畢繼才，崔震昆，張令文，林澤源，江海洋，莫海珍

(河南科技學(xué)院食品學(xué)院,河南新鄉(xiāng) 453003)

味覺是哺乳動(dòng)物尤其是人類的重要生理感覺之一[1]。哺乳動(dòng)物在選擇自己的食物源時(shí),食物的味道是一個(gè)關(guān)鍵篩選條件[2]。典型哺乳動(dòng)物的味蕾是由50～100個(gè)緊湊排列的味覺受體細(xì)胞(taste-receptor cells,TRCs)組成,每個(gè)成熟味覺受體細(xì)胞的壽命只有5～20 d,新的味覺受體細(xì)胞是通過味覺干細(xì)胞的不斷分化更新產(chǎn)生[3]。成熟味覺受體細(xì)胞可以感受到五種基本味型:苦味、咸味、鮮味、甜味和酸味[4]。有學(xué)者認(rèn)為最新研究發(fā)現(xiàn)一些新的味道應(yīng)該考慮進(jìn)去,如醇厚(kokumi)味[5]、脂肪味[6]和金屬味[7]。簡言之,味覺的傳遞過程就是食品中的風(fēng)味化學(xué)物質(zhì)激活了存在于味蕾中的一系列特異化味覺受體細(xì)胞,味覺受體細(xì)胞將這些刺激信號(hào)通過神經(jīng)傳導(dǎo)的方式呈遞到神經(jīng)中樞系統(tǒng)[8-9]。

食品中的風(fēng)味物質(zhì)種類很多,其中由蛋白質(zhì)水解產(chǎn)生的呈味肽(taste peptide)是由Juerg Solms在1969年第一次正式系統(tǒng)報(bào)道的[10]。呈味肽是一些對(duì)食品風(fēng)味有貢獻(xiàn)的蛋白質(zhì)序列片段,如多肽、二肽等。呈味肽不僅具有多種味型,如酸、甜、苦、咸等,還具有諸多生物活性功能,如降低血壓、抗氧化、抗菌、免疫調(diào)節(jié)、降低脂肪等[11]。其中,苦味肽往往是消費(fèi)者厭惡的一類呈味肽,它們通常在發(fā)酵、陳化和酶水解的食品產(chǎn)物中產(chǎn)生[12]。

本文系統(tǒng)地梳理了苦味的信號(hào)遞呈途徑,并對(duì)苦味肽的結(jié)構(gòu)特征和脫苦的方法等研究成果的研究進(jìn)展進(jìn)行了綜述。

1 味覺的生理學(xué)基礎(chǔ)和苦味傳遞機(jī)制

1.1 味蕾

舌頭是第一個(gè)與食品接觸并感知其味道的器官。味蕾(taste bud)是簇狀柱形感覺細(xì)胞,不均勻地分布在舌背部表面的乳突(papillae)結(jié)構(gòu)表面[13]。味覺細(xì)胞按照功能不同可以分為四種,分別是暗細(xì)胞(I型),亮細(xì)胞(Ⅱ型),中間細(xì)胞(Ⅲ型)和基細(xì)胞(Ⅳ型)[9,14](如表1)。其中Ⅱ型細(xì)胞是最為主要的味覺識(shí)別和傳導(dǎo)細(xì)胞。

表1 不同類型味蕾細(xì)胞及其功能Table 1 Different types of taste buds cell and their functions

大多數(shù)Ⅱ型細(xì)胞均含有細(xì)胞信號(hào)傳導(dǎo)中重要的擁有七次跨膜結(jié)構(gòu)域的G蛋白偶聯(lián)受體(G protein-coupled receptor,GPCR),包含兩種味覺受體,即1型味覺受體(T1R)和2型味覺受體(T2R),每種味覺受體相應(yīng)地僅響應(yīng)于一種味道(例如,甜味或苦味,但不能兩者兼有)。其中T2R特異性地結(jié)合苦味[15],而T1R可以分為三個(gè)亞基:T1R1,T1R2和T1R3,通?？梢栽谖独偌?xì)胞中共表達(dá)[16]。人受體T1R2和T1R3識(shí)別天然或合成的甜味劑,而T1R1和T1R3對(duì)L-谷氨酸等鮮味物質(zhì)進(jìn)行識(shí)別[17]。另外,唾液在味覺呈遞過程中的作用不容忽視。唾液可以為促味劑提供溶劑,將促味劑擴(kuò)散到受體細(xì)胞的功能位點(diǎn)。此外,唾液可以刺激味覺感受細(xì)胞(TRCs)從而影響味覺。Matsuo曾設(shè)想用人造唾液來幫助治療味覺功能障礙的療法[18]。

1.2 苦味受體

苦味、甜味、鮮味受體同屬于G蛋白偶聯(lián)受體大家族[19],分別被T2Rs受體、T1R2/T1R3異源二聚體和T1R1/T1R3異源二聚體所識(shí)別[20]。與T1Rs不同,苦味受體T2Rs一般認(rèn)為是單聚體,但最新研究表明，他們同樣也可形成同源二聚體和異源二聚體[21]。嚙齒動(dòng)物中有40多個(gè)T2R家族成員,人類有25條功能基因編碼T2Rs,但編碼T1R的基因只有3條[22]。人類和嚙齒動(dòng)物單獨(dú)的T2R僅僅可以辨別一種或幾種苦味化合物,如T2R3僅能感知一種化合物(94種不同的天然和合成化合物進(jìn)行測試),而T2R14至少可以感知33種化合物[23]。而單一的苦味物質(zhì)卻經(jīng)常能夠活化多種不同的T2Rs,如奎寧能夠活化九種不同的人類T2Rs受體[22]?？辔段镔|(zhì)與受體之間相互作用產(chǎn)生第二信號(hào),活化下游的一系列生理反應(yīng)。

1.3 苦味傳遞途徑

甜味、鮮味和苦味受體信號(hào)傳遞途徑盡管具有多樣性,但T1R和T2Rs卻擁有共同的細(xì)胞內(nèi)信號(hào)通路。不同的味覺受體GPCR均能與異源三聚體G蛋白偶聯(lián),包括Gβ3和Gγ13、Gαgus(也稱為味蛋白)、Gα14和Gαi[24-26]。早期學(xué)者認(rèn)為Gα亞基可以激活cAMP信號(hào),但最近研究表明，它主要是調(diào)節(jié)Gβγ亞基的作用。通過蛋白激酶A激活并維持信號(hào)蛋白處于響應(yīng)狀態(tài),cAMP也具有長期調(diào)節(jié)信號(hào)蛋白的功能[27]。當(dāng)T1Rs和T2Rs被呈味物質(zhì)激活時(shí),Gβγ二聚體被釋放,激發(fā)磷脂酶Cβ2以調(diào)節(jié)細(xì)胞內(nèi)Ca2+含量[24]。胞質(zhì)Ca2+水平升高導(dǎo)致瞬時(shí)受體電位陽離子通道TRPM5開放,TRPM5開放可有效地使味細(xì)胞的陽離子通道去極化[28-30]。目前,關(guān)于味道傳遞途徑中的許多細(xì)節(jié)問題還未有明確研究結(jié)論。

2 苦味肽來源和結(jié)構(gòu)特征

2.1 苦味肽來源

一些食品中的苦味被認(rèn)為是食品的典型味覺特征?？辔兑话愫茈y被人們接受,但在特定食品如啤酒、咖啡或干酪等的感官標(biāo)準(zhǔn)中卻是非常重要的呈味特性?？辔峨膬H是眾多呈苦味物質(zhì)中的一類,苦味肽除了能夠呈現(xiàn)出苦味之外,有些苦味肽還具有一定的生理功能,起到預(yù)防慢性疾病的作用[31-33]。Gordon[12]和Speck[34]等在1965年第一次報(bào)道了在乳清培養(yǎng)發(fā)酵過程中產(chǎn)生具有苦味的肽?？辔峨陌ㄓ卸?、三肽、六肽、八肽乃至幾十個(gè)氨基酸組成的肽,廣泛存在于天然食品和發(fā)酵食品中,尤其在蛋白質(zhì)含量豐富的食品發(fā)酵過程中會(huì)產(chǎn)生大量的苦味肽[35]。蛋白質(zhì)水解產(chǎn)物的味道可以因?yàn)榈鞍踪|(zhì)種類,水解條件和蛋白水解酶應(yīng)用的不同而發(fā)生變化[12]。

蛋白來源的苦味肽產(chǎn)生的途徑主要是用酶法水解的方式,實(shí)際中常用的蛋白質(zhì)水解的酶有三類:植物蛋白酶如木瓜蛋白酶[36]、菠蘿蛋白酶[37]等，動(dòng)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堿性蛋白酶[42]、風(fēng)味蛋白酶[43]、中性蛋白酶[42]、復(fù)合蛋白酶[43]、枯草桿菌蛋白酶[44]。目前用的較多的蛋白酶有胰蛋白酶、木瓜蛋白酶、中性蛋白酶和堿性蛋白酶。如用胃蛋白酶水解大豆蛋白可以獲得10種苦味肽,分別是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獲得三種苦味肽,分別是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]。利用枯草桿菌蛋白酶水解酪蛋白獲得的苦味肽也是三種,分別是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,隨著呈味肽發(fā)掘數(shù)量的猛增,研究單獨(dú)肽與其生物功能的關(guān)聯(lián)性十分耗時(shí)。因此,生物信息學(xué)數(shù)據(jù)庫對(duì)于發(fā)掘更多的呈味肽十分有用。如BIOPEP數(shù)據(jù)庫含有大量感官肽和氨基酸的信息,可為研究者提供一些感官肽的生物活性最新信息[49]。

2.2 苦味肽結(jié)構(gòu)特征

苦味肽是目前最容易描述的一類味覺感官肽,對(duì)苦味肽的科學(xué)評(píng)價(jià)和測量主要利用傅里葉轉(zhuǎn)換拉曼光譜技術(shù)進(jìn)行分析,研究確定苦味肽的相對(duì)苦度(Rcaf)。相對(duì)苦度Rcaf指是以1 mmol/L咖啡因溶液的閾值作為標(biāo)準(zhǔn),此時(shí)的相對(duì)苦度Rcaf為1.0[50]。

呈味肽的味型特征與肽的疏水性關(guān)系密切[51],而疏水性又與氨基酸組成、氨基酸序列、肽鏈長度、空間結(jié)構(gòu)等相關(guān)[52]。首先,肽的疏水性能由肽中氨基酸的組分和序列決定,其中脯氨酸(Pro)是主要的苦味肽的貢獻(xiàn)者[53]。多肽結(jié)構(gòu)包含Pro時(shí),可更容易地結(jié)合到T2Rs受體上[54]。此外,當(dāng)肽中出現(xiàn)甘氨酸(Gly),丙氨酸(Ala),纈氨酸(Val),亮氨酸(Leu),蘇氨酸(Tyr)和苯丙氨酸(Phe)時(shí)也可以增強(qiáng)肽的疏水性,從而影響與苦味受體的結(jié)合[55]。此外,肽的分子量、立體參數(shù)和空間結(jié)構(gòu)等同樣是苦味的影響因素[56]。如Wang等報(bào)道顯示，中度大小的肽通常比較大或者較小的肽段表現(xiàn)出的苦味更為突出。分子質(zhì)量(MW)在1.9～3.3 kDa之間的肽被稱為是高苦肽段,而較大或者較小分子質(zhì)量的肽段表現(xiàn)出較溫和的苦度[57]。多肽苦味還與肽中是否存在親水性基團(tuán)和堿性氨基酸殘基有關(guān)。有學(xué)者認(rèn)為，多肽中親水性基團(tuán)與疏水性基團(tuán)在空間相距0.3 nm時(shí)便會(huì)產(chǎn)生苦味[47]。

在探究苦味肽的結(jié)構(gòu)與苦味化學(xué)機(jī)理的研究中,學(xué)者們引用了Q值做為評(píng)估苦味的指標(biāo)。Q值表示肽的平均疏水性。Q=∑ΔGi/n,其中ΔGi指將氨基酸鏈(肽)從乙醇溶液中轉(zhuǎn)移到水溶液中所需平均自由能量(以cal/mol表示),n為肽鏈的氨基酸殘基數(shù)[58]。當(dāng)Q值大于1400 cal/mol時(shí)呈現(xiàn)苦味,Q值小于1300 cal/mol時(shí)不呈現(xiàn)苦味,Q值介于1300～1400 cal/mol之間則不能判斷肽是否具有苦味[58]。Q參數(shù)與作為苦味肽組分的氨基酸的疏水性有相關(guān)性,這種相關(guān)性稱為Q定則[12,59]。但Q定則只是經(jīng)驗(yàn)規(guī)律而并不是絕對(duì)的,有些肽如Glu-Val-Leu-Asn序列的Q值為1162.5 cal/mol,它同樣顯示出苦味[60]。Kim等用胰蛋白酶水解大豆11S抗蛋白原獲得的水解產(chǎn)物,水解產(chǎn)物中被描述為明顯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。他們的Q值都在80～1474 cal/mol之間。被描述為適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。他們的Q值是從0(如Glu-Gln-Gly-Gly-Glu-Gln-Gly)到1670 cal/mol(Arg-Pro肽)[61]。這些事實(shí)表明,肽的Q值盡管能提供一些參考信息,但苦味判斷并不能簡單地基于Q值來預(yù)測。值得注意是,血管緊張素轉(zhuǎn)換酶(Angiotensin Converting Enzyme,ACE)活性抑制所需要的結(jié)構(gòu)域與苦味肽的結(jié)構(gòu)相似,因此,很多苦味的二肽表現(xiàn)出ACE活性的抑制作用,如苦味三肽Phe-Phe-Phe[35,62-63]。

3 苦味肽脫苦技術(shù)研究

食品中的苦味往往是人們厭惡的味道,發(fā)酵食品中的苦味影響消費(fèi)者的接受度,制約著發(fā)酵食品的市場化。因此,對(duì)于苦味食品我們通過脫苦技術(shù)改良可以提高它的感官評(píng)定。食品工業(yè)中的脫苦技術(shù)需要考慮以下幾個(gè)因素:是否影響食品組分的活性(如苦味肽的保健功能);是否影響食品的消化率、吸收率和生物利用度;是否能高效地分離純化;是否是最經(jīng)濟(jì)的除去/掩蔽嫌忌味道的技術(shù)等。這里介紹幾種常見的苦味肽脫苦方法:

3.1 選擇分離法

選擇性分離脫苦主要是基于肽理化性質(zhì)的差異性,采用物理吸附、等電點(diǎn)沉淀、溶劑萃取和色譜分離等方法將蛋白水解液中的苦味肽分離出來的方式。如苦味的酪蛋白和明膠蛋白水解液可以用活性炭吸附達(dá)到降低苦味的目的[12]。疏水肽在其等電點(diǎn)(pI)附近時(shí)具有非常低的溶解度,通過將溶液pH調(diào)整到苦味肽的等電點(diǎn)附近,便可將其沉淀去除[64]。另有研究者用大孔樹脂分離制備出醬油中的多肽,結(jié)果表明XAD-16樹脂是吸附多肽最有效的樹脂[65]。另外,通過用仲丁醇、含水乙醇或異丙醇水溶液共沸萃取,使得酶促蛋白質(zhì)水解產(chǎn)物中的苦味肽在醇相中濃縮[66]。隨著現(xiàn)代分離技術(shù)的發(fā)展,用色譜的方法除去和制備呈味肽逐漸普及,如在發(fā)酵香腸中,肌原纖維蛋白發(fā)生強(qiáng)烈的蛋白水解,產(chǎn)生小的肽和游離氨基酸,用質(zhì)譜聯(lián)用的液相色譜法(Liquid Chromatograph Mass Spectrometer,簡稱LC-MS)進(jìn)行多肽的分離和鑒定,同樣可以進(jìn)行苦味肽的分離[67]。

3.2 掩蓋法

掩蓋法能夠有效地掩蓋食品感官味道,如在苦味食品中添加脫脂牛奶,大豆酪蛋白和酪蛋白水解產(chǎn)物可以掩蓋其原本的感官味道[68-69]。谷氨酸鹽和腺苷酸或者5′-核苷酸鈉鹽以及具有鮮味的多肽,如αGlu-Asp,αGlu-Glu,αGlu-Ser和αGlu-Glu-Glu同樣可以掩蓋苦味肽的苦味[70-71]。另外,味覺物質(zhì)的濃度和不同的味道刺激可以受到味道增強(qiáng)子和/或抑制子的作用,如低濃度時(shí),咸-酸和酸-苦可以互相增強(qiáng),但是在咸味環(huán)境下則可抑制苦味[17]。Kim是第一個(gè)發(fā)現(xiàn)并報(bào)道苦味和鮮味肽在受體水平相互作用的人。他們用五種大豆來源的鮮味肽Glu-Asp,Glu-Glu,Glu-Ser,Asp-Glu-Ser,Glu-Gly-Ser分別與具有明顯苦味的苦味劑水楊苷一起進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果顯示，五種具有鮮味的大豆鮮味肽(Glu-Asp,Glu-Glu,Glu-Ser,Asp-Glu-Ser和Glu-Gly-Ser)以非競爭性的方式抑制水楊苷誘導(dǎo)細(xì)胞內(nèi)鈣流,細(xì)胞內(nèi)鈣離子水平升高是苦味物質(zhì)結(jié)合苦味受體引起的一個(gè)重要指標(biāo),但對(duì)照組的無味肽Gly-Gly卻未見此結(jié)果[17]。

3.3 酶法

酶法脫苦與其他的脫苦方式相比具有突出的優(yōu)點(diǎn):更高的水解效率、反應(yīng)條件溫和、不破壞食品營養(yǎng)成分、水解過程便于控制等。因此用酶法脫苦是目前國內(nèi)外研究的熱點(diǎn)。Tamura等[69]用羧肽酶對(duì)苦味肽進(jìn)行水解或結(jié)構(gòu)修飾,最終能將苦味肽分子變?yōu)榉强辔冻煞?。Fujimaki[72]利用類蛋白反應(yīng)(即蛋白酶的轉(zhuǎn)肽作用)成功地降低了由胃蛋白酶水解的鱈魚、面筋、小球藻、酵母、大豆等帶來的苦味。Tchorbanov等[73]用食品級(jí)細(xì)胞內(nèi)氨肽酶(Aminopeptidases)可將高Q值的苦味酪蛋白和大豆分離蛋白水解物轉(zhuǎn)變成為低Q值沒有苦味的物質(zhì)。Fu等研究產(chǎn)自雅致放射毛霉(Actinomucorelegans)的羧肽酶(Carboxypeptidase)的酶活特性時(shí)發(fā)現(xiàn)該酶優(yōu)先分解疏水性氨基酸類底物,如Z-Phe-Leu,Z-Phe-Tyr-Leu和Z-Phe-Tyr。該羧肽酶是降低苦味肽的有效工具,在酶法脫苦技術(shù)應(yīng)用領(lǐng)域有廣闊前景[74]。在奶酪脫苦的研究過程中發(fā)現(xiàn),奶酪發(fā)酵初期苦味肽感受度較低,是因?yàn)榘l(fā)酵初期高活性的脫苦肽酶可以高效地降低奶酪中苦味物質(zhì)的含量[75]。另有研究者發(fā)現(xiàn)Lactobacillushelveticus在奶酪發(fā)酵中具有顯著的脫苦效果,主要是由于該菌能夠產(chǎn)生脯氨酰內(nèi)肽酶,如PepO2,PepO3和PepF可以將苦味肽水解[76]。

另外,可用γ-谷氨酰轉(zhuǎn)移酶(GGT)將苦味氨基酸如Phe、Val、Leu和His轉(zhuǎn)變成具有鮮味的γ-谷?；苌?。此方法不僅可以降低苦味氨基酸的濃度,而且產(chǎn)生的具有kokumi味或鮮味的γ-谷酰基肽又可以抑制苦味,從多個(gè)方面提高食品的風(fēng)味,是值得推廣的好方法[77]。

4 結(jié)論與展望

食品的味覺感受是食品質(zhì)量的重要部分。本文著重對(duì)苦味信號(hào)的傳遞機(jī)制、苦味肽的結(jié)構(gòu)特征和消除/降低苦味肽的方法等方面進(jìn)行綜述?？辔峨倪M(jìn)一步的研究方向可以有一下幾個(gè)領(lǐng)域:a苦味肽的苦味機(jī)制和生理信號(hào)傳導(dǎo)機(jī)制;b味覺與味覺相互關(guān)系的機(jī)制;c苦味肽的呈味科學(xué)評(píng)價(jià)體系;d苦味的降低或者消除的方法;e苦味肽生物信息數(shù)據(jù)庫建立和分析研究;f用多肽組學(xué)的方法研究苦呈味肽變化規(guī)律及機(jī)制;g苦味肽其他生物活性研究。

[1]張銘霞,陳思羽,李露,等. 食品中呈味肽類組分研究進(jìn)展[J]. 中國食品學(xué)報(bào),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.intern?tional 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]王麗華,王金鵬,金征宇,等. 呈味肽的風(fēng)味及調(diào)控[J]. 食品與發(fā)酵工業(yè),2014,40(6):104-109.

[20]曹英東,李方方,張勇,等. 苦味受體的生物學(xué)特征、信號(hào)轉(zhuǎn)導(dǎo)機(jī)制及苦味劑和苦味抑制劑對(duì)苦味受體的影響[J]. 動(dòng)物營養(yǎng)學(xué)報(bào),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]苗曉丹,劉源,仇春泱,等. 呈味肽構(gòu)效關(guān)系研究進(jìn)展[J].食品工業(yè)科技,2014,35(6):357-362.

[52]王知非,林璐,孫偉峰,等. 苦味肽和苦味受體研究進(jìn)展[J]. 中國調(diào)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. 含氨基酸結(jié)構(gòu)的濃厚感物質(zhì)及苦味抑制劑研究[D]. 武漢:華中農(nóng)業(yè)大學(xué),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 aminos?urezu-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]趙娜. 苦味評(píng)價(jià)細(xì)胞鈣成像模型的構(gòu)建[D]. 南京:南京大學(xué),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.