦味肽分子变为非苦味成分。Fujimaki[72]利用类蛋白反应(即蛋白酶的转肽作用)成功地降低了由胃蛋白酶水解的鳕鱼、面筋、小球藻、酵母、大豆等带来的苦味。Tchorbanov等[73]用食品级细胞内氨肽酶(Aminopeptidases)可将高Q值的苦味酪蛋白和大豆分离蛋白水解物转变成为低Q值没有苦味的物质。Fu等研究产自雅致放射毛霉(Actinomucorelegans)的羧肽酶(Carboxypeptidase)的酶活特性时发现该酶优先分解疏水性氨基酸类底物,如Z-Phe-Leu,Z-Phe-Tyr-Leu和Z-Phe-Tyr。该羧肽酶是降低苦味肽的有效工具,在酶法脱苦技术应用领域有广阔前景[74]。在奶酪脱苦的研究过程中发现,奶酪发酵初期苦味肽感受度较低,是因为发酵初期高活性的脱苦肽酶可以高效地降低奶酪中苦味物质的含量[75]。另有研究者发现Lactobacillushelveticus在奶酪发酵中具有显著的脱苦效果,主要是由于该菌能够产生脯氨酰内肽酶,如PepO2,PepO3和PepF可以将苦味肽水解[76]。

另外,可用γ-谷氨酰转移酶(GGT)将苦味氨基酸如Phe、Val、Leu和His转变成具有鲜味的γ-谷酰基衍生物。此方法不仅可以降低苦味氨基酸的浓度,而且产生的具有kokumi味或鲜味的γ-谷酰基肽又可以抑制苦味,从多个方面提高食品的风味,是值得推广的好方法[77]。

4 结论与展望

食品的味觉感受是食品质量的重要部分。本文着重对苦味信号的传递机制、苦味肽的结构特征和消除/降低苦味肽的方法等方面进行综述。苦味肽进一步的研究方向可以有一下几个领域:a苦味肽的苦味机制和生理信号传导机制;b味觉与味觉相互关系的机制;c苦味肽的呈味科学评价体系;d苦味的降低或者消除的方法;e苦味肽生物信息数据库建立和分析研究;f用多肽组学的方法研究苦呈味肽变化规律及机制;g苦味肽其他生物活性研究。

[1]张铭霞,陈思羽,李露,等. 食品中呈味肽类组分研究进展[J]. 中国食品学报,2016,16(2):209-217.

[2]Born S,Levit A,Niv M Y,et al. The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands[J]. The Journal of Neuroscience:the Official Journal of the Society for Neuroscience,2013,33(1):201-213.

[3]Barlow L A,Klein O D. Developing and regenerating a sense of taste[J]. Current Topics in Developmental Biology,2015,111:401-419.

[4]Lindemann B. Receptors and transduction in taste[J]. Nature,2001,413(6852):219-225.

[5]Amino Y,Nakazawa M,Kaneko M,et al. Structure-CaSR-activity relation of kokumi gamma-Glutamyl peptides[J]. Chemical and Pharmaceutical Bulletin,2016,64(8):1181-1189.

[6]Tucker R M,Mattes R D,Running C A. Mechanisms and effects of "fat taste" in humans[J]. Biofactors,2014,40(3):313-326.

[7]Omur-Ozbek P,Dietrich A M,Duncan S E,et al. Role of lipid oxidation,chelating agents,and antioxidants in metallic flavor development in the oral cavity[J]. Journal of Agricultural and Food Chemistry,2012,60(9):2274-2280.

[8]Iwaniak A,Minkiewicz P,Darewicz M,et al. Food protein-originating peptides as tastants-physiological,technological,sensory,and bioinformatic approaches[J]. Food Research International,2016,89(Pt 1):27-38.

[9]Roper S D,Chaudhari N. Taste buds:cells,signals and synapses[J]. Nature Reviews Neuroscience,2017,18(8):485-497.

[10]Solms J. The taste of amino acids,peptides and proteins[J]. Internationale Zeitschrift Für Vitaminforschung.internătional Journal of Vitamin Research[J].Journal International De Vitaminologie,1969,39(3):320-322.

[11]Udenigwe C C,Aluko R E. Food protein-derived bioactive peptides:production,processing,and potential health benefits[J]. Journal of Food Science,2012,77(1):11-24.

[12]Maehashi K,Huang L. Bitter peptides and bitter taste receptors[J]. Cellular & Molecular Life Sciences Cmls,2009,66(10):1661-1671.

[13]Cao X,Zhou X,Cao Y,et al. Expression of NUCB2/nesfatin-1 in the taste buds of rats[J]. Endocrine Journal,2016,63(1):37-45.

[14]Roper S D. Parallel processing in mammalian taste buds?[J]. Physiology & Behavior,2009,97(5):604-612.

[15]Sidhu C,Jaggupilli A,Chelikani P,et al. Regulation of Rac1 GTPase activity by quinine through G-protein and bitter taste receptor T2R4[J]. Molecular and Cellular Biochemistry,2017,426(1-2):129-136.

[16]Kokabu S,Lowery J W,Toyono T,et al. On the emerging role of the taste receptor type 1(T1R)family of nutrient-sensors in the musculoskeletal system[J]. Molecules,2017,22(3):469-472.

[17]Kim M J,Son H J,Kim Y,et al. Umami-bitter interactions:the suppression of bitterness by umami peptides via human bitter taste receptor[J]. Biochemical and Biophysical Research Communications,2015,456(2):586-590.

[18]Matsuo R. Role of saliva in the maintenance of taste sensitivity[J]. Critical Reviews in Oral Biology and Medicine,2000,11(2):216-229.

[19]王丽华,王金鹏,金征宇,等. 呈味肽的风味及调控[J]. 食品与发酵工业,2014,40(6):104-109.

[20]曹英东,李方方,张勇,等. 苦味受体的生物学特征、信号转导机制及苦味剂和苦味抑制剂对苦味受体的影响[J]. 动物营养学报,2017,29(3):769-775.

[21]Kuhn C,Bufe B,Batram C,et al. Oligomerization of TAS2R bitter taste receptors[J]. Chemical Senses,2010,35(5):395-406.

[22]Meyerhof W,Batram C,Kuhn C,et al. The molecular receptive ranges of human TAS2R bitter taste receptors[J]. Chemical Senses,2010,35(2):157-170.

[23]Lossow K,Hubner S,Roudnitzky N,et al. Comprehensive analysis of mouse bitter taste receptors reveals different molecular receptive ranges for orthologous receptors in mice and humans[J].The Journal of Biological Chemistry,2016,291(29):15358-15377.

[24]Huang L,Shanker Y G,Dubauskaite J,et al. Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium[J]. Nature Neuroscience,1999,2(12):1055-1062.

[25]Tizzano M,Dvoryanchikov G,Barrows J K,et al. Expression of Galpha14 in sweet-transducing taste cells of the posterior tongue[J]. BMC Neuroscience,2008,9(1):1-15.

[26]McLaughlin S K,McKinnon P J,Margolskee R F. Gustducin is a taste-cell-specific G protein closely related to the transducins[J]. Nature,1992,357(6379):563-569.

[27]Clapp T R,Trubey K R,Vandenbeuch A,et al. Tonic activity of Galpha-gustducin regulates taste cell responsivity[J]. FEBS Letters,2008,582(27):3783-3787.

[28]Perez C A,Huang L,Rong M,et al. A transient receptor potential channel expressed in taste receptor cells[J]. Nature Neuroscience,2002,5(11):1169-1176.

[29]Zhang Z,Zhao Z,Margolskee R,et al. The transduction channel TRPM5 is gated by intracellular calcium in taste cells[J].The Journal of Neuroscience,2007,27(21):5777-5786.

[30]Liu D,Liman E R. Intracellular Ca2+and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5[J]. Proceedings of the National Academy of Sciences of the United States of America,2003,100(25):15160-15165.

[31]Fiat A M,Jolles P. Caseins of various origins and biologically active casein peptides and oligosaccharides:structural and physiological aspects[J]. Molecular and Cellular Biochemistry,1989,87(1):5-30.

[32]Raikos V,Dassios T. Health-promoting properties of bioactive peptides derived from milk proteins in infant food:a review[J]. Dairy Science and Technology,2014,94(2):91-101.

[33]Yoshikawa M,Fujita H,Matoba N,et al. Bioactive peptides derived from food proteins preventing lifestyle-related diseases[J]. Biofactors,2000,12(1-4):143-149.

[34]Gordon D F,Jr,Speck M L. Bitter peptide isolated from milk cultures of streptococcus cremoris[J]. Applied Microbiology,1965,13(4):537-542.

[35]Upadhyaya J,Pydi S P,Singh N,et al. Bitter taste receptor T2R1 is activated by dipeptides and tripeptides[J]. Biochemical and Biophysical Research Communications,2010,398(2):331-335.

[36]Song S,Li S,Fan L,et al. A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction[J]. Food Chemistry,2016,208:81-88.

[37]Cheung I W,LiChan E C. Application of taste sensing system for characterisation of enzymatic hydrolysates from shrimp processing by-products[J]. Food Chemistry,2014,145:1076-1085.

[38]Vijaykrishnaraj M,Roopa B S,Prabhasankar P. Preparation of gluten free bread enriched with green mussel(Pernacanaliculus)protein hydrolysates and characterization of peptides responsible for mussel flavour[J]. Food Chemistry,2016,211:715-725.

[39]Maehashi K,Matsuzaki M,Yamamoto Y,et al. Isolation of peptides from an enzymatic hydrolysate of food proteins and characterization of their taste properties[J]. Bioscience,Biotechnology,and Biochemistry,1999,63(3):555-564.

[40]Schindler A,Dunkel A,Stahler F,et al. Discovery of salt taste enhancing arginyl dipeptides in protein digests and fermented fish sauces by means of a sensomics approach[J]. Journal of Agricultural and Food Chemistry,2011,59(23):12578-12588.

[41]Humiski L M,Aluko R E. Physicochemical and bitterness properties of enzymatic pea protein hydrolysates[J]. Journal of Food Science,2007,72(8):605-611.

[42]Saha B C,Hayashi K. Debittering of protein hydrolyzates[J]. Biotechnology Advances,2001,19(5):355-370.

[43]Wang L,Niu Q,Hui Y,et al. Assessment of taste attributes of peanut meal enzymatic-hydrolysis hydrolysates using an electronic tongue[J]. Sensors(Basel),2015,15(5):11169-11188.

[44]Nikolaev I V,Sforza S,Lambertini F,et al. Biocatalytic conversion of poultry processing leftovers:Optimization of hydrolytic conditions and peptide hydrolysate characterization[J]. Food Chemistry,2016,197(Pt A):611-621.

[45]Adler-Nissen J. Enzymatic hydrolysis of soy protein for nutritional fortification of low pH food[J]. Ann Nutr Aliment,1978,32(2-3):205-216.

[46]Cho M J,Unklesbay N,Hsieh F H,et al. Hydrophobicity of bitter peptides from soy protein hydrolysates[J]. Journal of Agricultural and Food Chemistry,2004,52(19):5895-5901.

[47]Bumberger E,Belitz H D. Bitter taste of enzymic hydrolysates of casein IIsolation,structural and sensorial analysis of peptides from tryptic hydrolysates of beta-casein[J]. Zeitschrift Fur Lebensmittel-Untersuchung und-Forschung,1993,197(1):14-19.

[48]Habibi-Najafi M B,Lee B H. Bitterness in cheese:a review[J]. Critical Reviews in Food Science and Nutrition,1996,36(5):397-411.

[49]Iwaniak A,Minkiewicz P,Darewicz M,et al.,BIOPEP database of sensory peptides and amino acids[J]. Food Research International,2016,85:155-161.

[50]Otagiri K,Miyake I,Ishibashi N,et al.,Studies of bitter peptides from casein hydrolyzate II Syntheses of bitter peptide fragments and analogs of BPIa(Arg-Gly-Pro-Pro-Phe-Ile-Val)from casein hydrolyzate[J]. Bulletin of the Chemical Society of Japan,2006,56(4):1116-1119.

[51]苗晓丹,刘源,仇春泱,等. 呈味肽构效关系研究进展[J].食品工业科技,2014,35(6):357-362.

[52]王知非,林璐,孙伟峰,等. 苦味肽和苦味受体研究进展[J]. 中国调味品,2016,41(9):152-156.

[53]Kim H O,Li-Chan E C. Quantitative structure-activity relationship study of bitter peptides[J]. Journal of Agricultural and Food Chemistry,2006,54(26):10102-10111.

[54]Ishibashi N,Kubo T,Chino M,et al.,Taste of proline-containing peptides[J]. Bioscience Biotechnology & Biochemistry,1973,52(1):95-98.

[55]段婷婷. 含氨基酸结构的浓厚感物质及苦味抑制剂研究[D]. 武汉:华中农业大学,2015.

[56]Hashizume K,Ito T,Shimohashi M,et al. Taste-guided fractionation and instrumental analysis of hydrophobic compounds in sake[J]. Bioscience,Biotechnology,and Biochemistry,2012,76(7):1291-1295.

[57]Wang W,De Mejia E G. A new frontier in soy bioactive peptides that may prevent age-related chronic diseases[J]. Comprehensive Reviews in Food Science and Food Safety,2005,4(4):63-78.

[58]Ney K H. Voraussage der bitterkeit von peptiden aus deren aminosäurezu-sammensetzung[J]. European Food Research & Technology,1971,147(2):64-68.

[59]Maehashi K,Matano M,Wang H,et al. Bitter peptides activate hTAS2Rs,the human bitter receptors[J]. Biochemical and Biophysical Research Communications,2008,365(4):851-855.

[60]Lemieux L,E Simard R. Bitter flavour in dairy products II:A review of bitter peptides from caseins:their formation,isolation and identification,structure masking and inhibition[J]. Dairy Science & Technology,1992,72(4):335-385.

[61]Kim I M R,Kawamura Y,Lee C H. Isolation and identification of bitter peptides of tryptic hydrolysate of soybean 11s glycinin by reverse-phase high-performance liquid chromatography[J]. Journal of Food Science,2003,68(8):2416-2422.

[62]Sharma P,Yi R,Nayak A P,et al. Bitter taste receptor agonists mitigate features of allergic asthma in mice[J]. Scientific Reports,2017,7:46166.

[63]Pydi S P,Sobotkiewicz T,Billakanti R,et al. Amino acid derivatives as bitter taste receptor(T2R)blockers[J]. The Journal of Biological Chemistry,2014,289(36):25054-25066.

[64]Adler-Nissen J. Control of the proteolytic reaction and of the level of bitterness in protein hydrolysis processes[J]. Journal of Chemical Technology and Biotechnology Biotechnology,1984,34(3):215-222.

[65]Zhuang M,Zhao M,Lin L,et al. Macroporous resin purification of peptides with umami taste from soy sauce[J]. Food Chemistry,2016,190:338-344.

[66]Lalasidis G. Four new methods of debittering protein hydrolysates and a fraction of hydrolysates with high content of essential amino acids[J]. Ann Nutr Aliment,1978,32(2-3):709-723.

[67]Berardo A,Devreese B,De Maere H,et al. Actin proteolysis during ripening of dry fermented sausages at different pH values[J]. Food Chemistry,2017,221:1322-1332.

[68]赵娜. 苦味评价细胞钙成像模型的构建[D]. 南京:南京大学,2015.

[69]Tamura M,Mori N,Miyoshi T,et al. Practical debittering using model peptides and related compounds[J]. Agricultural and Biological Chemistry,1990,54(1):41-51.

[70]Tokita K,Jr B J. Sweet-bitter and umami-bitter taste interactions in single parabrachial neurons in C57BL/6J mice[J].Journal of Neurophysiology,2012,108(8):2179-2190.

[71]Zhang Y,Venkitasamy C,Pan Z,et al. Novel umami ingredients:Umami peptides and their taste[J]. Journal of Food Science,2017,82(1):16-23.

[72]Yamashita M,Arai S,Tsai S J,et al. Plastein reaction as a method for enhancing the sulfur-containing amino acid level of soybean protein[J]. Journal of Agricultural and Food Chemistry,1971,19(6):1151-1154.

[73]Tchorbanov B,Marinova M,Grozeva L. Debittering of protein hydrolysates by lactobacillus lbl-4 aminopeptidase[J]. Enzyme Research,2011(10):538676.

[74]Fu J,Li L,Yang X Q. Specificity of carboxypeptidases from actinomucor elegans and their debittering effect on soybean protein hydrolysates[J]. Applied Biochemistry and Biotechnology,2011,165(5-6):1201-1210.

[75]Broadbent J R,Barnes M,Brennand C,et al. Contribution ofLactococcuslactiscell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat Cheddar cheese[J]. Applied and Environmental Microbiology,2002,68(4):1778-1785.

[76]Sridhar V R,Hughes J E,Welker D L,et al. Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides[J]. Applied and Environmental Microbiology,2005,71(6):3025-3032.

[77]Suzuki H,Kato K,Kumagai H. Enzymatic synthesis of gamma-glutamylvaline to improve the bitter taste of valine[J]. Journal of Agricultural & Food Chemistry,2004,52(3):577-580.