influÊncia dos compostos polifenÓlicos no … · influência dos compostos ... a nível sensorial...

INFLUÊNCIA DOS COMPOSTOS POLIFENÓLICOS NO SABOR DOS ALIMENTOS: RELAÇÃO ENTRE A SUA ESTRUTURA E A CAPACIDADE DE INTERAÇÃO COM PROTEÍNAS DA SALIVA E RECETORES DO SABOR

Susana Isabel Pinto T. P. SoaresPrograma Doutoral em QuímicaDepartamento de Química e Bioquímica2012

Orientador Victor de Freitas, Professor Catedrático, Faculdade de Ciências da Universidade do Porto

“A ciência nos traz conhecimento; a vida, sabedoria" Will Durant

“Não há nada que não se consiga com a força de vontade,…" Cícero

FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de

interação com proteínas da saliva e recetores do sabor

iii

AGRADECIMENTOS

Em primeiro lugar gostava de agradecer à Fundação Para a Ciência e Tecnologia

(FCT) pelo financiamento deste trabalho na forma de uma bolsa de Doutoramento

(SFRH/BD/41946/2007) sem a qual a realização deste trabalho seria impossível.

Gostava de agradecer ao Departamento de Química e Bioquímica da Faculdade de

Ciências da Universidade do Porto e à Linha de Investigação 2 do Centro de

Investigação em Química (CIQ) pelos diversos apoios logísticos e financeiros

concedidos.

Gostava de agradecer ao meu orientador Professor Doutor Victor de Freitas sem o

qual este trabalho não seria possível. Todas as “discussões” científicas, todo o

interesse, disponibilidade e compreensão foram fundamentais. Gostava também de

agradecer ao Professor Doutor Nuno Mateus, “co-orientador”. Sempre disponível,

atento, interessado e principalmente com um humor característico. Para além da

excelente orientação científica, os professores foram excelentes “companheiros” e

conselheiros nas fases menos boas desta etapa e também a nível pessoal, e não há

palavras para expressar esse reconhecimento.

Gostava de agradecer a alguns colegas investigadores e professores do

Departamento de Química e Bioquímica, nomeadamente à Isabel Sousa pela

disponibilidade e ajuda na utilização do DLS, à Dra. Zélia pela ajuda técnica nas

análises de LC-MS e à Professora Doutora Maria José Feio pela ajuda inicial na

realização da electroforese.

Gostava de agradecer a todas as pessoas que trabalharam e conviveram comigo

diariamente na realização deste trabalho. Ana Luísa, André, Iva, Joana Azevedo,

Joana Oliveira, Luís, Natércia e Rui. Sem dúvida que o excelente ambiente deste

laboratório é uma das mais-valias e fundamental para o sucesso científico de todas as

pessoas que por aqui passam. Espero que assim continue!

Já agora obrigada pelas dádivas de saliva e pelo “grande” sacrifício de estarem 1h

sem comer e beber…

Uma pequena palavra de apreço também ao Frederico Nave. Uma pessoa única e um

poço de conhecimento. Obrigada pelas pequenas discussões científicas.

Apesar de mais recentes na equipa, mas não menos importantes, a Natércia Brás e a

Virgínia. Obrigada pela boa disposição, companheirismo e disponibilidade.

iv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

Gostava de agradecer também à minha família, em particular aos meus queridos pais,

marido, irmão, sobrinhos e cunhada. Somos poucos, na verdade cada vez menos,

mas somos felizes e unidos. Vocês têm sido o meu corrimão na vida; quando a subida

está mais difícil tenho tido sempre em quem me apoiar! Não sei como será no dia em

que faltarem. Adoro-vos.

Uma palavra em especial ao meu marido porque é quem me atura todos os dias. Há

fases menos boas e sem dúvida que o que temos passado não tem sido fácil. A vida já

é complicada e cheia de contratempos e penso que conservar o bom humor é um

segredo de sobrevivência. E agradeço-te por isso, porque apesar de tudo estás

sempre bem disposto, tentas por-me bem disposta e dia-a-dia vamos caminhando

juntos e percorrendo a estrada da nossa vida. Obrigada pelas vezes em que levas ao

colo nessa estrada…

Gostava de agradecer a alguns amigos muito especiais. Debi, Ligia, Ricardo Lopes e

Raquel. Um bocado cliché e lamechas mas sem dúvida que os amigos, e vocês em

particular, são a família que me permitiram escolher. Nem toda a gente tem a sorte de

ser protegido e acarinhado pela família ou amigos sinceros, mas eu tive essa sorte.

Sem Deus não há vida, sem família não há base e sem amigos não há mundo

colorido. Obrigada por terem estado, por estarem e por “irem estar” presentes em

todas as fases cruciais da minha vida. Por todas as maluqueiras que já fizemos e as

que ainda vamos fazer. Sem dúvida que vocês são uma parte de mim.

Uma palavra especial para a Raquelita porque és sem dúvida a irmã que nunca tive e

estás no meu coração.

Gostava de agradecer a outras pessoas por também serem uma parte importante da

minha vida e pelos mais de 10 anos de amizade (em alguns casos já lá vão 20 anos!!)

e aventuras. Liliana, Carlos, Zé Manel, Cátia, Manuel André, Ricardo (Baroni) o que

nós temos é raro e precioso.

Gostava de agradecer a mais um grupo de amigos Iva, Ricardo, Joana (Pá), António

(Toli), Vanessa, Manuel, Rita (Mocas), João (Cucus) e Paulo (Xeno) por partilharem a

vossa vida comigo e fazerem parte da minha também.

Gostava de agradecer à Susie Kohl e Sophie Thallman pela enorme hospitalidade,

disponibilidade, simpatia e amizade aquando da minha estadia na Alemanhã. Apesar

de ter sido pouco tempo e corresponder a apenas uma pequena parte deste trabalho,

foi uma experiência muito enriquecedora quer a nível científico como a nível pessoal.

Sem dúvida que vocês deixaram saudade dos muitos bons momentos que passamos.

FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade

de interação com proteínas da saliva e recetores do sabor

v

Gostava de agradecer ao Professor Doutor Wolfgang Meyerhof do Deutsches Institut

für Ernährungsforschung Potsdam – Rehbrücke (DIFE) pela oportunidade que me

proporcionou de ir para o seu laboratório e crescer como investigadora. Uma pessoa

sempre extremamente disponível, interessada e atenciosa.

Gostava de agradecer ao Hugo Osório (IPATIMUP) pela incansável ajuda na

identificação das proteínas salivares. Uma excelente pessoa, sempre disponível,

interessada, muito trabalhadora e experiente. Gostava também de agradecer ao Rui

Vitorino (Universidade de Aveiro) pela valiosa e crucial ajuda na identificação das

proteínas salivares. Sempre disponível e interessado.

Gostava de agradecer ao Professor Massimo Castagnola pela valiosa contribuição na

interpretação e identificação das proteínas salivares por ESI-MS. Mostrou-se uma

pessoa sempre disponível e extremamente interessada.

vi FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a

capacidade de interação com proteínas da saliva e recetores do sabor

vii

RESUMO

Os polifenóis são metabolitos secundários das plantas e, por isso, estão

presentes em alimentos e bebidas de origem vegetal (e.g. vinho tinto, cerveja,

chá, sumos de frutas, etc). Estes compostos têm recebido muita atenção nos

últimos anos devido às suas propriedades biológicas (antioxidantes,

anticancerígena, etc) e às propriedades organoléticas que conferem aos

alimentos. Os taninos são um grupo específico de polifenóis que têm a

capacidade de interagir com as proteínas, nomeadamente as proteínas

salivares. Esta interação é importante não só no contexto biológico, mas também

a nível sensorial uma vez que está na origem da sensação da adstringência nos

alimentos. De facto, é aceite pela comunidade científica que a adstringência se

deve à complexação e/ou precipitação das proteínas salivares ricas em prolina

(PRPs) pelos taninos.

Enquanto a adstringência é uma sensação tátil percecionada na cavidade oral, o

amargor é um gosto resultante da interação de compostos com os recetores de

sabor amargo. Alguns compostos polifenólicos de baixo peso molecular são

amargos.

Este trabalho tem como objetivo global a compreensão das propriedades

sensoriais associadas a polifenóis (amargor e a adstringência), e de que modo é

que diferentes carboidratos (usados na indústria alimentar) influenciam a

adstringência. Para isso, este trabalho focou-se em determinar: (a), as principais

famílias de proteínas salivares que apresentam mais afinidade para interagirem

com os taninos numa solução modelo e diretamente no vinho tinto; (b), como é

que o enriquecimento de um vinho tinto em taninos influencia a perceção da

adstringência; (c), como é que as caraterísticas (solubilidade e tamanho) dos

complexos formados entre as diferentes famílias de proteínas salivares e dois

tipos de taninos (condensados e hidrolisáveis) podem influenciar o

desenvolvimento da adstringência (d), de que modo certos carboidratos

comerciais (goma arábica, β-ciclodextrina, pectina e ácido poligalacturónico)

inibem a interação taninos/proteínas, particularmente com as proteínas salivares

(e), quais os recetores de sabor amargo que são ativados por alguns polifenóis

vulgarmente presentes em alimentos de origem vegetal e produtos derivados.

As proteínas salivares presentes na saliva humana foram caraterizadas por uma

abordagem proteómica (HPLC-DAD, ESI-MS, SDS-PAGE e MALDI-TOF). Estas

técnicas foram então adaptadas para estudar a afinidade entre as diferentes

viii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

famílias de proteínas salivares e os taninos condensados. Os resultados

desmonstraram que os taninos condensados interagem inicialmente com as

PRPs acídicas e estaterina e só depois com as histatinas, PRPs glicosiladas e

PRPs básicas.

A análise da composição das proteínas salivares por HPLC após interação direta

com o vinho tinto, permitiu conhecer alguns fenómenos relacionados com a

adstringência de vinhos tintos enriquecidos em taninos condensados por

comparação com a análise sensorial dos mesmos vinhos. Os resultados

demonstraram que no caso de vinhos com concentrações baixas de taninos, a

complexação/precipitação de PRPs acídicas e estaterina está correlacionada

com a intensidade da adstringência. No entanto, para vinhos com concentrações

maiores, a adstringência parece correlacionar-se com a interação dos taninos

com as PRPs glicosiladas. Pela primeira vez, surgiram evidências de que as

diferentes proteínas salivares estão envolvidas em diferentes fases do fenómeno

fisiológico associado à perceção da adstringência.

Posteriormente, pretendeu-se comparar a interação das proteínas salivares com

um tanino hidrolisável (PGG) vs. tanino condensado (procianidina trimérica). Os

resultados obtidos por HPLC e dynamic light scattering (DLS) mostraram que

ambos os taninos interagem sobretudo com as proteínas aPRPs e estaterina

formando complexos insolúveis e solúveis. Por outro lado, as bPRPs interagem

pouco com a procianidina trimérica e as gPRPs só complexam com a PGG. Em

geral, a PGG apresentou mais tendência para a formação de complexos

insolúveis enquanto a procianidina trimérica mostrou mais tendência para a

formação de complexos solúveis (exceto com a estaterina). Os resultados

obtidos apresentam importantes evidências de que diferentes taninos e

proteínas salivares podem influenciar e ter mecanismos diferentes na sua

interação e consequentemente no desenvolvimento da adstringência.

O efeito dos carboidratos na interação taninos/proteínas foi estudado usando

dois modelos de proteínas diferentes: taninos condensados/α-amilase e taninos

condensados/proteínas salivares. No primeiro estudo, foram usadas as técnicas

de extinção da fluorescência, nefelometria e DLS; no segundo modelo,

monitorizou-se por HPLC as proteínas salivares que permaneciam solúveis na

presença dos taninos após a adição dos carboidratos e analisaram-se por SDS-

PAGE os precipitados formados.

Globalmente os resultados mostraram que os carboidratos estudados (goma

arábica, β-ciclodextrina, pectina e ácido poligalacturónico) reduziram a formação



ix

de agregados insolúveis e precipitação de complexos. A pectina foi sempre o

carboidrato mais eficiente, seguida da goma arábica. Na interação com a α-

amilase, os estudos por fluorescência permitiram conhecer os mecanismos mais

prováveis subjacentes à ação inibidora dos carboidratos: a goma arábica e a β-

ciclodextrina inibem a interação tanino/α-amilase pela sua associação aos

taninos, impedindo-os de interagir com a proteína (mecanismo de competição);

no caso da pectina observou-se a formação de um complexo ternário tanino/α-

amilase/pectina solúvel em meio aquoso (mecanismo complexo ternário). A

formação de complexos ternários envolvendo a goma arábica e a pectina

também foram evidenciadas nos estudos com as proteínas salivares.

Na parte final deste trabalho, estudou-se o sabor amargo de alguns polifenóis

pertencentes a diferentes classes [(-)-epicatequina, PGG, procianidinas dimérica

B3 e trimérica C2, malvidina-3-glucósido e cianidina-3-glucósido], identificando

quais dos 25 recetores de sabor amargo dos humanos (hTAS2Rs) são ativados

por estes compostos.

Os resultados obtidos mostraram que diferentes compostos ativam diferentes

hTAS2Rs. (-)-Epicatequina ativou três recetores hTAS2R4, hTAS2R5 e

hTAS2R39, enquanto a PGG só ativou dois recetores, hTAS2R5 e hTAS2R39.

Por outro lado, a malvidina-3-glucósido e o trímero C2 só ativaram um recetor,

hTAS2R7 e hTAS2R5, respetivamente. Um dos resultados mais notáveis é que

os taninos são os primeiros agonistas naturais encontrados para o hTAS2R5,

apresentando grande potência na ativação deste recetor. Para além disso, os

grupos catecol e/ou galhoilo parecem ser estruturalmente importantes na

mediação da interação dos polifenóis com este recetor.

Os valores obtidos de EC50 para os diferentes compostos variam cerca de 100-

vezes, sendo os valores mais baixos os da PGG e malvidina-3-glucósido. Isto

sugere que estes compostos podem ser significativamente importantes no

amargor de frutos, vegetais e produtos derivados, mesmo que estejam presentes

em concentrações muito baixas.

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xi

ABSTRACT

Polyphenols compounds are secondary metabolites of plants being present in

food and beverages derived from plants (e.g., red wine, beer, tea, fruit juices,

etc). A lot of interest has been paid to these compounds because of their

biological properties (antioxidants, anticancer, etc) and because of the

organoleptic properties that they confere to food and their. Tannins are a group of

polyphenols that have the ability to interact with proteins, particularly salivary

proteins. This interaction is important both in a biological context and also in a

sensorial context, since it is at the origin of astringency development in food.

Indeed, it is widely accepted by the scientific community that astringency is due

to the complexation and/or precipitation of salivary proteins rich in proline (PRPs)

by tannins.

While astringency is a tactile sensation perceived in the oral cavity, bitterness is a

taste that results by the interaction between compounds and bitter taste

receptors. Some low molecular weight polyphenols are known to taste bitter.

The main goal of this work was to understand and have insights about the

sensorial properties of polyphenols (bitter taste ans astringency) and to study in

which way different carbohydrates commonly used in food industry influence

astringency. So, this work as focused in determine: (a), the main families of

salivary proteins that have more affinity to interact with tannins in a model

solution and directly in red wine; (b), how supplementation of red wine with

tannins influence the perception of astringency; (c), how the characteristics

(solubility and size) of the formed complexes between the different families of

salivary proteins and two types of tannins (condensed and. Hydrolysable) could

affect astringency; (d), how some commercial carbohydrates (arabic gum, β-

ciclodextrina, pectin and polygalacturonic acid) inhibit the interaction

tannins/proteins, in particular with salivary proteins; (e), which human bitter taste

receptors are activated by several polyphenols commonly present in vegetal food

and derived products.

The salivar proteins present in human saliva were identified by proteomic

approache (HPLC-DAD, ESI-MS, SDS-PAGE and MALDI-TOF). These

techniques were then adapted to study the interaction between different families

of salivary proteins and condensed tannins. The results showed that condensed

tannins interact firstly with acidic PRPs ans statherin and only then with histatins

and glycosylated and basic PRPs.

xii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

The analysis of the salivary proteins by HPLC after interaction with red wine,

allowed to study some phenomena related to astringency of red wines enriched

with condensed tannins by comparison with the sensory analysis of the same

wines. The results showed that for red wines with low concentrations of

condensed tannins, the complexation/precipitation of acidic PRPs and statherin is

correlated with astringencency intensity. However, for red wines with higher

tannin concentrations, astringency seems to be correlated with tannins’

interaction with glycosylated PRPs. To our knowledge, for the first time there are

evidences that different families of salivary proteins are involved in different

stages of the physiological phenomena associated to astringency perception.

In the next step, it was compared the interaction between salivary proteins and a

hydrolizable tannin (PGG) vs. condensed tannin (procianidin trimer). The results

obtained by HPLC and dynamic light scattering (DLS) show that both tannins

interact mainly with aPRPS and statherin, forming both soluble as insoluble

complexes. On the other hand, bPRPs interact poorly with procyanidin trimer and

gPRPs only complex with PGG. In general, PGG showed more tendency to form

insoluble complexes while procyanidin trimer formed more soluble complexes

(except for statherin). The results present significant evidences that different

tannins and salivary proteins could influence and have different mechanisms in

their interactions, and consequently, in astringency development.

The effect of carbohydrates in tanins/proteins interaction was study based on two

models of different proteins: condensed tannins/α-amylase and condensed

tannins/salivary proteins. In the first study, it were used the following techniques

fluorescence, nephelometry and DLS; in the second model, the salivary proteins

that remain soluble in the presence of condensed tannins after addition of

carbohydrates were monitored by HPLC and the respective precipitates were

analyzed by SDS-PAGE.

Overall, the results showed that all the tested carbohydrates (arabic gum, β-

ciclodextrina, pectin and polygalacturonic acid) reduced the formation of insoluble

aggregates and complexes precipitation. Pectin was always the most efficient

carbohydrate followed by arabic gum. In the interaction with α-amylase,

fluorescence studies gave insights about the mechanism by which different

carbohydrates inhibit tannins/α-amylase interaction: arabic gum and β-

cyclodextrin inhibit this interaction competing with proteins for tannins association

(competition mechanism); pectin seems to inhibit this interaction by the formation

of a ternary complex tannin/α-amylase/pectin with increasing solublity in aqueous



xiii

medium (ternary complex mechanism). The formation of ternary complexes

involving arabic gum and pectin was also suggested in the study with salivary

proteins.

In the final part of this work, the bitterness of several polyphenols from different

classes [(-)-epicatechin, PGG, procyanidin dimer B3 and trimer C2, malvidin-3-

glucoside and cyanidin-glucoside] was studied by identification of the hTAS2Rs

that were activated by these compounds.

The results show that diferente compounds activated different subsets of

hTAS2Rs. (-)-Epicatechin activated three receptors hTAS2R4, hTAS2R5 and

hTAS2R39 while PGG activated only two receptors, hTAS2R5 and hTAS2R39.

Otherwise, malvidin-3-glucoside and trimer C2 only activated one receptor,

hTAS2R7 and hTAS2R5, respectively. One of the most important result is that

tannins are the first natural agonists for hTAS2R5, having high potency in its

activation. Futhermore, the catechol and/or galloyl groups seem to be structural

important in mediating polyphenols interaction with this receptor.

The EC50 values obtained for the several tested compounds vary around 100-

times fold, being the lowest for PGG and malvidin-3-glucoside. This suggests that

these compounds could be significant in the bitterness of fruits, vegetables and

derived products, even if they are present in very low concentrations.

xiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor



xv

ÍNDICE

Agradecimentos .................................................................................................. iii

Resumo .............................................................................................................. vii

Abstract ............................................................................................................... xi

Índice ................................................................................................................. xv

Lista de Quadros .............................................................................................. xvii

Lista de Figuras ................................................................................................. xix

Lista de Abreviaturas ....................................................................................... xxix

Publicações científicas e Organização da tese ............................................... xxxiii

Introdução Geral ................................................................................................ 35

1. Compostos não-flavonóides ....................................................................... 39

2. Compostos flavonóides .............................................................................. 39

2.1 Antocianinas ......................................................................................... 40

2.2 Flavan-3-óis ......................................................................................... 41

3. Taninos ...................................................................................................... 43

3.1 Taninos condensados (Proantocianidinas) ........................................... 44

3.2 Taninos hidrolisáveis ............................................................................ 46

4. Compostos polifenólicos nos alimentos. Ingestão e impacto na saúde humana ......................................................................................................... 47

5. Interação entre taninos e proteínas ............................................................ 52

5.1 Tipos de ligação envolvidos na interação tanino-proteína .................... 52

5.2 Modelos da interação tanino/proteína ................................................... 56

5.3 Fatores que influenciam a interação tanino/proteína ............................ 59

5.3.1 Estrutura dos taninos ..................................................................... 59

5.3.2 Estrutura das proteínas .................................................................. 62

5.3.3 Presença de carboidratos .............................................................. 64

5.3.4 Outros fatores (força iónica, pH, etanol e temperatura) .................. 70

6. Saliva humana ........................................................................................... 71

6.1 Proteínas ricas em prolina (PRPs) ....................................................... 71

6.1.1 PRPs acídicas (aPRPs) ................................................................. 73

6.1.2 PRPs básicas (bPRPs) e glicosiladas (gPRPs) .............................. 74

6.2 Histatinas ............................................................................................. 75

6.3 Estaterina ............................................................................................. 76

7. Polifenóis e sabor amargo ......................................................................... 76

xvi FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

7.1 Relação entre a estrutura dos polifenóis e o sabor amargo .................. 77

7.2 Recetores do sabor amargo (hTAS2Rs) ............................................... 78

Objetivos ............................................................................................................ 83

Resultados ......................................................................................................... 87

Capítulo 1 ...................................................................................................... 89

Capítulo 2 .................................................................................................... 123

Capítulo 3 .................................................................................................... 143

Capítulo 4 .................................................................................................... 161

Capítulo 5 .................................................................................................... 183

Capítulo 6 .................................................................................................... 203

Conclusão e Perspetivas futuras ...................................................................... 227

Referências Bibliográficas ................................................................................ 233



xvii

LISTA DE QUADROS

Tabela 1. Teor em polifenóis de diversos alimentos. Adaptado de Scalbert e

Williamson (2000).............................................................................................. 47

Tabela 2. Distribuição e grau de polimerização de taninos condensados em

alimentos. Adaptado de Gu et al. (2004). .......................................................... 48

Tabela 3. Resumo de alguns trabalhos que evidenciam o tipo e caraterísticas

importantes das interações tanino/proteína, indicando os taninos e proteínas

estudados, bem como as metodologias utilizadas no estudo. ........................... 55

Capítulo 1:

Table 1.1. Experimental Masses (Da) Detected in the Twelve Chromatographic

Fractions by RP-HPLC-ESI-MS, and MALDI-TOF/TOFa .................................. 102

Capítulo 2:

Table 2.1. Tannin specific activity (TSA) of 50 µL of whole (WS) and acidic saliva

(AS) toward red wines supplemented with several concentrations of oligomeric

procyanidins (OPC). For the same concentration of OPC, the results of WS and

AS are significantly different (P<0.05), except for the 2.0 g.L-1 concentration

(athese results are statistically equal). Astringency rating from the sensorial

evaluation. The orders with different letters are significantly different (P<0.1). 137

Capítulo 3:

Table 3.1 Percentage of salivary proteins involved in the formation of soluble and

insoluble complexes formed by the interaction with 399 µM of each tannin. These

results represent the average of three independent experiments. ................... 156

Capítulo 4:

Table 4.1 Average size of aggregates (Z) present in procyanidin fractions and

PPA (1 µM) solutions (100 mM acetate buffer with 12% ethanol/water (v/v) pH

xviii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

5.0) and polydispersity in absence and presence of carbohydrates, measured by

dynamic light scattering. .................................................................................. 175

Capítulo 5:

Table 5.1 Half maximal effective concentration (EC50) in inhibition of

tannin/salivary protein precipitation by three carbohydrates: AG, PGA and pectin.

The values with equal letters are not significantly different (P<0.05). ............... 195

Capítulo 6:

Table 6. 1 EC50 values (µM) for the test compounds and respective receptors.

The values with superscript equal letters are not significantly different (P < 0.05).

Values with ≥ are estimates because the dose-response curves did not saturate.

........................................................................................................................ 215

Table 6.2 Threshold concentrations (µM), defined as the lowest concentration

that resulted in calcium signals in receptor-transfected cells, for the test

compounds...................................................................................................... 215

Table 6.3 Signal amplitudes (given as relative fluorescence changes ∆F/F) for

the test compounds. ........................................................................................ 216



xix

LISTA DE FIGURAS

Fig.1 Estrutura química dos maiores grupos de polifenóis e alguns exemplos de

alimentos ricos em cada classe de compostos. ................................................. 38

Fig.2 Estrutura química do núcleo flavânico. A e B – anéis aromáticos, C – anel

heterocíclico pirano. .......................................................................................... 39

Fig. 3 Estrutura química do catião flavílio (3-glicósido) e dos respetivos derivados

pirúvicos (DP) de antocianinas. Substituintes das antocianinas (e dos derivados

pirúvicos) mais comuns na natureza. Glc – glucose. ......................................... 41

Fig.4 Estrutura química dos flavan-3-óis mais comuns nas plantas. .................. 42

Fig.5 Reação de decomposição das proantocianidinas em meio ácido – Reação

de Bate-Smith (Bate-Smith 1954). ..................................................................... 44

Fig.6 Estrutura química dos dois tipos de procianidinas diméricas e substituintes

de cada procianidina dimérica tipo B. ................................................................ 45

Fig.7 Estrutura das moléculas base dos taninos hidrolisáveis (ácidos gálhico e

elágico) e exemplo de um tanino hidrolisável β-1,2,3,4,6-pentagalhoil-O-D-

glucopiranose (pentagalhoilglucose, PGG). ...................................................... 46

Fig.8 Caracterização da dieta alimentar da população portuguesa para o período

entre 2003 e 2008 (com base na Balança Alimentar Portuguesa). O gráfico

superior apresenta o consumo da população em função dos grupos de

alimentos, estando salientados por rectângulos laranja os grupos de alimentos

que contêm polifenóis; o gráfico inferior apresenta os alimentos mais

consumidos dentro de cada grupo de alimentos rico em polifenóis (Balança

Alimentar Portuguesa, Instituto Nacional de Estatística).................................... 49

Fig.9 Vias de absorção dos polifenóis. Adaptado de Visioli et al. (2011) ........... 52

Fig.10 Esquema representativo das interações entre taninos e proteínas,

evidenciando o tipo de ligações mais relevante bem como os grupos de cada

molécula (do tanino e da proteína) envolvidos em cada tipo de ligação. Adaptado

de Asano et al. (1982). ...................................................................................... 54

xx FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

Fig.11 Modelo da interação tanino/proteína. Os taninos são exibidos com duas

extremidades as quais podem ligar-se às proteínas. As proteínas são

apresentadas como possuindo um número fixo de locais de ligação. Adaptado

de Siebert et al. (1996). ..................................................................................... 56

Fig.12 Esquema de uma curva de nefelometria para uma concentração fixa de

tanino e concentrações crescentes de proteína. Adaptado de Hagerman e

Robbins (1987). ................................................................................................. 57

Fig.13 Mecanismo molecular proposto para a interação das PRPs com os

taninos. Na fase inicial (1ª fase) as proteínas compactam-se, devido a ligações

apertadas múltiplas com os taninos multidentados. Na 2ª fase forma-se um

dímero com outra proteína recoberta de taninos, tornando o complexo insolúvel.

Na 3ª fase, há uma maior complexação e ocorre precipitação do complexo.

Adaptado de Jöbstl et al. (2004). ....................................................................... 58

Fig.14 Conformações da (-)-epicatequina, dímero B2 e seus derivados

galhoilados, obtidos por mecânica molecular e RMN. Adaptado de de Freitas et

al. (1998). .......................................................................................................... 61

Fig.15 Representação no modo space filling da estrutura terciária da albumina

sérica bovina (BSA), mioglobina e gelatina as quais apresentam conformações

diferentes. A mioglobina e a BSA têm estrutura terciária globular, sendo a

estrutura da mioglobina mais fechada e aparecendo aqui apresentada com o

grupo heme (grupo prostético essencial para a sua função). O colagénio tem

uma estrutura enrolada em tripla hélice apresentando uma conformação mais

aberta que as outras proteínas. ......................................................................... 64

Fig.16 Representação esquemática da (A) estrutura da parede celular primária

evidenciando os carboidratos mas importantes e a lamela média, e da (B)

degradação da pectina e da hemicelulose durante o amadurecimento dos frutos.

A degradação destes carboidratos reduz a integridade das paredes celulares,

diminuindo a rigidez dos frutos (adaptado de Wakabayashi (2000)). ................. 65

Fig.17 Mecanismos possíveis de inibição da agregação dos taninos com as

proteínas pelos carboidratos. P: proteína, T: tanino, C: carboidrato. Adaptado de

Mateus et al. (2004). ......................................................................................... 67



xxi

Fig.18 Percentagens aproximadas (p/p) das principais classes de proteínas e

péptidos salivares na saliva total. Adaptado de Messana et al. (2008). ASH,

albumina sérica humana; sIgA, imunoglobulina A secretora; IgG, imunoglobulina

G; aPRPs, proteínas-ricas em prolina acídicas; bPRPs, proteínas-ricas em

prolina básicas; gPRPs, proteínas-ricas em prolina glicosiladas. ...................... 71

Fig.19 Esquema representativo da estrutura das aPRPs com indicação da região

acídica responsável pela função atribuída a estas proteínas. ............................ 73

Fig.20 Esquema representativo da estrutura da estaterina com indicação das

diferentes regiões responsáveis pelas funções atribuídas a esta proteína. ....... 76

Fig.21 Representação esquemática dos hTAS2Rs. Os resíduos de aminoácidos

estão indicados por círculos coloridos. A similaridade da sequência entre os 25

hTAS2Rs está indicada pelo código de cores. Os círculos com a linha em pontos

correspondem aos resíduos que não estão presentes em todos os recetores. Os

quadrados magenta indicam as regiões que contêm resíduos adicionais em

alguns recetores (Meyerhof 2005). .................................................................... 80

Fig.22 Mecanismo proposto de transdução de sinal do sabor amargo para as

células recetoras de sabor. Os compostos amargos ativam os recetores de sabor

amargo, os quais ativam os heterotrimeros da gustaducina. A α-gustaducina

estimula a PDE (fosfodiesterase) a hidrolizar o cAMP (adenosina monofosfato

cíclica); a diminuição de cAMP pode disinibir os canais de iões inibidos por

nucleótidos, elevando o Ca2+. Por outro lado, as subunidades βγ-gustaducina

libertadas ativam a PLCβ2 (fosfolipase C2), gerando IP3 (inositol trisfosfato) que

leva à libertação de Ca2+ armazenado, contribuindo para o aumento do Ca2+

intracelular. Adaptado de Meyerhof (2005). ....................................................... 81

Capítulo 1:

Fig.1.1(A) Typical RP-HPLC profile detected at 214 nm of the acidic saliva (AS)

solution of whole human saliva. The pointed lines and numbers show the ranges

and names assigned to each HPLC fraction, with the outline of the main proteins

identified for each HPLC fraction (Table 1). At the bottom, there is the distribution

of the different families of salivary proteins along the chromatogram. *, fragments

of proteins; His, histatin; Stat, sthaterin; (di-, mono-, n-)-phosp, (di-, mono-, non-)-

phosphorylated; bPRP, basic proline-rich protein; gPRPs, glycosylated proline-

xxii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

rich proteins, aPRPs, acidic proline-rich proteins. (B) Total ion current (TIC)

profile of AS solution collected by the ion-trap mass spectrometer (ESI-MS)... 100

Fig.1.2 Deconvolution process for fraction 10. (A) ESI mass spectrum obtained

by the average of 77 mass spectra collected in the 38.49-40.39 min range during

the HPLC separation reported in the TIC profile (Fig.1.1B). (B) The bottom panel

reports the deconvolution of the upper ESI-MS (A). ......................................... 101

Fig.1.3 SDS-PAGE of the 12 HPLC fractions isolated from the HPLC after the

injection of the AS solution. The molecular weight markers were substituted by

lines, and the molecular mass is expressed in kDa as marked on the left side.

The gels were stained by with Imperial Protein Stain, a Coomassie R-250 dye-

based reagent. ................................................................................................ 104

Fig.1.4 SDS-PAGE of each HPLC fraction isolated from the HPLC after the

injection of the AS solution. The molecular weight markers were substituted by

lines and the molecular mass is expressed in kDa. The gels were stained by the

periodic acid Schiff procedure in order to visualize glycoproteins. ................... 107

Fig.1.5 RP-HPLC profile detected at 214 nm of the AS solution before (AS

control) and after the interaction with increasing concentrations of grape seed

fraction (GSF). ................................................................................................. 108

Fig.1.6 Percentages of area decrease of each HPLC fraction after the interaction

of AS solution with increasing concentrations of GSF (A, B, and C). Percentages

of area decrease for each family of salivary proteins (D). ................................ 109

Fig.1.7 SDS-PAGE of the AS solution before and after the interaction with two

concentrations of grape seed fraction (GSF) (0.133 mM and 0.232 mM). The

molecular weight markers were substituted by lines, and the molecular mass is

expressed in kDa. ........................................................................................... 110

Fig.1.8 SDS-PAGE of HPLC fractions 6 to 11 isolated from the HPLC after the

injection of the AS solution before (C) and after the interaction with two

concentrations of grape seed fraction (0.133 and 0.232 mM). The molecular

weight markers were substituted by lines, and the molecular mass is expressed

in kDa. ............................................................................................................. 110



xxiii

Capítulo 2:

Fig.2.1 Typical Reverse Phase HPLC profile detected at 214 nm of the AS

solution of human saliva. The dotted lines and numbers show the ranges and the

main SP family assigned to each HPLC peptide region. .................................. 131

Fig.2.2 (A) HPLC profile detected at 214 nm of the AS solution before (control)

and after the interaction with increasing volumes of red wine supplemented with

1.5 g.L–1 of OPC (condensed tannins). (B) Percentages of area decrease of each

HPLC salivary peptide region after the interaction of AS solution with increasing

volumes of red wine supplemented with 1.5 g.L–1 OPC. Values are expressed as

the arithmetic means of 3 experiments ± standard deviation. .......................... 132

Fig.2.3 Reverse Phase HPLC profile detected at 214 nm of the AS solution

before (control) and after the interaction with 10 µL of each red wine

supplemented with different concentrations of OPC. ....................................... 133

Fig.2.4 Percentages of area decrease of each HPLC peptide regions after the

interaction of AS solution with (A) 10 and (B) 20 µL of red wine supplemented

with increasing concentrations of OPC. Values are expressed as the arithmetic

means of 3 experiments ± standard deviation. ................................................ 134

Fig.2.5 Percentages of area decrease of each HPLC peptide regions after the

interaction of AS solution with 20 µL of OPC solutions in acetate buffer matrix.

Values are expressed as the arithmetic means of 3 experiments ± standard

deviation. ......................................................................................................... 135

Fig.2.6 Reverse Phase HPLC profile detected at 214 nm of the precipitates

resultant from the interaction of 10 or 20 µL of red wine supplemented with 0.5,

2.0, and 3.0 g.L–1 of OPC with the AS. ............................................................ 136

Capítulo 3:

Fig.3.1 A. Adapted from Hagerman and Robbins (1987). Titration of a plant

extract containing tannin with protein (BSA): region A, excess tannin; region B,

equivalence point; region C, excess protein. B. Adapted from Siebert and co-

workers (Siebert, Troukhanova et al. 1996). Model for protein/polyphenol

interaction that explains the results observed in Fig. 3.1A. Polyphenols are

xxiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

depicted as having two ends that can bind to protein. Proteins are depicted as

having a fixed number (three) of polyphenol binding sites. .............................. 146

Fig.3.2 Scheme of the experimental approach used to study protein/tannin

interaction indicating the supposed kind of complexes formed and removed in

each step. ....................................................................................................... 149

Fig.3.3 RP-HPLC profile detected at 214 nm of the AS solution before (control

saliva) and after the interaction with 133 and 399 µM of A, PGG and B,

procyanidin trimer. All these solutions were filtered (0.22 µm) prior to HPLC

analysis. .......................................................................................................... 151

Fig.3.4 RP-HPLC profiles detected at 214 nm of the AS solution after the

interaction with 399 µM of procyanidin trimer and 133 µM of PGG. These

solutions were non-filtered and filtered (0.22 µm) prior to HPLC analysis. ....... 153

Fig.3.5 The graphics represent the variation of the chromatographic peaks area

of the several salivary proteins studied with the increase in tannins concentration.

The results are expressed as percentage of the area of each protein relatively to

the control saliva (100%) analyzed by HPLC, before and after solutions filtration

(0.22 µm). Statherin graphic was used as an example indicating the percentage

of protein involved in the formation of soluble and insoluble complexes. These


Fig.3.6 Influence of tannin concentration on the average size of salivary soluble

proteins/tannins complexes present in solution. These results represent the

average of three independent experiments. .................................................... 155

Capítulo 4:

Fig.4.1 Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) and

procyanidin fraction IV (17 µM; mean MW = 2052) solution, in the absence (full

line) and presence of increasing concentrations of Arabic gum (0.1, 0.2, 0.3, 0.4,

0.5, 0.6 g.L–1). The experiments were performed in 100 mM acetate buffer (pH

5.0) with 12% ethanol/water (v/v). ................................................................... 169

Fig.4.2 A. Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM)

(solid spectra) and in the presence of the highest polysaccharide concentration

[arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g.L-1, - - -; and pectins 80 mg.L-1



xxv

(27% MD, ; 58-63% MD, ; and 72% MD, ]. B. Several fluorescence

emission spectra of procyanidin fractions alone and in the presence of the

highest polysaccharide concentration (arabic gum 0.6 g.L-1, ; β-cyclodextrin

4.4 g L-1, ; and pectin 58-63% MD 80 mg.L-1, ). The experiments were

performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v). .... 170

Fig.4.3 Influence of increasing concentrations of arabic gum in (upper data)

variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution

and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates

measured by nephelometry. Mean MWs of fractions II, III, and IV are 950, 1512,

and 2052, respectively. All experiments were performed in 100 mM acetate buffer

(pH 5.0) with 12% ethanol/water (v/v). ............................................................. 171

Fig.4.4 Influence of increasing concentrations of β-cyclodextrin in (upper data)

variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution

and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates

measured by nephelometry. Mean MWs of procyanidin fractions II, III, and IV are

950, 1512, and 2052, respectively. All experiments were performed in 100 mM

acetate buffer (pH 5.0) with 12% ethanol/water (v/v). ...................................... 172

Fig.4.5 Percentage of procyanidin/PPA (1 µM) insoluble aggregates in the

presence of increasing concentrations of different pectins measured by

nephelometry. Mean MWs of procyanidin fractions II (a), III (b), and IV (c) are

950, 1512, and 2052, respectively. All experiments were performed in 100 mM

acetate buffer (pH 5.0) with 12% ethanol/water (v/v). ...................................... 173

Fig.4.6 Possible mechanisms (i and ii) involved in the inhibition of the

aggregation of tannins and proteins by polysaccharides. P, protein; T, tannin; C,

polysaccharide/carbohydrate. .......................................................................... 176

Capítulo 5:

Fig.5.1Typical RP-HPLC profile detected at 214 nm of the AS solution of human

saliva in the absence (— ) and presence (- - -) of 300 µM GSF. The vertical

dotted lines show the ranges and the main salivary proteins family assigned to

each HPLC peptide region: bPRPs, gPRPs, and aPRPs. ................................ 191

xxvi FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

Fig.5.2 Part of the chromatograms of the AS solution after the interaction with

GSF (300 µM) in the absence (AS solution + 300 µM GSF) and presence of the

several tested carbohydrates (pectin, 5.0 g.L–1; AG, 10.0 g.L–1; and PGA, 20.0

g.L–1). .............................................................................................................. 193

Fig.5.3 Influence of carbohydrate concentration on salivary proteins precipitation

by condensed tannins (GSF, 300 µM). (A) AG, (B) PGA, and (C) pectin. These


Fig.5.4 SDS-PAGE of the pellets that resulted from the interaction between AS

and GSF in the absence (C) and presence of the several carbohydrates (pectin,

10.0 g.L−1; AG, 25.0 g.L−1; and PGA, 25.0 g.L−1). The molecular weight markers

were substituted by lines, and the molecular mass marked on the left side is

expressed in kDa. The gels were stained with Imperial Protein Stain, a

Coomassie R-250 dye-based reagent. The table shows the ratio between the

densitometry values obtained for total salivary proteins present in AS (control AS)

and in each experiment. The values with equal letters are not significantly

different (P < 0.05). ......................................................................................... 194

Fig.5.5 Possible mechanism (i and ii) involved in the inhibition of the aggregation

of tannins and proteins by carbohydrates. P, protein; T, tannin; and C,

carbohydrate.40 ................................................................................................ 196

Capítulo 6:

Fig.6.1 Chemical structures of the four polyphenol compounds studied. ......... 208

Fig.6.2 Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells

expressing the TAS2Rs indicated in the graphs following administration of 10.0

mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or

10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are

indicated by the thinner solid line. Fluorescence changes of Fluo4-AM loaded

HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs

following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-

glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-

transfected cells (empty plasmid) are indicated by the thinner solid line. ......... 213



xxvii

Fig.6.3 Concentration-response curves for HEK293T-Gα16gust44 cells

transfected with DNA for the indicated TAS2R following stimulation with the

indicated test compound. Error bars represent the confidential interval (P = 0.05).

(a) Dose dependent activation was observed for TAS2R4, TAS2R5, and

TAS2R39 by (-)-epicatechin (a), for TAS2R5 and TAS2R39 by PGG (b), for

TAS2R7 by malvidin-3-glucoside (c), and for TAS2R5 by procyanidin trimer (d).

........................................................................................................................ 214

xxviii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor



xxix

LISTA DE ABREVIATURAS

aa – aminoácido

ACN – acetonitrilo

AG – Arabic gum (goma arábica)

AGPs – arabinogalactana-proteínas

aPRP(s) – proteínas ricas em prolina acídicas

AS – acidic saliva (saliva acídica)

ASH – albumina sérica humana

bPRP(s) – proteínas ricas em prolina básicas

BSA – bovine serum albumin (albumina sérica bovina)

cAMP – cyclic adenosine mono-phosphate (adenosina monofosfato cíclica)

cNMP – cyclic nucleoside 5’ mono-phosphate (nucleósido monofosfato cíclico)

DAG – diacilglicerol

DC – dicroísmo circular

DLS – dynamic light scattering

DP – derivado pirúvico

EC50 – half maximal effective concentration (concentração efectiva na inibição de

metade da actividade)

ECG – galhato de epicatequina

EGCG – galhato de epigalhocatequina

EGC – epigalhocatequina

ESI – electrospray ionization (ionização por “electrospray”)

ESI-MS – electrospray ionization-mass spectrometry (espectrometria de massa

com ionização por “electrospray”)

FDR – false discovery rate (taxa de descobertas falsas)

FRET – fluorescence resonance energy transfer (fluorescência por transferência

de energia por ressonância)

Glc – glucose

xxx FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

gPRP(s) – proteínas ricas em prolina glicosiladas

GSF – grape seed fraction (fração extraída de grainhas de uvas)

HPLC-DAD ou RP-HPLC-DAD – reverse phase-high performance liquid

chromatography-diode array detector (cromatografia de fase reversa de elevada

eficiência com detector por díodo)

hTAS2Rs – human bitter taste receptors (recetors de sabor amargo do homem)

His – histatin (histatina)

IgG – imunoglobulina G

IP3 – inositol trisfosfato

LC-MS – liquid chromatography mass spectrometry (cromatografia liquída

seguida de análise por espectrometria de massa)

IUP – intrinsically unstructured proteins (proteínas não-estruturadas

intrinsecamente)

MALDI-TOF/TOF – matrix assisted light desorption ionization – time-of-

flight/time-of-flight (ionização e dessorção a laser assistida por matriz com

analisador do tempo de vôo)

MD – methoxylation degree (grau de metil esterificação)

MS – mass spectrometry (espectrometria de massa)

MW – molecular weight (peso molecular)

OPC – oligomeric procyanidins (procianidinas oligoméricas)

PAS – periodic acid Schiff’s staining (coloração de Schiff com ácido periódico)

PDE – phosphodiesterase (fosfodiesterase)

PGG – pentagalhoilglucose

PGA – polygalacturonic acid (ácido poligalacturónico)

pI – ponto isoeléctrico

PLCβ2 – phospholipase C2 (fosfolipase C2)

PMF – peptide mass fingerprint (identificação de proteínas pela identificação da

massa de péptidos característicos)

PPA – α -amylase from porcine pancreas (α-amilase de pâncreas de porco)



xxxi

PRP(s) – proteínas ricas em prolina

RMN ou NMR – ressonância magnética nuclear (nuclear magnetic ressonance)

RGII – ramnogalacturonanas do tipo II

SDS – sodium dodecylsulfate (dodecilsulfato de sódio)

SDS-PAGE – sodium dodecylsulfate-polyacrylamide gel electrophoresis

(electroforese em gel de dodecilsulfato de sódio)

sIgA – imunoglobulina A secretora

SNPs – single nucleotide polymorphisms (polimorfismos de um nucleótido)

SP – salivary proteins (proteínas salivares)

Stat – statherin (estaterina)

tetraGG - tetragalhoilglucose

TFA – trifluoroacetic acid (ácido trifluoracético)

TIC – total ion current (corrente iónica total)

TRC – taste receptor cells (células recetoras do sabor)

triGG - trigalhoilglucose

Tris – tris(hydroxymethyl)aminomethane [tris(hidroximetil)aminometano]

TSA – tannin-specific activity (actividade específica dos taninos)

UV – ultravioleta

UV-Vis – ultravioleta-visível

WS – whole saliva (saliva total)

XIC – extracted ion current (corrente iónica extraída)

xxxii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor



xxxiii

PUBLICAÇÕES CIENTÍFICAS E ORGANIZAÇÃO DA TESE

Esta tese de doutoramento foi organizada com base nestas seis publicações

científicas.

A parte inicial da tese é uma introdução global que visa elucidar o leitor acerca

dos compostos utilizados neste trabalho (polifenóis e proteínas, em particular as

proteínas salivares), bem como da relevância quer destes compostos quer do

objecto de estudo deste trabalho (interação tanino/proteína).

A segunda parte da tese inclui os resultados sob a forma de seis capítulos,

correspondendo cada capítulo a uma das publicações referida. A organização

dos resultados não foi efectuada por ordem cronológica das publicações mas foi

dividida em dois temas (interação tanino/proteína e efeito dos carboidratos na

interação tanino/proteína). Salvo pequenas excepções, toda a parte

experimental descrita foi realizada por mim.

Capítulo 1: Reactivity of Human Salivary Proteins Families Toward Food Polyphenols Soares, S., Vitorino, R., Osório, H., Fernandes, A., Venâncio, A., Mateus, N., Amado, F., de Freitas, V.

J. Agric. Food Chem. 2011, 59, 5535–5547

Com a exceção do manuseamento do MALDI e do LC-ESI-MS, e a análise de dados com o Global Protein Server Workstation, toda a parte experimental descrita neste artigo foi realizada por mim.

Capítulo 2: Effect of Condensed Tannins Addition on the Astringency of Red Wines

Soares, S., Sousa, A., Mateus, N., de Freitas, V.

Chem. Senses 2011, 37, 191-198

Toda a parte experimental descrita neste artigo foi realizada por mim.

Capítulo 3: Interaction of Different Classes of Salivary Proteins with Food Tannins Soares, S., Mateus, N., de Freitas V.

Food Research International, 2012, 49, 807-813

Com exceção da síntese da procianidina trimérica C2, toda a parte experimental descrita neste artigo foi realizada por mim.

xxxiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

Capítulo 4: Mechanistic Approach by Which Polysaccharides Inhibit α-Amylase/Procyanidin Aggregation Soares, S., Gonçalves, R. M., Fernandes, I., Mateus, N., de Freitas, V.

J. Agric. Food Chem. 2009, 57, 4352–4358

Toda a parte experimental descrita neste artigo foi realizada por mim.

Capítulo 5: Carbohydrates Inhibit Salivary Proteins Precipitation by Condensed Tannins Soares, S., Mateus, N., de Freitas, V.

J. Agric. Food Chem. 2012, 60, 3966–3972

Com exceção da análise da pectina, toda a parte experimental descrita neste artigo foi realizada por mim.

Capítulo 6: Different Phenolic Compounds Activate Distinct Subsets of Human Bitter Taste Receptors Soares, S., Kohl, S., Thalmann, S., Mateus, N., Meyerhof, W., de Freitas, V.

Submetido ao J. Agric. Food Chem.

Com exceção da síntese da procianidina dimérica B3 e trimérica C2 e da construção dos constructos, toda a parte experimental descrita neste artigo foi realizada por mim.

INTRODUÇÃO GERAL



37

Os compostos polifenólicos (vulgo polifenóis, designação utilizada ao longo da

tese) são uma grande família de metabolitos secundários sintetizados pelas

plantas e, consequentemente, estão presentes em alimentos e bebidas de

origem vegetal. Actualmente, já foram identificados milhares de polifenóis

naturais, apesar de só alguns estarem presentes em níveis significativos na dieta

alimentar (Shahidi e Naczk 1995). Nas plantas, estes compostos têm diversas

funções biológicas, sendo responsáveis pela cor, protecção contra a radiação

ultra-violeta, defesa contra ataques microbiológicos e contra predadores, entre

outras. Nos alimentos e bebidas de origem vegetal, os polifenóis são

responsáveis pelas suas principais caraterísticas organoléticas, nomeadamente

propriedades do sabor (amargor e adstringência), cor, odor e estabilidade

oxidativa (Naczk e Shahidi 2006).

O interesse nos polifenóis existentes nos alimentos, nomeadamente em frutas,

vegetais e bebidas derivadas de plantas (e.g. cerveja, chá e vinho tinto), tem

aumentado exponencialmente ao longo dos últimos anos. Isto deve-se ao facto

destes compostos estarem associados aos benefícios para a saúde humana das

dietas ricas nesses alimentos, em particular a famosa dieta Mediterrânica. Na

verdade, os polifenóis têm sido alvo de inúmeros estudos biológicos e

epidemiológicos, tendo-lhes sido atribuídas várias propriedades biológicas

benéficas como ação anti-mutagénica (Shim et al. 1995, Morley et al. 2005),anti-

cancerígena (Khafif et al. 1998, Faria et al. 2006), anti-oxidante (Faria et al.

2006, Wolfe et al. 2008, Azevedo et al. 2010), anti-alérgica (Akiyama et al. 2000,

Singh et al. 2011) e anti-bacteriana (Funatogawa et al. 2004, Taguri et al. 2004).

De facto, os polifenóis são os antioxidantes naturais mais abundantes na nossa

dieta alimentar e quase diariamente surgem evidências do seu papel na

prevenção de doenças degenerativas como o cancro e doenças

cardiovasculares.

Quimicamente, os polifenóis apresentam um ou mais aneis aromáticos com um

ou mais grupos hidroxilo com uma grande diversidade estrutural que

compreende desde moléculas fenólicas simples até polímeros de elevado peso

molecular. Os polifenóis são geralmente divididos em duas grandes famílias: os

flavonóides e os não-flavonóides (Fig.1).

38 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor

Fig.1 Estrutura química dos maiores grupos de polifenóis e alguns exemplos de alimentos ricos em cada classe de compostos.



39

1. Compostos não-flavonóides

Os não-flavonóides são uma vasta família de compostos, sendo sobretudo

moléculas simples como ácidos benzóicos, ácidos cinâmicos e estilbenos (Fig.1),

mas incluem também moléculas mais complexas derivadas destes,

nomeadamente galhotaninos e elagitaninos. Os ácidos benzóicos e cinâmicos

são denominados ácidos fenólicos e encontram-se principalmente em formas

conjugadas. Estes ácidos existem numa variedade de produtos vegetais, desde

frutos a cereais.

2. Compostos flavonóides

A família de polifenóis mais relevante nos alimentos é a dos flavonóides, sendo

também a família mais diversificada estruturalmente. Atualmente já foram

descritos mais de 4000 flavonóides e esta listagem continua a crescer.

A estrutura básica dos flavonóides é o núcleo flavânico, que consiste numa

estrutura C6-C3-C6 constituída por dois anéis aromáticos (A e B) ligados por um

anel heterocíclico pirano (C) (Fig.2). As diferentes classes de flavonóides diferem

entre si no grau de oxidação e padrão de substituição do anel C. Para além

disso, dentro de uma classe, os compostos diferem entre si no número e posição

dos grupos hidroxilo, metoxilo e glicosilo (Fig.1).

Os flavonóides são divididos em várias classes, como apresentado na Fig.1. A

razão para uma diversidade tão elevada advém da ocorrência de numerosos

padrões de substituição em que os substituintes primários (e.g. grupos hidroxilo,

metoxilo ou glicosilo) podem ser adicionalmente substituídos (e.g. glicosilados ou

acilados), originando por vezes estruturas muito complexas.

Fig.2 Estrutura química do núcleo flavânico. A e B – anéis aromáticos, C – anel heterocíclico pirano.

Sendo a família de polifenóis mais abundante nos alimentos é também a família

de polifenóis mais conhecida pelas suas propriedades antioxidantes. Em 1990

(Bors et al. 1990), foram descritas pela primeira vez as caraterísticas químicas

O8

5

7

6

43

2

2'3'

4'

5'

6'

1'

A C

B


dos flavonóides que determinam o seu potencial de neutralizar radicais livres

e/ou a sua capacidade antioxidante: a estrutura o-di-hidroxilo (catecol) do anel B,

a conjugação da ligação dupla C2-C3 com o grupo 4-oxo do anel C e a presença

adicional de grupos hidroxilo nas posições 3 e 5.

2.1 Antocianinas

As antocianinas (do grego anthos que significa flor e kyanos que significa azul)

são os pigmentos mais abundantes e importantes das plantas, frutas e vegetais,

sendo responsáveis pelas variadíssimas cores apresentadas por estes produtos.

A estrutura das antocianinas deriva de glicósidos do catião flavílio poli-

hidroxilados e/ou metoxilados (Fig. 3). Nos frutos, as antocianinas encontram-se

na forma glicosilada, no entanto, noutros contextos podem existir sob a forma

não glicosilada denominando-se antocianidinas (agliconas). A diversidade

estrutural das antocianidinas depende do número e posição dos grupos hidroxilo

e metoxilo ligados aos anéis aromáticos e esta diversidade reflete-se na cor de

cada molécula. Para além da diversidade relacionada com os substituintes do

catião flavílio, as antocianinas podem diferir no tipo, número e posição dos

glicósidos ligados à molécula (pentoses, hexoses e metilpentoses; mono-, di- e

trissacáridos); e na presença e tipo de ácidos esterificados na molécula de

açúcar. Na maioria dos casos, os açúcares ligam-se na posição O-3, mas

também pode ocorrer esterificação nas posições O-5 e O-7. Os açúcares mais

comuns são a glucose, a ramnose, a galactose, a xilose e a arabinose. As

antocianidinas mais frequentemente encontradas em frutas são pelargonidina,

cianidina, delfinidina, peonidina, petunidina e malvidina, cujas estruturas estão

apresentadas na Fig. 3. Devido a todas estas possibilidades de substituição

estão identificadas na natureza um elevado número de antocianinas.

Dependendo da sua estrutura estes compostos exibem cores desde vermelha a

azul (Brouillard 1983). Por exemplo, a pelargonidina tem cor laranja-

avermelhada enquanto a delfinidina tem cor violeta-azulada a pH ácido.

Recentemente foram detectados no vinho novos pigmentos derivados de

antocianinas, em particular os derivados pirúvicos de antocianinas (Fig. 3)

(Fulcrand et al. 1998, Mateus et al. 2003). Estes compostos exibem uma cor

alaranjada e pela sua natureza química são muito estáveis, apresentando maior

intensidade de cor a pH mais elevado comparativamente aos seus derivados

antociânicos (Oliveira et al. 2006, Carvalho et al. 2010).



41

Catião flavílio Derivado pirúvico (DP)

Fig. 3 Estrutura química do catião flavílio (3-glicósido) e dos respetivos derivados pirúvicos (DP) de antocianinas. Substituintes das antocianinas (e dos derivados pirúvicos) mais comuns na natureza. Glc – glucose.

A diversidade de cores apresentada por estes pigmentos naturais e seus

derivados é a razão pela qual eles têm sido tão estudados, pois existe uma vasta

possibilidade de aplicação tecnológica, especialmente na indústria alimentar. No

entanto, um dos maiores obstáculos para a aplicação das antocianinas em

matrizes alimentares advém da sua instabilidade a variações de pH e

temperatura.

Dentro da família dos compostos flavonóides, as antocianinas são o grupo de

compostos que mais se destaca pela capacidade antioxidante devido à sua

biodisponibilidade (Rio et al. 2010), a qual será abordada em mais detalhe na

secção 4.

2.2 Flavan-3-óis

Os flavan-3-óis são dos grupos de flavonóides mais abundantes na natureza.

Estes compostos diferem entre si na estereoquímica do carbono 3 do anel C e

grau de hidroxilação do anel B (Fig.4). Têm uma unidade monomérica básica de

(+)-catequina ou (-)-epicatequina, e podem encontrar-se sob a forma de

monómeros ou polímeros (procianidinas). Ao contrário das outras classes de

flavonóides (que existem principalmente sob a forma de glicósidos), os flavanóis

encontram-se normalmente sob a forma de agliconas ou esterificados com o

OOH

OH

R1

R2

OH

O-Glc

+OOH

R1

R2

OH

O-Glc

O

COOH

+

Antocianina R1 R2 Malvidina-3-glucósido OCH3 OCH3

Cianidina-3-glucósido OH H

Delfinidina-3-glucósido OH OH

Pelargonidina-3-glucósido H H

Petunidina-3-glucósido OCH3 OH


ácido gálhico, formando por exemplo, epigalhocatequina e epigalhocatequina

galhato.

Fig.4 Estrutura química dos flavan-3-óis mais comuns nas plantas.

Têm uma unidade monomérica básica de (+)-catequina ou (-)-epicatequina, e

podem encontrar-se sob a forma de monómeros ou polimeros (procianidinas).

Ao contrário das outras classes de flavonóides (que existem principalmente sob

a forma de glicósidos), os flavanóis encontram-se normalmente sob a forma de

agliconas ou esterificados com o ácido gálhico, formando por exemplo,

epigalhocatequina e epigalhocatequina galhato.

Os carbonos C2 e C3 desta unidade são assimétricos constituindo centros

quirais. Nos isómeros (+)-catequina e (-)-epicatequina o grupo 3,4-hidrofenilo

ligado ao carbono C2 e o grupo hidroxilo no carbono C3 surgem em posição

trans (2R, 3S) e nos restantes isómeros em posição cis (2R, 3R).

Tal como a maioria dos compostos polifenólicos, vários estudos mostram que os

flavanóis têm propriedades antioxidantes devido a várias caraterísticas

importantes, nomeadamente grande estabilização das formas oxidadas devido a

uma deslocalização eletrónica extensa, quelatação de metais e captura de

radicais livres e espécies reativas de oxigénio (Faria et al. 2006, De La Iglesia et

al. 2010). Estes compostos podem também inibir a agregação plaquetária (Jin et

al. 2008), a inflamação vascular (Oizumi et al. 2010, Hu et al. 2011) e proteger

contra a neurodegeneração (Guo et al. 1996, Bastianetto et al. 2006,

Ramassamy 2006).

A importância desta classe de compostos reside também no facto de serem a

unidade estrutural constituinte das procianidinas (vulgo taninos condensados)

muito abundantes na natureza.

Flavan-3-ol R1 R2 R3 (+)-catequina OH H H

(-)-epicatequina H OH H

(+)-galhocatequina OH H OH

(-)-epigalhocatequina H OH OH

(-)-epigalhocatequina galhato H O-galhoilo OH

O

OH

OH

R1R2

OH

OH

R3A C

B

2

3



43

3. Taninos

Os taninos representam um grupo de compostos polifenólicos muito

heterogéneo, sendo difícil uma definição química rigorosa. O termo tanino foi

inicialmente usado para descrever compostos de origem vegetal responsáveis

pela conversão das peles de animais em couro, através da formação de

complexos estáveis entre esses compostos e o colagénio da pele animal. No

entanto, em 1962, foi proposta por Bate-Smith e Swain (1962) a definição

química de tanino, que diz que os taninos são “todos os compostos fenólicos

solúveis em água, com um peso molecular situado entre 500 e 3000 Dalton, cuja

principal propriedade (para além das reacções caraterísticas dos compostos

fenólicos) é a de formarem complexos insolúveis com os alcalóides, gelatina e

outras proteínas”.

Desde então, têm vindo a ser descobertos taninos com pesos moleculares muito

elevados na ordem das dezenas de milhares de Dalton (Prieur et al. 1994,

Souquet et al. 1996, Guyot et al. 1997).

Como referido anteriormente, uma das principais caraterísticas dos taninos é a

capacidade para complexar e precipitar proteínas. Esta propriedade é

duplamente a origem de atributos positivos e negativos atribuídos a estes

compostos. Por um lado, é aceite que a interação dos taninos com as proteínas

salivares está na origem da sensação de adstringência, que poderá ser, numa

certa dose, atributo positivo da qualidade de certas bebidas, como o vinho tinto,

cerveja e chá. A adstringência traduz-se numa sensação equilibrada de secura,

constrição e aspereza percebida na cavidade oral aquando da ingestão de

certos alimentos (Mcrae e Kennedy 2011). Por outro lado, a interação dos

taninos com enzimas digestivas pode inibi-las originando problemas

gastrointestinais e pode levar à diminuição do ganho de peso corporal (Griffiths

1986, Goncalves et al. 2007).

Classicamente os taninos são divididos em dois grandes grupos: os taninos

condensados (proantocianidinas), que são oligómeros de catequinas ligados por

ligações C-C; e os taninos hidrolisáveis, que são ésteres de monossacáridos

com ácido gálhico ou oligómeros de ácido gálhico/elágico.


3.1 Taninos condensados (Proantocianidinas)

Os taninos condensados são mais frequentes na alimentação do que os taninos

hidrolisáveis, estando presentes em concentrações significativas no chocolate,

frutos (uvas, maçãs, entre outros) e bebidas derivadas (Santos-Buelga e

Scalbert 2000).

Estruturalmente, os taninos condensados são polímeros de unidades flavanol, e

a sua estrutura varia de acordo com o número de grupos hidroxilo no anel B e

com a estereoquímica do carbono 3 do heterociclo C. Estes compostos dividem-

se globalmente em dois grandes grupos: as procianidinas, que são polímeros de

catequinas ((+)-catequina ou (-)-epicatequina); e as prodelfinidinas, que são

derivadas de galhocatequina e epigalhocatequina. As prodelfinidinas distinguem-

se das procianidinas por apresentarem um grupo OH na posição 5 do anel B. A

designação de proantocianidinas resulta do facto destes compostos libertarem

antocianidinas por decomposição em meio ácido – reação de Bate-Smith (Bate-

Smith 1954) (Fig.5).

Fig.5 Reação de decomposição das proantocianidinas em meio ácido – Reação de Bate-Smith (Bate-Smith 1954).

As unidades fundamentais das proantocianidinas estão geralmente ligadas entre

si por ligações do tipo C-C (ligação interflavânica que resulta do acoplamento

oxidativo entre monómeros) entre o carbono C4 de um flavanol e os carbonos

C8 (4→8) ou C6 (4→6) do flavanol seguinte. Consoante a ligação interflavânica,

as proantocianidinas diméricas são divididas em tipo A e tipo B (Fig.6). As de

tipo A possuem uma ligação 4→8 acrescida de uma ligação éter entre o grupo

hidroxilo do carbono C5 ou C7 do anel A de uma unidade e o carbono C2 do

anel pirânico da outra unidade. As de tipo B possuem apenas ligações

O

OH

OH

OH

ROH

OH

O

OH

OH

OH

ROH

OH

O

OH

OH

OH

ROH

OH

+

O

OH

OH

OH

OH

OH

R

H / Qcal

Oxigénio

+R=H CianidinaR=OH Delfinidina

R=H CatequinaR=OH Galhocatequina

+



45

interflavânicas 4→8 ou 4→6. Os dois tipos de proantocianidinas diméricas são

classificados numericamente, de acordo com a estereoquímica do carbono C3

do anel C de cada unidade. As proantocianidinas triméricas pertencem ao tipo C

e as de tipo D são as proantocianidinas triméricas que têm uma ligação de tipo A

e outra de tipo B.

Tipo A Tipo B Procianidina A2 Dímero C4-C8 Dímero C4-C6 (C4-C8 e ligação éter C2-O7)

R1 R2 R3 R4 Dímeros Tipo B C4-C8 C4-C6

OH H H OH B1 B5 OH H OH H B2 B6 H OH H OH B3 B7 H OH OH H B4 B8

Fig.6 Estrutura química dos dois tipos de procianidinas diméricas e substituintes de cada procianidina dimérica tipo B.

O grau de polimerização dos taninos condensados pode variar, podendo-se

encontrar oligómeros (dímeros a hexâmeros) e polímeros (que podem atingir

graus de polimerização muito elevados) (Prieur et al. 1994). Na verdade, já

foram identificados nas uvas taninos condensados com diferentes graus de

polimerização. Nas grainhas, o grau de polimerização situa-se entre 2 e 16,

enquanto na película é superior, entre 3 e 83 unidades de flavanol (Souquet et

al. 1996).

Em suma, os taninos condensados podem diferir nas unidades constituintes,

posições das ligações interflavânicas, comprimento da cadeia e presença de

substituintes (e.g. grupos galhoilo ou glicosilo) o que se traduz numa diversidade

muito elevada de compostos.

O

O

OH

OH

OH

OH

OOH

OH

OH

OH

OH

4

2

8

O

R4R3

OH

OH

OH

OH

O

R2R1

OH

OH

OH

OH

4

8

O

R2R1

OH

OH

OH

OH

O

OH

OH

R3R4

OHOH6

4


3.2 Taninos hidrolisáveis

Os taninos hidrolisáveis estão presentes sobretudo nas partes não comestíveis

das plantas. No entanto, estes compostos podem ser introduzidos na dieta

alimentar devido a operações tecnológicas de transformação dos alimentos. No

caso do vinho tinto, por exemplo, estes compostos podem passar da madeira

para o vinho durante o envelhecimento em barrica.

Este grupo de taninos tem como moléculas base o ácido gálhico (taninos

gálhicos) e o ácido elágico (taninos elágicos). Os taninos gálhicos estão quase

sempre na forma de ésteres múltiplos de D-glucose e podem formar estruturas

complexas como a pentagalhoilglucose (β-1,2,3,4,6-pentagalhoil-O-D-

glucopiranose) (PGG) (Fig.7). A PGG tem cinco ligações éster idênticas que

envolvem os grupos hidroxilo alifáticos da molécula de açúcar. Tal como a maior

parte dos galhotaninos, a PGG tem muitos isómeros entre os quais variam as

propriedades (bio)químicas, nomeadamente susceptibilidade de hidrólise e

capacidade para precipitar proteínas.

Ácido gálhico Ácido elágico β-1,2,3,4,6-pentagalhoil-O-D-glucopiranose (Pentagalhoilglucose, PGG)

Fig.7 Estrutura das moléculas base dos taninos hidrolisáveis (ácidos gálhico e elágico) e exemplo de um tanino hidrolisável β-1,2,3,4,6-pentagalhoil-O-D-glucopiranose (pentagalhoilglucose, PGG).

Nos taninos isolados das árvores ou produtos derivados de sumagre e de

carvalho, são frequentemente encontrados galhotaninos contendo até 12 grupos

galhoilo esterificados com uma glucose central. Por exemplo, o ácido tânico

comercial (utilizado na indústria alimentar, cosméticos, bebidas e tintas) é

composto por uma mistura de galhotaninos destas árvores.

OH

OH

OHO

OH

O

O

OH

OH

O

OH

OH

O OH

OHOH

OHOH OH

OHOH

OH

O

O

O

O

O

OHOH

OHO

O

OH

OHOH

OOO

O



47

4. Compostos polifenólicos nos alimentos. Ingestão e impacto na saúde

humana

Diversos alimentos de origem vegetal são ricos em polifenóis. Como tem vindo a

ser referido ao longo do texto, os alimentos e bebidas conhecidos pelo seu

elevado teor em polifenóis incluem frutos, legumes, cereais e produtos

derivados, como cerveja, sumos de fruta, vinho, chá e chocolate. A Tabela 1

apresenta o teor em polifenóis de alguns alimentos frequentemente ingeridos na

dieta alimentar.

Tabela 1. Teor em polifenóis de diversos alimentos. Adaptado de Scalbert e Williamson (2000).

Alimento (100 g ou mL)

Família de polifenóis (mg) Fenóis totais

(mg) Ácidos fenólicos

Flavanóis

Antocianinas Outros Monómeros catequina Proantocianidinas

Batata 14,5 14,5 - 28,5 Tomate 8 0,5 8 - 37 Alface 8 1 9 - 23 Maçã 5,5 10,5 100 7 239 - 440 Chocolate preto 80 430 510 - 840 Sumo laranja 22 22 - 75 Café 75 2 75 – 89,5 Vinho tinto 9 27,2 36 3 77,6 - 180 Chá preto 65 4 69 - 100

A maioria dos estudos sobre a composição polifenólica das plantas ou alimentos

foca-se apenas em monómeros e estruturas oligoméricas de pequena dimensão,

devido à ausência de técnicas eficazes na separação de estruturas mais

complexas. Geralmente, os polímeros são negligenciados, apesar de serem

frequentemente os polifenóis principais das plantas. Assim, a maior parte das

quantificações em polifenóis diz respeito a um número reduzido de compostos

e/ou a estimativas aproximadas do conteúdo total de polifenóis.

No entanto, apesar destas limitações, é de salientar o trabalho de Gu e seus

colaboradores (Gu et al. 2004) que determinaram a concentração e o grau de

polimerização de taninos condensados de vários alimentos e bebidas

usualmente ingeridos (Tabela 2).


Tabela 2. Distribuição e grau de polimerização de taninos condensados em alimentos. Adaptado de Gu et al. (2004).

Grau de polimerização 1 2 3 4-6 7-10 > 10 Total Alimento (mg/100 g peso fresco ou mg/L de bebida) Maçã 9,6 ± 0,9 13,8 ± 0,6 9,3 ± 0,4 30,2 ± 1,2 25,4 ± 1,2 37,6 ± 2,6 125,8 ± 6,8 Morango 4,2 ± 0,7 6,5 ± 1,3 6,5 ± 1,2 28,1 ± 6,5 23 ,9 ± 3,5 75,8 ± 13,4 145,0 ± 24,9 Pêra 2,7 ± 1,5 2,8 ± 1,3 2,3 ± 0,9 6,5 ± 1,9 4,6 ± 1,0 13,1 ± 11,3 31,9 ± 7,8 Chocolate preto

31,4 ± 0,2 31,2 ± 0,9 21,1 ± 0,8 55,5 ± 3,5 38,5 ± 3,0 68,2 ± 8,8 246,0 ± 0,3

Cerveja 4,0 11,0 ± 1,0 3,0 4,0 ND ND 23,0 ± 2,0 Vinho tinto 20,0 ± 1,0 40,0 ± 1,0 27,0 ± 1,0 67,0 ± 2,0 50,0 ± 1,0 110,0 ± 2,0 313,0 ± 5,0 Feijão vermelho

10,6 ± 0,0 19,4 ± 0,8 18,1 ± 0,6 80,0 ± 2,7 75,7 ± 2,4 252,9 ± 0,8 456,6 ± 7,5

Canela 23,9 ± 1,3 256,3 ± 11,3 1252,2 ± 62,2 2608,6 ± 140,3 1458,3 ± 116,1 2508,8 ± 92,9 8108,2 ± 424,2 Pistachio 10,9 ± 4,3 13,3 ± 1,8 10,5 ± 1,2 42,2 ± 5,2 37,9 ± 4,9 122,5 ± 37,1 237,3 ± 52,0

ND – Não detectado

Dependendo dos hábitos alimentares, uma pessoa pode consumir desde 100 ng

a vários gramas de compostos polifenólicos diariamente, principalmente se tiver

uma alimentação rica em frutas e legumes (Macheix et al. 1990).

Em Portugal, de acordo com a Balança Alimentar Portuguesa, no período entre

2003 e 2008 acentuaram-se os desequilíbrios da dieta alimentar portuguesa em

comparação com a década de 90. Verificou-se um consumo excessivo de

calorias e gorduras saturadas, disponibilidades deficitárias em frutos, hortícolas

e leguminosas secas e recurso excessivo aos grupos alimentares de carne,

pescado e de óleos e gorduras. A caracterização da alimentação da população

portuguesa nesse período está apresentada na Fig.8. Os grupos de alimentos

que contêm polifenóis estão salientados com rectângulos laranja e o gráfico

inferior discrimina os alimentos que são consumidos em maior quantidade em

cada grupo salientado.

Apesar de, tal como referido, se acentuarem os desequilíbrios da dieta alimentar

portuguesa, verifica-se que os grupos de alimentos ricos em polifenóis

constituem ainda uma importante parcela da dieta portuguesa. O vinho, a

cerveja, os frutos e as hortícolas ainda são ingeridos em quantidades

importantes. Em última instância, esta monitorização da dieta alimentar e as

acções sociais de incentivo ao consumo destes grupos de alimentos residem

numa questão de saúde pública.



49

Fig.8 Caracterização da dieta alimentar da população portuguesa para o período entre 2003 e 2008 (com base na Balança Alimentar Portuguesa). O gráfico superior apresenta o consumo da população em função dos grupos de alimentos, estando salientados por rectângulos laranja os grupos de alimentos que contêm polifenóis; o gráfico inferior apresenta os alimentos mais consumidos dentro de cada grupo de alimentos rico em polifenóis (Balança Alimentar Portuguesa, Instituto Nacional de Estatística).

Há muito que a associação da dieta mediterrânica a uma baixa incidência de

doenças cardiovasculares é conhecida e suportada por vários estudos

epidemiológicos (Hertog et al. 1993, Hollman e Katan 1999, Kaur e Kapoor

2001). Esta dieta carateriza-se pelo baixo consumo de alimentos ricos em

gordura saturada e de grande valor calórico, e consumo elevado de alimentos

ricos em hidratos de carbono complexos, fibras, vitaminas, minerais e

numerosos antioxidantes, nomeadamente, alimentos ricos em polifenóis. Deste

Bebidas n

ão-a

lcoó lic

as

Lac tic

ínio

s

Cerea

is

Bebida

s alco

ólica

s

Frutos

Hortícu

l as

Raíze

s e T

ubér

culos

Carne

Pesca

do

Óleos , a

zeite

e m

argar

ina

Produ

tos e

stimul

antes

0

100

200

300

400

500

g ou

mL/

pess

oa/d

ia

Bebida

s não-a

lcoó lic

as

Bebida

s alco

ó licas

Frutos

Hortic

ulas

Óleos,

azeit

e e m

argar

ina

Produt

os est

imula

ntes

0

100

200

300

(Cho

cola

te, c

afé)

g ou

mL/

pess

oa/d

ia


modo, neste tipo de alimentação predominam os cereais e seus derivados,

legumes, hortaliças, frutos e azeite.

O Paradoxo Francês é também um exemplo clássico desta associação: a

população de uma região francesa apresentava uma baixa incidência de

doenças cardiovasculares, apesar do elevado consumo de gorduras saturadas e

hábitos tabágicos. Este facto foi atribuído ao consumo moderado e regular de

vinho tinto (Renaud e De Lorgeril 1992).

Do total de polifenóis ingeridos diariamente estima-se que 1/3 é composto por

ácidos fenólicos e 2/3 por flavonóides (Scalbert e Williamson 2000). Os

flavonóides consumidos em maior extensão são os taninos condensados e as

antocianinas existindo então um grande interesse nestes compostos.

Relativamente à ação benéfica dos polifenóis na saúde humana uma das

questões principais é a sua biodisponibilidade. Isto porque um composto pode

ter grandes capacidades antioxidantes e outras atividades biológicas importantes

in vitro, as quais não serão significativas in vivo se pouco ou nenhum composto

chegar aos tecidos/órgãos alvo. Assim, após a estimativa da ingestão de

alimentos ricos em polifenóis, é importante estimar e estudar a sua

biodisponibilidade.

Dos diversos grupos de polifenóis, a biodisponibilidade das antocianinas tem

sido das mais estudadas (Fig.9) e actualmente sabe-se que a absorção de

alguns destes compostos ocorre no intestino delgado (Rio et al. 2010). Tem sido

aceite que a absorção das antocianinas ocorre na forma de agliconas, após

hidrólise dos glicósidos. Para explicar esta hidrólise e o aparecimento das

agliconas nas células epiteliais, têm sido propostas duas vias, a ‘difusão passiva

após hidrólise pela lactase floridizina hidrolase’ e ‘hidrólise pela β-glicosidade

citosólica’. No caso da primeira via, a hidrólise das antocianinas em agliconas

torna as moléculas mais apolares, aumentando o seu carácter lipofilico e a sua

proximidade com a membrana celular, levando a uma difusão passiva das

moléculas (Day et al. 2000). A última via pressupõe o transporte dos glicósidos

polares para as células, envolvendo possivelmente o transportador dependente

de Na+ GLUT-1, e a hidrólise ocorre após as antocianinas chegarem ao

citoplasma das células (Gee et al. 2000). Antes de passarem para a corrente

sanguínea, as agliconas são metabolizadas por enzimas sulfotransferases, UDP-



51

glucuronosiltransferases e catecol-O-metiltransferases originando

respetivamente metabolitos sulfatados, glucurinados e/ou metilados.

Os compostos que não são absorvidos no intestino delgado podem ser

absorvidos no intestino grosso onde a microflora intestinal pode clivar as

moléculas conjugadas. As agliconas resultantes podem sofrer ainda abertura

dos anéis, levando à formação de ácidos fenólicos e hidroxicinâmicos os quais

podem ser absorvidos e sujeitos ao metabolismo de fase II no fígado antes de

serem excretados.

Relativamente à biodisponibilidade das proantocianidinas a literatura existente

não é tão vasta, mas é um pouco controversa. Alguns estudos sugerem que

apenas os oligómeros de baixo peso molecular e grau de polimerização

(monómeros, dímeros e trimeros) são absorvidos intatos no trato gastrointestinal

(Holt et al. 2002, Deprez et al. 2004, Rasmussen et al. 2005) e as estruturas

mais complexas seriam degradadas nas condições do estômago. No entanto

outros estudos suportam que as proantocianidinas poliméricas passariam

inalteradas pelo intestino delgado, sendo depois degradadas em ácidos fenólicos

pela microflora do intestino grosso (Déprez et al. 2000, Manach et al. 2004). Os

produtos de degradação incluiriam os ácidos fenilacético, fenilpropiónico e

fenilvalérico.

Após a passagem da barreira intestinal pelas proantocianidinas ou seus

derivados originados na fermentação, estes compostos chegam ao fígado pela

veia porta, onde são metabolizados, provavelmente hidroxilados, metilados ou

conjugados a ésteres de sulfato ou glucuronilados tal como acontece com outros

flavonóides (e.g. antocianinas).


Fig.9 Vias de absorção dos polifenóis. Adaptado de Visioli et al. (2011)

Relativamente às acções benéficas para a saúde humana já foram reportados

diversos estudos que atestam esta relação. As capacidades antioxidante e

anticancerígena dos polifenóis já foram comprovadas inúmeras vezes (Faria et

al. 2006, Fernandes et al. 2009, Faria et al. 2010). Para além disto já foi

observada a diminuição do risco de doenças cardiovasculares (pela redução da

oxidação das LDL e diminuição do colesterol plasmático (Green e Jucha 1986,

Porto et al. 2003), redução do risco de desenvolvimento de doenças

neurodegenerativas, em particular doença de Alzheimer e Parkinson (Ehrnhoefer

et al. 2008, Wang et al. 2012) e retardamento do envelhecimento celular (Howitz

et al. 2003).

5. Interação entre taninos e proteínas

Uma das propriedades mais importantes dos taninos é, como já foi referido

anteriormente, a capacidade de interação e precipitação de proteínas. Esta

propriedade está na origem de várias atividades associadas a estes compostos,

nomeadamente as suas atividades biológica e organolética, quer positivas quer

nocivas. Devido à sua relevância, esta propriedade tem sido o âmbito de estudo

de diversos grupos de investigação (De Freitas e Mateus 2012).

5.1 Tipos de ligação envolvidos na interação tanino -proteína

O núcleo polifenólico tem uma estrutura molecular favorável à interação com

proteínas (Fig.10), pois apresenta:

ConjugaçãoMetilaçãoSulfatação

Glucuronilação

Células e tecidos

FÍGADO

Excreção urinária

VEI

A P

OR

TA

Glicósidos AgliconasÁcidos

fenólicos

CÓLON

INTE

STIN

O D

ELG

AD

OAgliconas

Glicósidos

ESTÔMAGO

Polifenóis oligoméricos

Monómeros



53

⇒ Regiões hidrofóbicas (apolares) que podem interagir com regiões

hidrofóbicas das proteínas, tais como cadeias laterais dos resíduos de

aminoácidos alanina, leucina, isoleucina e prolina, entre outros;

⇒ Regiões hidrofílicas que podem estabelecer pontes de hidrogénio com os

grupos carbonilo e amina das proteínas.

Apesar da estrutura dos taninos permitir a existência de vários tipos de ligações

com as proteínas, grande parte da literatura sugere que a maioria das interações

tanino/proteína são governadas por ligações não-covalentes, em particular

ligações hidrofóbicas (Oh et al. 1980, Baxter et al. 1997, Jöbstl et al. 2006) e

pontes de hidrogénio (Hagerman e Butler 1980, Hagerman e Klucher 1986)

(Fig.10). As ligações iónicas são as menos significativas pois os polifenóis são

ácidos fracos, com pKa de 9-10, e praticamente não apresentam grupos

carregados a valores de pH ácidos e neutros.

Diversos autores verificaram a existência de interações hidrofóbicas em

complexos entre taninos e proteínas, em particular em proteínas ricas em prolina

(PRPs) ou péptidos similares, sendo também as ligações por pontes de

hidrogénio apontadas como ligações importantes (Tabela 3). Na verdade, vários

estudos sugerem que as interações tanino/proteína são inicialmente governadas

por interações hidrofóbicas e os complexos são, então, estabilizados por pontes

de hidrogénio (Oh et al. 1980, Haslam 1996). As ligações de hidrogénio são

forças que, apesar de serem relativamente fracas individualmente, no seu

conjunto são importantes para a estabilização dos complexos tanino/proteína.


Fig.10 Esquema representativo das interações entre taninos e proteínas, evidenciando o tipo de ligações mais relevante bem como os grupos de cada molécula (do tanino e da proteína) envolvidos em cada tipo de ligação. Adaptado de Asano et al. (1982).

.

O

OH

OH

OH

OH

OH

Região

hidrofílica

Região

hidrofóbica

Tanino

NH3 CH

O

N CH

HCH3 CH2

N

O

CH

COO

CHCH3 CH3

CH2 CH2

CH2

H+ _

Regiões

hidrofóbicas

Regiões

hidrofílicas

Proteína

N

NHCO

CO

NHC O

NHCO

NCO

COO

NH3

OOH

OH

OHOH

HOH

OOH

OH

OHOH

HOH

+

_

OOH

OH

OHOH

HOH

OOH

OH

OHOH

HOH

NH

N

O

C

NHC

O

O

NC

NHC

O

O

NH3

COO

+

_TaninoProteína Proteína

Pontes de hidrogénio Ligações hidrofóbicas



55

Tabela 3. Resumo de alguns trabalhos que evidenciam o tipo e caraterísticas importantes das interações tanino/proteína, indicando os taninos e proteínas estudados, bem como as metodologias utilizadas no estudo.

Tanino Proteína/Técnica Conclusão Referência Taninos condensados de grainhas de uva

Poli-aa de Pro, hidroxiprolina e Lys, BSA, gelatina, citocromo c / Cromatografia

Interações hidrofóbicas entre a gelatina ou a poli-prolina e os taninos

Oh et al. (1980)

PGG Dois péptidos (19 e 22 aa) com sequência repetitiva rica em Pro, Gli e Glu / RMN

Locais de ligação maioritários são, não só os resíduos de prolina, mas também a ligação amida e o aa precedentes

Murray e Williamson (1994)

Catequina e seu dimero

Poli-Pro / RMN Interações hidrofóbicas entre os anéis benzénicos da catequina e os resíduos hidrofóbicos conformacionalmente acessíveis, mas sem preferência para a prolina

Hatano e Hemingway (1996)

(-)-EC, PGG, tetraGG, triGG

PRP salivar humana (IB-5) / RMN

A IB-5 enrola-se no tanino aumentando as interações cooperativas intramoleculares. A rigidez das moléculas é importante para estas interações, sendo maior a interação para moléculas relativamente rígidas

Charlton et al. (1996)

(-)-Epicatequina, B2, galhoilglucoses, galhato de propilo

Péptido (19 aa) igual a um fragmento de PRP salivar de rato / RMN

Interações hidrofóbicas do tipo “stacking” do anel benzénico do polifenol com a face do anel pirrolidina da prolina

Baxter et al. (1997)

EGCG Heptapéptido rico em Pro / RMN As prolinas estão quase sempre envolvidas em interações hidrofóbicas do tipo “stacking” com preferência para envolver a primeira prolina de um grupo de duas

Charlton et al. (2002)

B3 Fragmento de 14 aa de PRP salivar humana (IB-7) / RMN em fase sólida, MS em MALDI e ESI, DC

A B3 liga-se à região hidrofílica do péptido, sugerindo que a interação é dominada por pontes de hidrogénio entre os grupos carbonilo dos resíduos de prolina e os grupos OH fenol e catecol

Simon et al. (2003)

EGCG β-caseína / Single Molecule Force Microscopy

Interações hidrofóbicas multivalentes Jöbstl et al. (2006)

EGC, ECG, EGCG, B2 e derivado galhato

PRP salivar humana (IB-5) / ESI-MS e tandem MS

Na fase gasosa, as interações são governadas por pontes de hidrogénio

Canon et al. (2010)

aa – aminoácidos, BSA – albumina sérica sovina, CD – dicroismo circular, ECG – galhato de epicatequina, EGC – galhato de epigalhocatequina, ESI – electrospray ionization, MALDI – matrix assisted laser desorption ionization, MS – espectroscopia de massa, PGG – pentagalhoilglucose, PRP – proteína rica em prolina, RMN – ressonância magnética nuclear, tetraGG – tetragalhoilglucose, triGG - trigalhoilglucose

Em suma, estes estudos indicam que tanto as interações hidrofóbicas como as

ligações por pontes de hidrogénio podem estar presentes entre taninos e

proteínas, fortalecendo-se mutuamente e sendo afetadas por diversos fatores,

em particular a estrutura da proteína e do tanino, e as condições do meio

(solvente, pH, entre outros).

As interações hidrofóbicas incluem interações de Van der Waals ou π-π stacking

entre o anel B dos taninos rico em electrões ou o grupo galhoilo e a face planar

pro-S das ligações amida heterocíclicas da prolina.

Nas PRPs, os principais locais de ligação são os resíduos de prolina, sendo um

local particularmente favorável à ligação o primeiro resíduo de prolina de uma

sequência Pro-Pro. O resíduo de prolina pode ser considerado como um bom


local de ligação já que proporciona uma superfície plana, rígida e hidrofóbica

favorável para interações com outras superfícies planas e hidrofóbicas como os

anéis benzénicos. As principais caraterísticas e propriedades dos resíduos de

prolina e das PRPs serão abordadas mais à frente.

5.2 Modelos da interação tanino/proteína

Estudos da interação da BSA (Hagerman e Robbins 1987) e de um fragmento

sintético de 19 resíduos de uma PRP com taninos (Baxter et al. 1997) sugerem

que os taninos se comportam como ligandos multidentados, isto é, diferentes

anéis fenólicos da mesma molécula podem interagir com a proteína. Isto permite

aos taninos estabelecerem ligações cruzadas entre duas ou mais moléculas

proteicas, ou ligar-se a mais do que um ponto da cadeia peptídica de uma

proteína. Deste modo, a estequiometria e tamanho dos complexos

tanino/proteína dependem das suas concentrações relativas, i. e., da razão

tanino/proteína.

Hagerman e Robbins (1987) verificaram que adicionando quantidades

crescentes de BSA a uma quantidade fixa de tanino, existia uma razão

tanino/proteína óptima para a qual ocorria precipitação máxima (concentração

estequiométrica). Para razões tanino/proteína superiores ou inferiores, a

quantidade de complexos insolúveis diminuía. Um modelo proposto para explicar

estas observações surgiu mais tarde (Siebert et al. 1996) e está esquematizado

na Fig.11.

Fig.11 Modelo da interação tanino/proteína. Os taninos são exibidos com duas extremidades as quais podem ligar-se às proteínas. As proteínas são apresentadas como possuindo um número fixo de locais de ligação. Adaptado de Siebert et al. (1996).

Considerando que cada molécula de tanino e de proteína têm um número fixo de

locais de ligação, a situação em que ocorre a maior interação possível é aquela

em que o número de locais de ligação dos taninos iguala o número de locais de

ligação da proteína, resultando na formação de agregados maiores e um

máximo de turvação (plateau). Se houver excesso de proteína relativamente ao



57

tanino, cada tanino é capaz de estabelecer pontes entre duas moléculas de

proteína, mas será improvável que estas proteínas formem mais pontes com

outras proteínas. Isto resultará principalmente em dímeros proteicos, ou seja,

agregados mais pequenos. Com tanino em excesso, todos os locais de ligação

da proteína estarão ocupados, mas a probabilidade de se formarem pontes será

baixa porque cada extremidade livre do tanino terá poucas hipóteses de

encontrar um local de ligação de uma proteína livre. Isto também resultará em

agregados mais pequenos (Fig.12). Este modelo faria prever o aumento de

agregação à medida que se aumentam simultaneamente as concentrações de

proteína e tanino, o que na prática é o que é observado (Siebert et al. 1996).

Deste modo, na maioria dos casos, a agregação está hiperbolicamente

relacionada tanto com a concentração de proteína como com a de tanino

(Fig.12). O tipo de agregados tanino-proteína formado depende das suas

concentrações relativas. Nas concentrações estequiométricas (plateau), os

taninos são capazes de atuar como ligandos multidentados, estabelecendo

pontes entre proteínas ou complexos tanino-proteína, correspondendo a um

máximo de agregação com agregados enormes. Para razões tanino-proteína

mais baixas ou mais elevadas, formam-se agregados mais pequenos.

Fig.12 Esquema de uma curva de nefelometria para uma concentração fixa de tanino e concentrações crescentes de proteína. Adaptado de Hagerman e Robbins (1987).

No entanto, aparentemente, este modelo pode não se aplicar a todas as

proteínas. Por exemplo, para o caso das PRPs o comportamento parece ser

distinto. A adição de quantidades crescentes de uma PRP salivar a uma

quantidade fixa de tanino levou à formação de complexos insolúveis

tanino/proteína, os quais permaneceram insolúveis independentemente da

Tur

vaçã

o

Concentração de proteína

Excesso de tanino

Excesso de proteína

Estequiometria Plateau


quantidade de proteína adicionada. Deste modo, foi proposto um mecanismo

molecular alternativo para a interação dos taninos com as PRPs (Fig.13).

PRP Tanino

1ª fase 2ª fase 3ª fase

Proteína com Compactação da Uma segunda Agregação das enrolamento proteína após proteína é revestida proteínas revestidas aleatório ligação aos por taninos formando complexos

(PRP) taninos extensos

Fig.13 Mecanismo molecular proposto para a interação das PRPs com os taninos. Na fase inicial (1ª fase) as proteínas compactam-se, devido a ligações apertadas múltiplas com os taninos multidentados. Na 2ª fase forma-se um dímero com outra proteína recoberta de taninos, tornando o complexo insolúvel. Na 3ª fase, há uma maior complexação e ocorre precipitação do complexo. Adaptado de Jöbstl et al. (2004).

Os princípios gerais deste mecanismo são similares ao mecanismo

anteriormente descrito. A associação dos taninos com as proteínas é um

fenómeno principalmente superficial. Numa primeira fase, para concentrações

baixas de tanino, este associa-se à superfície das PRPs ligando-se

simultaneamente várias moléculas de tanino a uma molécula de proteína e o

mesmo tanino a vários locais da proteína. A proteína, que inicialmente

apresentava uma conformação aleatória, envolve os taninos enrolando-se e

tornando a sua estrutura mais compacta, ficando como que revestida pelos

taninos. Com o aumento da concentração de tanino (2ª fase), os taninos

estabelecem ligações cruzadas entre diferentes moléculas proteicas, tornando o

complexo insolúvel e a solução coloidal. Na terceira fase, ocorrem mais ligações

cruzadas aumentando a agregação e levando à precipitação (Luck et al. 1994,

Jobstl et al. 2004).

A observação de que o comportamento dos taninos com as PRPs é distinto do

comportamento com as outras proteínas tem implicações interessantes na

função defensiva proposta para as PRPs (Luck et al. 1994), o que será abordado

na secção 6.1.



59

5.3 Fatores que influenciam a interação tanino/prot eína

A interação tanino/proteína depende de vários fatores e, obviamente das

caraterísticas do tanino e da proteína. O tipo de tanino e proteína (tamanho

molecular, grupos funcionais, conformação, entre outros), bem como as

condições do meio (solvente, força iónica, pH, temperatura e presença de outros

compostos, entre outros) afetam esta interação. A influência dos parâmetros

mais importantes para este trabalho vai ser discutida nas próximas secções.

5.3.1 Estrutura dos taninos

Vários aspetos estruturais dos taninos influenciam a capacidade de ligação às

proteínas, nomeadamente o peso molecular e a estereoquímica da molécula, o

tipo de ligação interflavânica, projeção dos grupos hidroxilo e presença de

grupos galhoilo (Okuda et al. 1985, Ricardo-Da-Silva et al. 1991, Kawamoto et

al. 1995, Baxter et al. 1997, De Freitas e Mateus 2001).

De uma forma geral, a interação dos taninos com as proteínas aumenta com o

grau de polimerização, peso molecular e extenção de galhoilação, resultando

provavelmente da presença de mais anéis fenólicos e grupos o-fenólicos.

A influência do peso molecular do tanino é evidenciada por vários estudos

(Baxter et al. 1997, Sarni-Manchado et al. 1999, De Freitas e Mateus 2001,

Canon et al. 2010). Baxter e os colaboradores (1997) estudaram por RMN a

interação de vários taninos (e.g procianidina dimérica B2, PGG e (-)-

epicatequina propil galhato) com um fragmento sintético (19 resíduos) de uma

PRP e concluíram que os compostos maiores e mais complexos interagem mais

com o péptido pela seguinte ordem: (-)-epicatequina propil galhato << PGG <

dímero B2. Os compostos mais pequenos podem ligar apenas um anel fenólico

a um resíduo de prolina enquanto os compostos maiores ocupam dois ou três

resíduos de prolina consecutivos. Os polifenóis mais complexos interagem como

ligandos multidentados.

Sarni-Machado e seus colaboradores (1999) obtiveram também a mesma

tendência estudando a interação de taninos condensados isolados de grainhas

de uvas e proteínas salivares; eles analisaram o sobrenadante e o precipitado

resultante da interação por SDS-PAGE e tiólise ácida. Neste estudo observaram

que as procianidinas de elevado peso molecular, o que neste caso correspondia

a um elevado grau de polimerização, foram selectivamente precipitadas pelas


proteínas salivares, enquanto as procianidinas diméricas e triméricas

permaneceram em solução.

De Freitas e Mateus (2001) estudaram a interação de taninos condensados (e.g.

(+)-catequina, procianidinas diméricas e triméricas, galhato de (-)-epicatequina,

procianidina B2 galhato) com proteínas salivares humanas, nomeadamente α-

amilase e PRPs, por nefelometria. Neste estudo observaram o efeito do peso

molecular, bem como o efeito da extenção de galhoilação e da estereoquímica.

Relativamente ao efeito do peso molecular os resultados obtidos são

concordantes com os restantes estudos, sendo os dímeros e os trímeros mais

reativos que os monómeros. Relativamente à estereoquímica observaram que a

(+)-catequina era mais reativa com as PRPs do que a (-)-epicatequina e que os

dímeros com ligação interflavânica C4-C8 eram mais reactivos que os

correspondentes dímeros com ligação interflavânica C4-C6. Isto possivelmente

deve-se a restrições conformacionais impostas pelas ligações do tipo C4-C6.

A presença de grupos galhoilo também afeta a interação dos taninos com as

proteínas pois induz um aumento de locais no tanino aptos a ligarem-se à

mesma proteína ou a proteínas diferentes. De facto, de Freitas e Mateus (2001)

observaram que a presença de um grupo galhoilo aumentava a reatividade

comparativamente com os correspondentes não galhoilados. No entanto, esse

aumento de reatividade foi maior no caso dos monómeros do que no caso dos

dímeros. Este facto foi explicado com base na estrutura fechada que o dímero

B2 -3”-O-galhato apresenta como resultado de uma possível interação “π-π

stacking” entre os anéis benzénicos do grupo galhoílo e catecol (Fig.14).

Também Okuda e seus colaboradores (1985) observaram o efeito da presença

de grupos galhoilo na interação tanino/proteína. Eles determinaram

colorimetricamente a ligação da hemoglobina e azul de metileno a diversos

taninos isolados de plantas (e.g. ácido gálhico, PGG, (-)-epicatequina,

procianidina dimérica B2 e derivados galhoilados) de peso molecular e grau de

galhoilação diferentes. Neste estudo observaram que os compostos de baixo

peso molecular tinham uma afinidade de ligação nula ou muito baixa, e que o

aumento do peso molecular, sobretudo induzido pela extensão de galhoilação,

levou a um aumento da ligação de 35%.



61

Fig.14 Conformações da (-)-epicatequina, dímero B2 e seus derivados galhoilados, obtidos por mecânica molecular e RMN. Adaptado de de Freitas et al. (1998).

Este efeito da presença de grupos galhoilo também é visível nos taninos

hidrolisáveis. Kawamoto e seus colaboradores (1995) observaram que a

afinidade de uma série de galhoilglucoses para a BSA aumenta com a extensão

da galhoilação: monogalhoilglucose < di- < tri- < tetra- < penta. Para além do

número de grupos galhoilo também a sua posição nos taninos hidrolisáveis afeta

a interação com proteínas. Os mesmos autores observaram que a afinidade da

2,3,6- é superior à da 2,3,4-tri-O-galhoilglucose e a afinidade da 4,6- é superior à

da 2,3-di-O-galhoilglucose.

De acordo com alguns autores, os taninos hidrolisáveis interagem com as

proteínas de modo similar aos taninos condensados regendo-se pelos mesmos

princípios gerais (Hagerman et al. 1998, Tang et al. 2003, Gyémánt et al. 2009) e

revelando, em alguns casos, mais afinidade para as proteínas (Bacon e Rhodes

1998, 2000, Soares et al. 2007, Obreque-Slier et al. 2010).

Por exemplo, Gyémánt e colaboradores (2009) demonstraram que a PGG inibe

a amilase salivar humana e esta interação pode envolver os anéis aromáticos da

PGG, sugerindo um stacking com as cadeias laterais aromáticas dos resíduos

da proteína. Este tipo de interação é semelhante às ligações stacking sugeridas

para a interação dos taninos condensados com as proteínas.

Bacon e Rhodes (2000) observaram que as várias famílias de PRPs (acídicas,

básicas e glicosiladas) apresentavam elevada afinidade para os taninos


hidrolisáveis (afinidade igual ou superior a monómeros de taninos condensados

estudados anteriormente (Bacon e Rhodes 1998)) e que essa afinidade estava

relacionada com a estrutura dos taninos, em particular com a extenção de

galhoilação e grau de polimerização.

5.3.2 Estrutura das proteínas

Outro fator que influencia a interação tanino/proteína é a estrutura das proteínas.

A estrutura primária, i.e., a sequência de resíduos de aminoácidos, bem como a

estrutura terciária das proteínas, o tamanho e a carga da proteína são aspectos

importantes na interação com os taninos.

Uma das primeiras observações de que a capacidade para os taninos

precipitarem proteínas diferentes varia consideravelmente foi feita por Hagerman

e Butler (1981). Estes autores estudaram a especificidade desta interação por

um ensaio de competição em que mediram a percentagem de precipitação de

uma proteína (BSA) marcada radioativamente na presença e ausência de outra

proteína (competidora). Estes autores observaram que a afinidade relativa das

proteínas para os taninos condensados pode variar mais de 100 vezes e que é

uma interação muito específica. As proteínas globulares com enrolamento mais

compacto têm afinidades muito inferiores para os taninos do que as proteínas

conformacionalmente mais abertas.

Um dos fatores que afecta a interação tanino/proteína é a estrutura primária das

proteínas. Wroblewski e seus colaboradores (2001) verificaram por RMN que a

ligação de taninos a histatinas modificadas (i.e., com os mesmos resíduos de

aminoácidos, mas em que os resíduos de aminoácidos foram dispostos

aleatoriamente) foi significativamente diminuída. Esta observação é suportada

por evidências recentes de que os taninos se ligam a locais muito específicos

das proteínas (Cala et al. 2010), os quais são alterados se houver modificação

da sequência.

Uma caraterística comum às proteínas e péptidos com elevada afinidade para os

taninos parece ser um elevado teor em resíduos de prolina. A gelatina contém

18% de prolina e de hidroxiprolina e a PRP da parótida de rato contém 40% de

prolina e ambas as proteínas são conhecidas por uma elevada afinidade para os

taninos. No entanto, a afinidade das proteínas não é intrínseca aos anéis

heterocíclicos de prolina, já que a prolina e seus derivados apresentam



63

afinidades para os taninos semelhantes à mostrada por outros aminoácidos

acíclicos como a glicina ou a alanina (Hagerman e Butler 1981).

Para além disto, o trabalho de Richard e seus colaboradores (2005) apresenta

evidências de que pode não existir interação entre os polifenóis e os resíduos de

prolina se o ambiente circundante a esses resíduos não for adequado à

interação. Richard e seus colaboradores estudaram a interação entre polifenóis

(trans-resveratrol e PGG) e a neurotensina (um péptido biológico de 13 resíduos

de aminoácidos com conformação semelhante às PRPs) por RMN estrutural e

modelação molecular. Estes autores demonstraram que a sequência primária

tem um papel importante na interação com os polifenóis. Neste trabalho, os

autores não observaram stacking hidrofóbico entre os polifenóis e os resíduos de

prolina, mas em estudos anteriores com complexos de bradiquinina-PGG (Vergé

et al. 2002) e noutro trabalho com um péptido rico em prolina (Charlton et al.

2002) essas interações foram observadas. Com base nisto, Richard e seus

colaboradores sugeriram que o ambiente estereoquímico dos resíduos de prolina

é muito importante. No caso da neurotensina, os resíduos de prolina estão

desarranjados e desorientados pelos resíduos adjacentes de arginina, tirosina e

lisina e as suas cadeias laterais volumosas previnem a aproximação dos anéis

do polifenol à prolina. Por outro lado, no caso da bradiquinina e do péptido rico

em prolina, a presença de dois resíduos consecutivos de prolina liberta do

esqueleto peptídico os anéis da prolina, permitindo assim uma aproximação

mais fácil dos anéis fenólicos. Esta aproximação é ainda reforçada pela

presença de um resíduo de glicina em ambos os casos, dado que este

aminoácido não é volumoso e confere um certo grau de flexibilidade ao

esqueleto peptídico. Deste modo, o ambiente que envolve os resíduos de prolina

parece ter um papel determinante na interação destes resíduos com os

polifenóis. Aparentemente, os resíduos de prolina têm de estar posicionados

correctamente na sequência concedendo uma certa flexibilidade para dar origem

a uma interação forte com os polifenóis.

Como tem vindo a ser referido, os resíduos de prolina, para além de poderem

ser um local de ligação, são resíduos que induzem aos péptidos uma

conformação aberta, levando a uma maior exposição de locais de ligação aos

taninos comparativamente a proteínas com uma estrutura mais compata e

globular. Proteínas que têm estruturas globulares compactas, tais como a

ribonuclease A, lisozima e mioglobina têm afinidades inferiores para os taninos

do que proteínas globulares menos fechadas e estruturadas como a BSA e a


histona F1 (Fig.15). Isto ocorre provavelmente por causa da acessibilidade

acrescida do esqueleto peptídico nas proteínas menos compactas. Por exemplo,

a ribonuclease A tem metade da afinidade para os taninos que a mesma

proteína oxidada pelo ácido perfórmico, porque esta última tem uma estrutura

com enrolamento aleatório enquanto a proteína nativa tem uma estrutura mais

compacta (Hagerman e Butler 1981).

BSA Mioglobina Colagénio

Fig.15 Representação no modo space filling da estrutura terciária da albumina sérica bovina (BSA), mioglobina e gelatina as quais apresentam conformações diferentes. A mioglobina e a BSA têm estrutura terciária globular, sendo a estrutura da mioglobina mais fechada e aparecendo aqui apresentada com o grupo heme (grupo prostético essencial para a sua função). O colagénio tem uma estrutura enrolada em tripla hélice apresentando uma conformação mais aberta que as outras proteínas.1

Este conjunto de dados permite afirmar que a estrutura primária, a qual se reflete

na estrutura terciária das proteínas, são fatores importantes e determinantes na

interação com os taninos.

5.3.3 Presença de carboidratos

A interação tanino/proteína pode ser influenciada pela presença de outros

compostos, nomeadamente, carboidratos.

As primeiras conceções de que a presença de carboidratos poderia inibir esta

interação surgiram no âmbito da compreensão do amadurecimento dos frutos,

que se traduz por uma importante diminuição da sensação de adstringência.

1 As imagens da estrutura terciária das proteínas foram retiradas de: BSA: http://www.pdb.org/pdb/education_discussion/molecule_of_the_month/images/1e7i.gif Mioglobina: http://courses.washington.edu/conj/protein/myo.gif Gelatina: http://www.chm.bris.ac.uk/pt/polymer/images/johnmarshall/gelatin.gif

Grupo heme



65

5.3.3.1 Amadurecimento de frutos

Durante o amadurecimento dos frutos surgem várias alterações de natureza

físico-químicas a sensoriais. Duas das principais alterações consistem na

diminuição da adstringência e na alteração da textura, com os frutos a tornarem-

se mais moles e macios.

A alteração da textura tem sido explicada com base na degradação enzimática

de carboidratos estruturais da parede celular (pectina, hemicelulose e celulose) e

de carboidratos de armazenamento (Wakabayashi 2000, Prasanna et al. 2007).

Neste processo têm particular importância as pectinas dado que são os

constituintes principais da parede celular e praticamente os únicos constituintes

da lamela média (Fig.16).

Para além da degradação de carboidratos, o amolecimento dos frutos é

acompanhado de uma redução da adesão celular, como resultado da

degradação da lamela média. Portanto, globalmente, a degradação dos

carboidratos leva a uma diminuição da integridade da parede celular que por fim

resulta na perda de firmeza dos tecidos da fruta, como esquematizado na Fig.16.

A

B

Fig.16 Representação esquemática da (A) estrutura da parede celular primária evidenciando os carboidratos mas importantes e a lamela média2, e da (B) degradação da pectina e da hemicelulose durante o amadurecimento dos frutos. A degradação destes carboidratos reduz a integridade das paredes celulares, diminuindo a rigidez dos frutos (adaptado de Wakabayashi (2000)).

2 Adaptado de http://upload.wikimedia.org/wikipedia/commons/thumb/5/53/Plant_cell_wall_diagram.svg/250px-Plant_cell_wall_diagram.svg.png

Lamela média

Parede celular

primária

Membrana

plasmática

Pectina

Microfibrila

Celulose

Hemicelulose

Proteínas solúveis

Enzimas que hidrolisam hemicelulose

HemicelulosePectina

Amadurecimento

Microfibrila de celulose

Enzimas que hidrolisam pectina


Relativamente à diminuição da adstringência, duas linhas de pensamento

principais foram propostas ao longo dos anos para explicar esta alteração.

Uma das hipóteses consiste na modificação da composição polifenólica dos

frutos ao longo do amadurecimento, em particular na alteração do peso

molecular/grau de polimerização dos taninos (Goldstein e Swain 1963, Haslam

1998). Alguns estudos associam a diminuição de adstringência à diminuição da

concentração em taninos nos frutos. De acordo com Goldstein e Swain (1963) só

os polifenóis de tamanho médio são eficazes na interação com proteínas: os de

baixo peso molecular são muito pequenos e os de elevado peso molecular ou

não são suficientemente solúveis ou têm uma estrutura muito grande impedindo-

os de encaixar na matriz das proteínas salivares. Estes autores observaram que

na maturação de alguns frutos houve um declínio destes polifenóis. Estes

mesmos autores também avançaram a hipótese de que a perda de adstringência

se deve a um aumento do grau de polimerização dos taninos condensados,

afectando a sua solubilidade e extração. Para além disso, outros estudos sobre

a evolução da composição polifenólica de uvas tintas ao longo do processo de

maturação mostram que o teor em proantocianidinas totais diminui (Mateus et al.

2001).

No entanto, apesar de alguns estudos suportarem esta teoria, em alguns frutos

não há qualquer alteração da sua composição polifenólica e portanto a

diminuição de adstringência tem de ser explicada por outros factos.

Outra linha de pensamento para explicar a diminuição da adstringência durante

o amadurecimento reside na capacidade dos carboidratos presentes ou

libertados da parede celular interagirem com as proteínas salivares e/ou com os

taninos. Esta hipótese tem as suas origens há aproximadamente 100 anos e tem

vindo a ser suportada pela literatura até aos dias de hoje (Joslyn e Goldstein

1964, Ozawa et al. 1987, Haslam et al. 1992, Luck et al. 1994, Carvalho et al.

2006). As primeiras observações apontavam para que os taninos dos frutos se

ligassem à celulose. Mas, foi com o trabalho de Ozawa e colaboradores que

apareceram as evidências que suportaram esta teoria. Ozawa e seus

colaboradores (Ozawa et al. 1987) averiguaram a capacidade dos polifenóis

inibirem a enzima β-glucosidase na presença de outros compostos,

nomeadamente dos carboidratos poligalacturonato de sódio e ciclodextrinas.

Este trabalho demonstrou que estes compostos foram capazes de inibir a

interação do polifenol com a proteína e assim recuperar a actividade enzimática

da β-glucosidase. Os autores justificaram a ação destes compostos pela

capacidade dos carboidratos formarem bolsas hidrofóbicas em solução capazes



67

de encapsular e complexar os polifenóis, impedindo-os de interagir com a

enzima.

5.3.3.2 Mecanismos de inibição da interação tanino/proteína pelos

carboidratos

Com base nestes trabalhos e nas suas próprias observações, Haslam e seus

colaboradores (1992, Luck et al. 1994) propuseram dois mecanismos pelos

quais os carboidratos poderiam inibir a interação tanino/proteína (Fig.17):

(a) Os carboidratos são geralmente polielectrólitos (macromoléculas que

contêm grupos carregados) e, portanto, podem formar complexos

ternários com o complexo tanino/proteína, tornando-o solúvel em meio

aquoso – mecanismo do complexo ternário;

(b) Os carboidratos, devido à estrutura terciária (tipo gel) adquirida em

solução, têm capacidade para encapsular os polifenóis, ou total ou

parcialmente e, assim inibir a sua interação com as proteínas –

mecanismo de competição.

Fig.17 Mecanismos possíveis de inibição da agregação dos taninos com as proteínas pelos carboidratos. P: proteína, T: tanino, C: carboidrato. Adaptado de Mateus et al. (2004).

Esta capacidade dos carboidratos inibirem a interação tanino/proteína tem sido

documentada por diversos trabalhos, apesar de não ser uma investigação fácil

devido à escassez de carboidratos solúveis em água com estrutura e peso

molecular bem definidos. A grande maioria dos trabalhos foca-se principalmente

na ligação de polifenóis a carboidratos das paredes celulares das uvas pois este

é um aspecto muito importante no processo vinícola e que afeta a extração dos

Complexo ternário solúvel carboidrato/tanino/proteína

Complexo insolúvel tanino/proteína

Complexo solúvel carboidrato/tanino proteína

+

(a)

(b)


taninos das uvas. Na verdade, as evidências principais que suportam a interação

dos taninos com os carboidratos da parede celular surgiram em experiências

utilizando carboidratos modelo como a ciclodextrina, pectina, xantana, entre

outros e géis de carboidratos (Mcmanus et al. 1985, Luck et al. 1994, De Freitas

et al. 2003, Carvalho et al. 2006, Hanlin et al. 2010).

Luck e seus colaboradores (1994) verificaram a existência de diversos

carboidratos que inibiram a interação entre a gelatina e um tanino hidrolisável, a

PGG. Estes autores observaram que a eficiência de inibição varia com as

caraterísticas do carbohidrato, sendo a pectina, a goma arábica, a xantana e a

gelana os mais eficazes a prevenir a interação.

De Freitas e seus colaboradores (2003) também observaram a importância da

estrutura dos carboidratos na inibição da interação tanino/proteína. Estes

autores estudaram a influência de vários carboidratos na interação da BSA com

procianidinas oligoméricas por nefelometria. No geral, todos os carboidratos

testados foram eficientes em inibir esta interação, sendo observado um

comportamento diferente para os carboidratos iónicos e neutros. Os carboidratos

aniónicos, como a pectina, a xantana, o ácido poligalacturónico e a goma

arábica, foram muito mais eficientes na sua ação inibidora, o que sugere que as

interações hidrofílicas são dominantes na sua ação. Por outro lado, os

carboidratos neutros, como a glucose, dextrana, β-ciclodextrina e

arabinogalactana, foram menos eficientes na inibição da interação

tanino/proteína. Isto foi explicado com base no baixo carácter iónico destes

carboidratos, o que leva a uma baixa afinidade para eles complexarem com os

polifenóis.

Também Carvalho e seus colaboradores (2006) observaram que o carácter

iónico dos carboidratos é importante para a sua ação. Estes autores estudaram

o efeito de carboidratos isolados do vinho tinto (arabinogalactana-proteinas,

AGPs e ramnogalacturonanas do tipo II, RGII) na interação de uma PRP ou da

α-amilase com procianidinas oligoméricas. Este trabalho mostrou que a inibição

da agregação tanino/proteína com ambas as proteínas estava relacionada com

as cargas dos carboidratos: os mais eficientes eram os que apresentavam maior

carácter iónico (AGPs).

Com estes trabalhos verifica-se que é importante a interação entre os taninos e

os carboidratos.

Deste modo, torna-se relevante que os carboidratos têm de ter uma estrutura e

composição (carácter iónico) adequadas, bem como tamanho molecular e



69

flexibilidade capazes de complexar os polifenóis ou de interagir com os

complexos tanino/proteína aumentando a sua solubilidade.

Alguns estudos indicam que a estrutura do polifenol também é importante para o

efeito dos carboidratos. McManus e seus colaboradores (1985) estudaram a

interação entre os taninos e carboidratos por cromatografia usando gel de

dextrana. Estes autores observaram que os compostos aromáticos, em particular

os taninos, tinham uma grande afinidade para o gel de dextrana, a qual foi

aumentada pela presença de grupos hidroxilo na estrutura aromática. Apesar do

mecanismo pelo qual esta associação acontece ser ainda desconhecido, foi

proposto que os taninos são adsorvidos nos poros do gel devido a interações

entre os grupos hidroxilo dos taninos e os átomos de oxigénio envolvidos nas

ligações glicosídicas.

O efeito da pectina na adstringência de catequinas também já foi estudado em

soluções modelo. Hayashi e seus colaboradores (2005) estudaram o efeito da

pectina na adstringência de catequinas por espectroscopia de RMN e por um

“sensor de adstringência” (medição de diferenças de potencial entre a solução

de tanino e uma solução de referência). Estes autores observaram que a pectina

diminuiu a adstringência de catequinas com grupos galhoilo (EGCG e ECG)

enquanto nas catequinas não galhoiladas (EGC e epicatequina) não foi

observado nenhum efeito. Estes autores também observaram com base nas

alterações do deslocamento químico nos espetros de RMN que as catequinas

galhoiladas têm maior afinidade do que as não galhoiladas para interagir com a

pectina. Estes resultados apontam que a formação de complexos catequina-

pectina pode ser um fator importante na redução da adstringência.

Em suma, as conclusões principais dos diversos trabalhos apresentados são

que os polifenóis e os carboidratos podem formar complexos, sendo esta

interação governada pelos mesmos princípios que a interação com as proteínas.

A interação dos carboidratos com os polifenóis pode inibir a interação destes

com as proteínas ou pode ocorrer quando estes já formaram complexos com as

proteínas. A adsorção dos polifenóis aos carboidratos é mediada por interações

hidrofóbicas e pontes de hidrogénio, sendo a primeira favorecida pela existência

de cavidades hidrofóbicas, como por exemplo a cavidade interna das

ciclodextrinas. A afinidade é fortemente influenciada pelo peso molecular e

flexibilidade conformacional do polifenol. Para além disto, o estado e

conformação dos carboidratos também são importantes: os polifenóis ligam-se a


géis de dextrana mas não se ligam a oligómeros ou pequenos polímeros de

dextrana (Williamson et al. 1995).

5.3.4 Outros fatores (força iónica, pH, etanol e temperatura)

Para além dos carboidratos, vários outros fatores afetam a interação

tanino/proteína, nomeadamente a força iónica, o pH, o etanol e a temperatura.

Carvalho e seus colaboradores (2006) observaram um efeito oposto da força

iónica para a interação de duas proteínas diferentes com um tanino; enquanto

para uma PRP o aumento da força iónica levou a um grande aumento da

interação, para a α-amilase levou a uma ligeira diminuição da interação com o

tanino. A explicação sugerida assentou no ponto isoelétrico (pI) das proteínas;

ao pH a que se efetuaram as experiências (pH 5) a α-amilase provavelmente

estaria menos carregada positivamente que a PRP e por isso a força iónica não

provocou nenhuma alteração. Por outro lado, no caso da PRP houve

provavelmente uma maior hidratação dos iões com o aumento da força iónica, o

que removeu a água da estrutura dos agregados promovendo as interações

tanino/proteína.

No entanto, os estudos nesta área são controversos e aparentemente o efeito da

força iónica também depende da proteína e tanino em questão (Oh et al. 1980,

Siebert et al. 1996, De Freitas et al. 2003, Rawel et al. 2005).

O pH do meio está diretamente relacionado com a interação tanino/proteína

dado que ele afeta o grau de ionização de ambos os componentes. Em geral, a

interação e precipitação diminuem a pH baixos (<2) e elevados (>8) e é máxima

a pH próximo do pI da proteína onde as repulsões entre as proteínas são

minimizadas (Hagerman e Butler 1981, Yan e Bennick 1995, Naczk et al. 1996).

Relativamente ao efeito da temperatura, a literatura existente também não é

coerente. Por exemplo, Hagerman e seus colaboradores (1998) observaram que

a precipitação da BSA pela PGG aumenta com a temperatura. Também Siebert

e seus colaboradores (1996) estudaram a formação de precipitados entre

diversas proteínas (e.g. gelatina, lisozima, péptido rico em prolina, gliadina) e

taninos observaram que o aumento de temperatura conduzia a um aumento de

precipitação. Por outro lado, Rawel e seus colaboradores (2005) observaram em

estudos de ligação de diferentes taninos à BSA e HSA que o aumento da

temperatura levou a uma diminuição da interação. Portanto, também este fator

parece depender quer da proteína quer do tanino em questão.



71

6. Saliva humana

A saliva humana é uma mistura aquosa complexa de proteínas e minerais e tem

diversas funções fisiológicas. Devido às suas ações de lubrificação,

antibacteriana e tamponante contribui para a manutenção da integridade da

cavidade oral, mas também funciona como primeira etapa da digestão devido à

presença das enzimas α-amilase e lipase. A saliva é também importante na

perceção e sabor dos alimentos, dado que é o meio através do qual estas

propriedades são percebidas.

O fluído oral, vulgarmente referido como “saliva total (whole saliva)”, é composto

maioritariamente pelas salivas das glândulas maiores (parótida, submandibular,

sublingual), das glândulas salivares menores e também pelos fluidos das

gengivas.

A composição proteica da saliva tem sido alvo de interesse nos últimos anos, e o

conhecimento do proteoma salivar tem avançado muito, em particular na última

década com o desenvolvimento de técnicas de proteómica novas e potentes. O

avanço e o recurso a estas técnicas permitiu identificar diferentes classes de

proteinas e péptidos, muitas delas específicas da cavidade oral. As principais

classes estão apresentadas na Fig.18 e as mais importantes para este trabalho

serão abordadas nas secções seguintes.

Fig.18 Percentagens aproximadas (p/p) das principais classes de proteínas e péptidos salivares na saliva total. Adaptado de Messana et al. (2008). ASH, albumina sérica humana; sIgA, imunoglobulina A secretora; IgG, imunoglobulina G; aPRPs, proteínas-ricas em prolina acídicas; bPRPs, proteínas-ricas em prolina básicas; gPRPs, proteínas-ricas em prolina glicosiladas.

6.1 Proteínas ricas em prolina (PRPs)

As PRPs são, como o próprio nome indica, ricas no aminoácido prolina e, como

observado na Fig.18, são das classes mais abundantes na saliva humana.


Recentemente, estas proteínas têm sido consideradas uma classe da família das

proteínas intrinsecamente desestruturadas (intrinsically unstructured proteins,

IUP) (Boze et al. 2010). Estas proteínas caracterizam-se pela ausência de uma

estrutura terciária, permanecendo no entanto funcionais apesar da ausência de

uma estrutura bem definida.

Aproximadamente 25 a 42% dos resíduos de aminoácidos das PRPs são

prolina. Para além deste resíduo estas proteínas têm também um elevado

conteúdo em glutamina e glicina. Em conjunto, estes três resíduos

correspondem a cerca de 70 a 88% do total de resíduos destas proteínas

(Bennick 1975, Levine e Keller 1977) .

O aminoácido prolina é um aminoácido particular pois a sua cadeia lateral é

cíclizada com a amida do esqueleto peptídico principal. Esta caraterística tem

três consequências importantes (Williamson 1994): primeiro, a conformação do

próprio esqueleto da prolina é restrita (MacArthur, M. W. and Thornton, J. M.

1991 J. Mol. Biol. 218, 397; Nicholson, H., Tonrad, H., Becktel, W. J. and

Matthews, B. W. 1992 Biopolymers 32, 1432); segundo, o volume do grupo N-

metileno dá origem a restrições na conformação do resíduo de aminoácido

anterior à prolina; terceiro, dado que o azoto do grupo amida não tem um

hidrogénio é incapaz de atuar como dador de pontes de hidrogénio. Para além

disto, a prolina também é única na sua capacidade para formar ligações

peptídicas cis.

Estas caraterísticas conferem algumas restrições nas conformações acessíveis a

um dipéptido X-Pro, que como resultado tem tendência a ter uma conformação

bastante rígida e extendida. Um dipéptido Pro-Pro é mais restrito e existem

várias evidências de que uma sequência de quatro ou mais resíduos de prolina

em solução adoptam uma conformação de hélice poliprolina II. Tem sido

amplamente assumido que o elevado nível de resíduos de prolina nas PRPs é

apenas importante porque se traduz em estruturas abertas e flexíveis nestas

proteínas correspondendo a um potencial para complexação mais elevado.

Globalmente, as PRPs salivares são classificadas em dois grandes grupos: as

acídicas (aPRPs), e as básicas (bPRPs). Esta classificação reflete apenas a

carga global das proteínas, mas no final também se reflete em diferenças

funcionais entre as duas classes. Para além disto, as bPRPs ainda são

tipicamente divididas em PRPs glicosiladas (gPRPs) e não-glicosiladas.



73

6.1.1 PRPs acídicas (aPRPs)

As aPRPs são codificadas pelos genes PRH1-2 que estão no cromossoma

12p13.2 e a sua tradução resulta em proteínas que apresentam dois domínios

estruturais distintos. A região C-terminal é estruturalmente equivalente às bPRPs

e compreende cerca de 70-80% da molécula. Por outro lado, a região N-terminal

contêm resíduos fortemente acídicos (ácidos aspártico e glutâmico) e poucos

resíduos de prolina (Fig.19). A existência desta região acídica traduz-se num

ponto isoeléctrico (pI) baixo (4-5).

Na saliva humana podem ser detetadas 5 isoformas desta classe de proteínas.

Todas apresentam um N-terminal piroglutâmico e são normalmente di-

fosforiladas nas Ser-7 e Ser-22, apesar de serem também detetadas

quantidades menores de aPRPs mono- e não-fosforiladas. Também já foram

detetadas menores percentagens de isoformas tri-fosforiladas (Ser-17). Destas

isoformas, quatro (PRP-1, PRP-2, PIF-s e Dbs) podem ser parcialmente clivadas

próximo do C-terminal libertando um péptido comum de 44 resíduos (péptido P-

C) e quatro isoformas truncadas chamadas PRP-3, PRP-4, PIF-f e Db-f. A

isoforma Pa não é clivada, mas encontra-se frequentemente na saliva na forma

dimérica devido à presença específica de um resíduo de cisteína (C-103) na sua

estrutura.

Relativamente à função destas proteínas, está descrito que contribuem

principalmente para a manutenção da homeostasia do cálcio e seus sais na

cavidade oral. Na verdade, a atividade e os locais de ligação associados à

manutenção do equilíbrio do cálcio em solução está confinada à região acídica

N-terminal destas proteínas.

Fig.19 Esquema representativo da estrutura das aPRPs com indicação da região acídica responsável pela função atribuída a estas proteínas3.

3 Este esquema foi adaptado da base de dados UniprotKB

Péptido sinal

PRP1/PRP2

PRP3/PRP4

Péptido P-C

Região responsável pela inibiação da formação de hidroxiapatite; liga-se à hidroxiapatite e ao cálcio

N-terminal acídico C-terminal ~bPRPs


6.1.2 PRPs básicas (bPRPs) e glicosiladas (gPRPs)

As PRPs básicas e glicosiladas são os grupos mais complexos das PRPs, sendo

expressas por quatro genes diferentes (PRB1-PRB4) que estão agrupados no

cromossoma 12p13.2 perto dos genes PRH1-2. Devido há existência de uma

grande homologia e de um crossing-over desigual entre as repetições do terceiro

exão, isto produz frequentemente polimorfismos. Para além disto, as múltiplas

PRPs podem ser produzidas a partir do mesmo gene através de variações

alélicas, splicing diferencial e modificações pós-tradução.

Ao contrário das aPRPs, que estão contemporaneamente presentes na saliva

como isoformas completas ou truncadas, todas as bPRPs são detetadas apenas

sob a forma de múltiplos fragmentos peptídicos originados de maiores pro-

proteínas. A estrutura de vários péptidos já foi estabelecida [IB1, IB4 (P-H), IB5

(P-D), IB6, IB7, IB8a, IB8b, IB8c (P-F), IB9 (P-E), II-1 e II-2] mas ainda faltam

muitas peças para a definição total desta classe. Por exemplo, no perfil de LC-

MS da saliva é sempre detectado um péptido designado por P-J com massa de

5945 Da, mas que ainda não foi caracterizado definitivamente. Para além disso,

pelo menos três potenciais bPRPs de estrutura não conhecida, com massas de

10434, 23462 e 29415 Da, são recorrentemente detetadas. Uma propriedade

bastante interessante desta classe de proteínas é a elevada homologia das

sequências das diferentes proteínas.

As bPRP são compostas por resíduos que não têm carga a pH básico ou neutro.

Deste modo, o seu pI (pI > 9) é bastante mais elevado do que para as aPRPs. A

sua função ainda não é totalmente compreendida apesar de existirem evidências

que apontam para funções protetoras. Mehansho e seus colaboradores (1987)

propuseram que uma das suas funções seria ligar os taninos, prevenindo assim

os seus efeitos tóxicos no trato gastro-intestinal. Mais recentemente, outros

autores descobriram que estas proteínas ligam-se a vírus, em particular a uma

proteína do HIV-1, tendo assim uma ação anti-vírica (Robinovitch et al. 2001).

Relativamente às gPRPs, como o próprio nome indica, esta classe de proteínas

caracteriza-se pela presença de glicósidos na sua estrutura. Devido à

complexidade destes substituintes, não existem muitos dados na literatura

acerca destas proteínas. Atualmente, começam a surgir as primeiras

informações no que diz respeito ao tipo de açúcar e à posição de glicosilação

destas proteínas (Vitorino et al. 2011). Na verdade, estes dados são



75

fundamentais na análise destas proteínas por espectrometria de massa, pois só

sabendo a sua estrutura global é possível saber o seu peso molecular.

A função principal atribuída às gPRPs é a de lubrificação. Na verdade, as gPRPs

têm uma actividade lubrificante superior às formas não-glicosiladas (Hatton et al.

1985). No entanto, esta propriedade é afetada pelo tipo de carboidratos, bem

como pela extenção da glicosilação (Mcarthur et al. 1995). A glicosilação das

PRPs ocorre por ligação dos açúcares aos resíduos de asparagina (Hatton et al.

1985), treonina e serina (Oppenheim et al. 1985) o que permite frequentemente

antecipar os potenciais locais de glicosilação.

Para além desta função está também descrito que estas proteínas ligam-se às

bactérias da cavidade oral (Carpenter e Proctor 1999).

6.2 Histatinas

As histatinas são proteínas salivares ricas em histidina. Na verdade, este

aminoácido constitui cerca de 18-29% destas proteínas o que é invulgar pois a

histidina é, em conjunto com a prolina, um dos aminoácidos mais raros de

encontrar nas proteínas (Oppenheim et al. 1988).

Até à data, foram identificados dois genes responsáveis pela síntese das

histatinas, HTN1 (HIS1) e HTN2 (HIS2), os quais estão localizados no

cromossoma 4q13 (Vanderspek et al. 1989). Os produtos destes genes são a

histatina 1 e 3, respetivamente. Enquanto a primeira é um péptido de 38

resíduos de aminoácidos, fosforilado na Ser-2, a última apesar de ter uma

sequência semelhante à histatina 1, tem apenas 32 resíduos e não é fosforilada

(Oppenheim et al. 1988). Para além destas histatinas, existem muitas outras que

resultam maioritariamente de clivagem proteolítica da histatina 3 (Castagnola et

al. 2004).

Estas proteínas são consideradas como os principais percursores da estrutura

proteica que protege a superfície dos dentes (esmalte). Para além disto, as

histatinas têm actividade antibacteriana e anti-fúngica. A histatina 5 é

especialmente eficaz contra C.albicans (Oppenheim et al. 1988) e também é um

inibidor potente das metaloproteinases MMP2 e MMP9 (Gusman et al. 2001).


6.3 Estaterina

A estaterina é um fosfopéptido salivar de 43 resíduos de aminoácidos muito

específico e multifuncional (Fig.20). Este péptido acídico é secretado por várias

glândulas salivares e tem um invulgar conteúdo elevado em resíduos de tirosina,

prolina e glutamina (Schlesinger et al. 1989). O seu gene (STATH) está também

localizado no cromossoma 4q13.3 (Sabatini et al. 1987).

Esta proteína contribui sobretudo para o equilíbrio dos minerais na superfície dos

dentes. Na verdade, esta proteína tem uma grande afinidade para o fosfato de

cálcio, inibindo a sua precipitação de soluções supersaturadas (Schlesinger et al.

1989). Deste modo, a estaterina mantém a supersaturação da saliva humana em

cálcio, inibindo o depósito de minerais na superfície dos dentes e contribuindo,

em conjunto com outras proteínas salivares, para a estabilização do esmalte

(Hay et al. 1984). Para além disto, a estaterina contribui para a colonização

bacteriana (Gibbons e Hay 1988) e atua como lubrificante (Douglas et al. 1991).

A região N-terminal da estaterina é muito negativa, sendo o domínio responsável

pela ligação aos sais de cálcio, mas o nível de fosforilação da proteína é também

crucial para esta função (Raj et al. 1992).

Fig.20 Esquema representativo da estrutura da estaterina com indicação das diferentes regiões responsáveis pelas funções atribuídas a esta proteína4.

7. Polifenóis e sabor amargo

Muitos dos polifenóis presentes na nossa dieta alimentar, tais como as

antocianinas e os flavan-3-óis, são amargos, tornando o sabor das plantas e

produtos derivados desagradável (Drewnowski e Gomez-Carneros 2000). Na

verdade, entre as cinco sensações de sabor básicas, o sabor amargo é

particularmente importante no sentido de evitar substâncias perigosas (Ames et

al. 1990). Numerosas substâncias venenosas têm sabor amargo sendo repelidas

por várias espécies do reino animal. A rejeição instintiva do sabor amargo tem

4 Este esquema foi adaptado da base de dados UniprotKB

Péptido sinal

Estaterina

Região de ligação da hidroxiapatite;

Inibe o crescimento de cristais

Região hidrofóbica;

Inibe a precipitação dos

sais de fosfato de cálcio



77

sido crucial para a sobrevivência das espécies, apesar de não estar estabelecida

uma correlação evidente entre o amargor e a toxicidade (Glendinning 1994,

Hladik e Simmen 1996).

Por causa desta rejeição instintiva do sabor amargo, há muito tempo que a

remoção dos compostos responsáveis por este sabor dos alimentos é um

desafio sensorial crucial na indústria alimentar (Drewnowski e Gomez-Carneros

2000). Para isso, é importante a identificação dos compostos que tornam os

alimentos e bebidas derivadas de plantas amargos.

7.1 Relação entre a estrutura dos polifenóis e o sa bor amargo

A adstringência e o amargor são dois atributos sensoriais importantes no vinho

tinto, para os quais certos polifenóis têm um papel importante (Delcour et al.

1984, Noble 1998, Drewnowski e Gomez-Carneros 2000, Hufnagel e Hofmann

2008, Rudnitskaya et al. 2010). Tal como já foi referido anteriormente, enquanto

a adstringência aparenta ser uma sensação tátil de secura, constrição e

aspereza percecionada na cavidade oral, o amargor é um sabor que se

desenvolve pela ativação dos respetivos recetores de sabor.

Apesar de os taninos serem bem conhecidos pela indução da adstringência, eles

também têm sido frequentemente associados ao sabor amargo do vinho tinto.

No entanto, a literatura disponível relativamente à relação estrutura/amargor dos

taninos é inconsistente (Arnold et al. 1980, Robichaud e Noble 1990, Kallithraka

e Bakker 1997, Noble 1998, Kielhorn e Thorngate Iii 1999, Peleg et al. 1999).

Em geral, o número reduzido de estudos que avaliaram o amargor de vários

polifenóis, tais como frações poliméricas de ácido tânico e taninos, bem como de

monómeros, dímeros e trímeros de flavan-3-óis, demonstraram que as

moléculas maiores tendem a ser menos amargas e mais adstringentes. Peleg e

seus colaboradores (1999) observaram que a (-)-epicatequina era mais amarga

que o seu estereoisómero (+)-catequina e que ambos eram mais amargos que

os trímeros de procianidinas, catequina-(4-8)-catequina-(4-8)-catequina e

catequina-(4-8)-catequina-(4-8)-epicatequina. Robichaud e Noble (1990)

observaram que o ácido tânico, um tanino hidrolisável comercial rico em

pentagalhoilglucose, era mais amargo do que a (+)-catequina e do que um

extrato de grainhas de uva rico em procianidinas poliméricas. No entanto, outros

estudos mostraram que o amargor dos polifenóis aumenta com o peso

molecular.


Um outro estudo recente (Hufnagel e Hofmann 2008) demonstrou claramente

que concentrações inferiores ao limite de deteção de ácidos fenólicos de ésteres

etílicos e dos flavan-3-óis [(+)-catequina, (-)-epicatequina, procianidinas B1,B2,

B3 e C1] são responsáveis pelo amargor do vinho tinto estudado (Amarone della

Valpolicella DOC). Estes autores identificaram 82 compostos presentes nesse

vinho tinto (incluindo açúcares, polifenóis, aminoácidos e sais) e fizeram diversas

análises sensoriais para determinar a intensidade do amargor de misturas

incluindo todos os compostos ou frações de um tipo de compostos. O objetivo foi

determinar que combinação de compostos apresentava a mesma intensidade de

amargor e de adstringência que o vinho original. Noutro trabalho, os mesmos

autores (Hufnagel e Hofmann 2008), eles observaram que o limite de deteção do

amargor das procianidinas diméricas era aproximadamente metade dos

respetivos monómeros, ou seja, estas últimas mesmo em concentrações baixas

serão mais importantes pelo sabor amargo do vinho tinto.

Para além das inconsistências referidas, não há conhecimento suficiente acerca

do sabor amargo de muitas famílias de polifenóis, tais como as antocianinas.

Apenas Vidal e seus colaboradores (2004) observaram que as antocianinas

monoglicosiladas e respetivos derivados cumaroílo, não influenciavam a

adstringência e o amargor. Para além disso, estes autores observaram que os

polifenóis pigmentados semelhantes a taninos isolados do vinho apresentaram

resultados semelhantes às antocianinas.

Deste modo, torna-se importante a avaliação do amargor de diversos compostos

polifenólicos por um método fidedigno, reprodutível e objectivamente mensurável

por forma a esclarecer que compostos são responsáveis pelo amargor do vinho

tinto.

7.2 Recetores do sabor amargo (hTAS2Rs)

Os compostos amargos são detectados por um conjunto específico de células

recetoras de sabor (taste receptor cells, TRC) localizadas na cavidade oral em

grupos de células chamadas corpúsculo gustativo, as quais se encontram no

epitélio das papilas gustativas da língua e palato. Estas TRC caracterizam-se

pela expressão de membros da família do gene TASTE 2 Receptor (TAS2R) que

codifica recetores do sabor amargo (Adler et al. 2000, Chandrashekar et al.

2000, Matsunami et al. 2000, Meyerhof et al. 2009, Shi e Zhang 2009). Nos

seres humanos, esta família genética codifica cerca de 25 recetores de sabor

amargo (hTAS2Rs).



79

Os hTAS2Rs são proteínas com aproximadamente 290–330 resíduos de

aminoácidos. Apesar de serem estruturalmente diversos, os genes paralogos5

apresentam aproximadamente 17-90% de similaridade na sequência de

aminoácidos, sugerindo que diferentes membros da família podem reconhecer

compostos com estruturas muito diferentes (Matsunami et al. 2000). Na prática é

realmente necessário que assim seja, dado que os seres humanos têm apenas

25 hTAS2Rs para a detecção de um número enorme de compostos amargos

(Meyerhof 2005). Os compostos amargos são não só numerosos mas também

estruturalmente muito diversos, incluindo péptidos, aminoácidos, lactonas,

terpenóides, fenóis e flavonóides, entre outros, o que torna a previsão do

amargor difícil. Até à data, já foram identificados compostos amargos que

ativavam 20 destes recetores (Behrens e Meyerhof 2006).

Em geral, estes recetores têm em comum uma estrutura em hepta-hélice e

outros motivos sequenciais, que os classifica como membros da subfamília dos

recetores acoplados à proteína G.

A Fig.21 mostra que, em geral, a região citoplasmática dos segmentos putativos

transmembranares e os loops intracelulares são bem conservados. Os 20

aminoácidos que estão presentes em quase todos os hTAS2Rs (assinalados a

vermelho) estão, ou nos domínios intracelulares ou região inferior dos

segmentos transmembranares, provavelmente envolvidos no acoplamento à

proteína G, ativação do recetor e no enrolamento tri-dimensional dos recetores.

Os loops extracelulares e as partes superiors dos segmentos transmembranares

são menos conservados. Isto não é imprevisível dado que são estas regiões que

provavelmente formam os motivos heterogéneos de ligação aos inúmeros

compostos amargos. A região C-terminal dos hTAS2Rs também é muito variável,

o que reflecte possíveis diferenças na regulação dos recetores e no tráfego

celular (Meyerhof 2005).

É também interessante que a existência de numerosos polimorfismos de um

nucleótido (single nucleotide polymorphisms, SNPs) não-sinónimos nos genes

TAS2Rs, origine a codificação de variantes dos recetores (haplotipos), as quais

são funcionalmente distintas e são a base da alteração da perceção do amargor

na população (Drayna 2005, Kim et al. 2005, Pronin et al. 2007).

5 i.e. dois genes existentes em cromossomas diferentes do mesmo organismo mas que têm similaridades estruturais o que indica que derivaram do mesmo gene ancestral.


Fig.21 Representação esquemática dos hTAS2Rs. Os resíduos de aminoácidos estão indicados por círculos coloridos. A similaridade da sequência entre os 25 hTAS2Rs está indicada pelo código de cores. Os círculos com a linha em pontos correspondem aos resíduos que não estão presentes em todos os recetores. Os quadrados magenta indicam as regiões que contêm resíduos adicionais em alguns recetores (Meyerhof 2005).

Os hTAS2Rs, tal como referido anteriormente, pertencem à família dos recetores

acoplados à proteína G. Esta família compreende uma família de recetores

transmembranares que ao serem ativados por moléculas extracelulares,

desencadeiam uma cascata de transdução de sinal (a qual ativa a proteína G)

que resulta no final numa resposta celular.

No caso dos hTAS2Rs a proteína G associada à sua ativação é a gustaducina

(Margolskee 2002), nomeadamente a subunidade α e mais particularmente

serão os últimos 37 resíduos do C-terminal desta subunidade que interagem

com os hTAS2Rs (Ueda et al. 2003). Como qualquer proteína G, a gustaducina

é uma proteína heterotrimérica com as subunidades α, β e γ (Fig.22).

Inicialmente, após a ativação dos recetores ocorre a dissociação das

subunidades da gustaducina do recetor; a α-gustaducina induz uma diminuição

no cNMPs (nucleósido 5’-monofosfato cíclico) e o dímero βγ-gustaducina eleva o

IP3 (inositol trisfosfato)/DAG(diacilglicerol). As etapas seguintes da cascata de

sinalização não estão confirmadas, mas suspeita-se que a diminuição do cNMPs

pode atuar nas proteínas cinases as quais podem regular a atividade dos canais

iónicos das células recetoras do sabor. A cascata do dímero continua com a

ativação dos recetores do IP3, levando à libertação de Ca2+ intracelular seguida

pela libertação de neurotransmissores.



81

Fig.22 Mecanismo proposto de transdução de sinal do sabor amargo para as células recetoras de sabor. Os compostos amargos ativam os recetores de sabor amargo, os quais ativam os heterotrimeros da gustaducina. A α-gustaducina estimula a PDE (fosfodiesterase) a hidrolizar o cAMP (adenosina monofosfato cíclica); a diminuição de cAMP pode disinibir os canais de iões inibidos por nucleótidos, elevando o Ca2+. Por outro lado, as subunidades βγ-gustaducina libertadas ativam a PLCβ2 (fosfolipase C2), gerando IP3 (inositol trisfosfato) que leva à libertação de Ca2+ armazenado, contribuindo para o aumento do Ca2+ intracelular. Adaptado de Meyerhof (2005).

Para compreender melhor a relação estrutura/amargor é necessário um método

objetivo, robusto e fiável que permita analizar o amargor de vários compostos.

Nesse sentido, surgiu recentemente uma metodologia que consiste na medição

da ativação dos hTAS2Rs pelos compostos a testar, após a expressão

heteróloga dos recetores em células humanas, normalmente em células

embriónicas de rim (HEK-293). Esta metodologia baseia-se precisamente na

deteção do aumento do cálcio intracelular que ocorre após a ativação dos

recetores (Fig.22). Esta metodologia tem sido usada para testar compostos

pressupostamente amargos presentes em alimentos e bebidas, e.g., vegetais,

produtos de soja e cerveja (Bufe et al. 2002, Maehashi et al. 2008, Intelmann et

al. 2009, Roland et al. 2011).

Um ponto importante nesta metodologia, é a correlação dos dados

experimentais obtidos com os recetores in vitro, com os dados sensoriais obtidos

por experiências in vivo. Este aspecto foi considerado por Bufe e seus

colaboradores (2002), os quais observaram que as propriedades dos recetores

estudadas in vitro se aproximavam bastante dos resultados obtidos nos estudos

psicofísicos com humanos. Deste modo, a expressão heteróloga dos hTAS2Rs

aparenta ser uma metodologia in vitro viável e fidedigna para obter dados

relevantes do sabor amargo de composto.

TAS2R

Extracelular

Intracelular

PLC β2

DAG IP3α-gustaducina

GDP

α-gustaducina

GTP

Gβ1/3Gγ13

Gβ1/3Gγ13

Ca2+

ArmazénsInternos Ca2+

IP3

Ca2+

Catiões

Despolarizaçãocelular

Libertaçãoneurotransmissores

PDE

cNMP NMP

?

OBJETIVOS



85

Perante o que foi introduzido acerca da interação entre os taninos e as

proteínas, em particular as proteínas salivares, várias questões/objetivos foram

abordadas ao longo deste trabalho:

Capítulo 1

A interação tanino/proteína tem sido objecto de estudo de inúmeros grupos de

investigação, resultando na publicação de numerosos artigos científicos. No

entanto, em geral estes estudos debruçam-se em apenas uma ou duas

proteínas salivares as quais são postas a reagir individualmente com um dado

tanino. Deste modo, a questão inicial que se coloca é:

a) Qual/quais das principais famílias de proteínas salivares têm mais afinidade

para os taninos condensados, quando estas estão presentes em simultâneo?

Capítulo 2

É conhecido que a interação tanino/proteína é afetada por diversos fatores,

nomeadamente, pH, etanol, carboidratos, força iónica, entre outros. Para além

disto, é também conhecido que são várias as práticas vinícolas usadas no

sentido de aumentar a concentração de taninos no vinho tinto. Deste modo, vai-

se procurar saber:

a) De que forma é afetada a interação dos taninos condensados com as

proteínas salivares numa matriz complexa de vinho tinto.

b) De que forma é que o enriquecimento do vinho em taninos condensados

influencia a adstringência percebida.

Capítulo 3

Como já foi referido nos capítulos anteriores é conhecido que a interação

tanino/proteína é afetada por diversos fatores, nomeadamente, a estrutura do

polifenol. Para além disto, a interação entre taninos condensados e/ou

hidrolisáveis e as proteínas salivares também está pouco caracterizada, em

particular no que diz respeito à afinidade da interação, solubilidade e tamanho

dos complexos formados. Deste modo, várias questões se levantam:


a) Qual dos taninos (condensado vs. hidrolisável) tem mais afinidade para as

diferentes famílias de proteínas salivares, quando estas estão presentes em

simultâneo?

b) Qual o tipo de agregados (solúvel e/ou insolúvel) e tamanho dos complexos

formados entre os taninos e os diferentes tipos de proteínas salivares?

c) De que forma, a interação entre os taninos e as proteínas salivares se podem

correlacionar com a adstringência de vinhos tintos?

Capítulo 4

A capacidade dos carboidratos inibirem a interação tanino/proteína tem sido

documentada por diversos trabalhos. No entanto, existe pouca informação sobre

o mecanismo desta inibição. Assim, pretende-se saber:

a) De que forma carboidratos frequentemente utilizados na indústria alimentar

afectam a interação, a nível molecular, dos taninos com proteínas?

b) Qual o mecanismo pelo qual diferentes carboidratos exercem o seu efeito

inibidor da interação tanino/proteína?

Capítulo 5

No seguimento do Capítulo 4, mas com o foco na interação taninos/proteínas

salivares, a principal questão que se pretende estudar neste capítulo é:

a) De que forma carboidratos frequentemente utilizados na indústria alimentar

afectam a interação de taninos condensados com as proteínas salivares?

Capítulo 6

Vários estudos de análise sensorial têm atribuído o sabor amargo como

caraterística sensorial associada a vários polifenóis. No entanto, os resultados

dos vários estudos são contraditórios, e até à data não existem dados referentes

de quais dos 25 recetores de sabor amargo humanos são ativados por alguns

polifenóis. Assim, neste trabalho vai-se procurar saber:

a) Quais os recetores de sabor amargo que são ativados por alguns compostos

polifenólicos pertencentes a diferentes classes?

b) Quais os compostos que ativam mais eficientemente os recetores?

RESULTADOS

Capítulo 1

Reactivity of Human Salivary Proteins Families Toward Food Polyphenols

Soares, S., Vitorino, R., Osório, H., Fernandes, A., Venâncio, A., Mateus, N., Amado, F., de Freitas, V.

J. Agric. Food Chem. 2011, 59, 5535–5547



91

Reactivity of Human Salivary Proteins Families Towa rd Food Polyphenols

Susana Soares,† Rui Vitorino,‡ Hugo Osório,§ Ana Fernandes,† Armando

Venâncio,|| Nuno Mateus,† Francisco Amado,‡ and Victor de Freitas†

†Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

‡Department of Chemistry, University of Aveiro, Aveiro, Portugal

§Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal

||IBB, Center of Biological Enginneering, University of Minho, Campus de Gualtar, Braga, Portugal

Tannins are well-known food polyphenols that interact with proteins, namely,

salivary proteins. This interaction is an important factor in relation to their

bioavailability and is considered the basis of several important properties of

tannins, namely, the development of astringency. It has been generally accepted

that astringency is due to the tannin-induced complexation and/or precipitation of

salivary proline-rich proteins (PRPs) in the oral cavity. However, this

complexation is thought to provide protection against dietary tannins.

Neverthless, there is no concrete evidence and agreement about which PRP

families (acidic, basic, and glycosylated) are responsible for the interaction with

condensed tannins. In the present work, human saliva was isolated, and the

proteins existing in saliva were characterized by chromatographic and proteomic

approaches (HPLC-DAD, ESI-MS, sodium dodecyl sulfatepolyacrylamide gel

electrophoresis (SDS-PAGE), and MALDI-TOF). These approaches were also

adapted to study the affinity of the different families of salivary proteins to

condensed tannins by the interaction of saliva with grape seed procyanidins. The

results obtained when all the main families of salivary proteins are present in a

competitive assay, like in the oral cavity, demonstrate that condensed tannins

interact first with acidic PRPs and statherin and thereafter with histatins,

glycosylated PRPs, and bPRPs.

KEYWORDS: Astringency, condensed tannins, proline-rich proteins, salivary proteins


� INTRODUCTION

Tannins are polyphenols commonly found in plant-derived foods. Tannins are

classically divided into two major classes: condensed tannins

(proanthocyanidins), which are polymers of catechin, and hydrolyzable tannins,

which are gallic or ellagic esters of glucose. Although tannins have benefits for

human health regarding their antioxidant properties,1-3 they can have a number of

harmful effects including decrease in growth and body weight gain,4 and inhibition

of digestive enzymes.5 Regarding their antioxidant properties, an in vitro study1

showed that independently of the degree of polymerization, condensed tannins

provide protection of lipidic peroxidation, but this effect is increased for the less

complex structures. The same behavior was shown for condensed tannins

against tumor cell viability and proliferation.

One of the characteristics of tannins is their ability to precipitate certain proteins,

and this ability has been considered the basis of the sensation of astringency on

the human palate. Astringency has been defined as a complex group of

sensations involving dryness and tightening of the oral surface and puckering

sensations of the oral cavity.6,7 It was proposed by Bate-Smith7,8 that astringency

results from the interaction of tannins with salivary proteins (SP) in the mouth,

and since then, it has been generally accepted and supported by the literature9-12

that astringency is due to the tannin-induced interaction and/or precipitation of

salivary proline-rich proteins (PRPs) in the oral cavity. However, astringency is a

very complex sensory experience, and the possible mechanisms for its

development are presently controversially discussed in the scientific

community.13 Some studies point to the importance of the interaction of salivary

glycoproteins with tannins with consequent modifications of their viscous

properties and oral cavity delubrication,14,15 and also, the salivary flow rate

appears to be correlated with astringency intensity.16 The involvement of the cells

of the oral cavity in the development of astringency has also been suggested.17

Whole saliva represents a mixture of the secretions of the major (submandibular,

sublingual, and parotid) and minor salivary glands, together with the crevicular

fluid, bacteria, and cellular debris. The secretions from the different glands have

been shown to differ considerably and to be affected by different forms of

stimulation, day time, diet, age, gender, several disease states, and

pharmacological agents.18

In general, saliva is composed of proteins, electrolytes, and small organic

compounds. With respect to the proteinaceous component, saliva similarly to



93

other bodily fluids, presents a wide range of small molecular weight components

that are assigned by several authors as salivary peptides, all species with a m/z

below 20 kDa. Salivary peptides have been grouped into six structurally19 related

major classes, namely, histatins, basic proline- rich proteins (bPRPs), acidic

proline-rich proteins (aPRPs), glycosylated proline-rich proteins (gPRPs),

statherin, and cystatins. These peptides have important biological functions in

saliva associated with calcium binding to enamel, maintenance of ionic calcium

concentration (PRPs and statherin), associated with antimicrobial action

(histatins and cystatins), or protection of oral tissues against degradation by

proteolytic activity such as cystatins.20-28

There is a lot of detailed information for all these families of SP. The family of

PRPs is divided into three classes: acidic, basic, and glycosylated. More than 11

human basic-PRPs and five acidic PRP isoforms have been identified.29,30

Several closely related acidic proline-rich phosphoproteins (aPRPs) were

identified by isolation (proteins A and C,31 PRP1, 2, 3, and 432) or by studies of

protein polymorphism (identified as PIF-s, PIF-f, Db-s, Db-f, and Pa). Although

the nomenclature of this family of proteins is somehow confusing, in general, all

these proteins are isoforms.33 In general, the designations PRP1, 2, 3, and 4 will

be used here including the following alternative names: PRP1/PRP2-PIF-s, Pa,

and Db-s; PRP3/PRP4-PIF-f and Db-f. Histatins are a family of small, histidine-

rich proteins secreted by the parotid and submandibular glands.21 Statherin is

secreted by the parotid gland and is abundant in tyrosine residues.20 Cystatins

are natural inhibitors of cysteine proteinases.34

In the past years, several research groups studied the molecular basis of the

development of astringency using models bioassays with pure/isolated PRPs (or

similar proteins) and tannins.35-43 However, only few works had used whole saliva

to study the onset of astringency.44,45

Several studies have been carried out to evaluate interactions between

polyphenols and SP using different techniques, namely, SDS-PAGE,35,42,46

spectrophotometry,37,43 nephelometry,38,40,47 NMR,36,48 DLS (dynamic light

scattering),15,49 and mass spectrometry.50 Despite the several techniques applied,

there are some difficulties in correlating the perceived astringency to a single

physicalchemical phenomenon. To our knowledge, there are no experimental

evidence about the relative affinity of different PRP families (acidic, basic, and

glycosylated) individually or in a competitive/associative medium such as whole

saliva. In fact, the first studies that analyzed salivary protein (SP) interaction with


polyphenols by HPLC were done by Kallithraka and co-workers.44,51 They

analyzed human saliva before and after the interaction with polyphenols ((+)-

catechin, (-)-epicatechin, procyanidin B2, or procyanidin C1) by HPLC. However,

the SP involved in the interaction were not identified. In the present work,

chromatographic and proteomic approaches (HPLC-DAD, ESI-MS, sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and MALDI-

TOF-MS) were developed in order to study the affinity of different families of SP

by the interaction of saliva with grape seed procyanidins.

� MATERIALS AND METHODS

Materials. All reagents used were of analytical grade or better.

Hydrochloric acid and acetonitrile were purchased from Panreac Quimica, acetic

acid was purchased from Carlo Erba Reagents, sodium acetate and trifluoracetic

acid (TFA) were purchased from Fluka Biochemica, and ethanol was purchased

from AGA, Álcool e Géneros Alimentares, SA. Trizma base, glycerol, sodium

dodecyl sulfate, β-mercaptoethanol, acrylamide, N’,N’-methylenebisacrylamide,

tricine, tetramethylethylenediamine, ammonium persulfate, ammonium

bicarbonate, formic acid, SigmaMarker Wide Range, molecular weight 6.5-200.0

kDa, and sodium thiosulfate were purchased from Sigma. Bromophenol blue,

periodic acid, Schiff’s reagent, and Fuchsin-sulfite reagent were purchased from

Sigma-Aldrich. Imperial Protein Stain was purchased from Thermo Scientific

(United Kingdom). Potassium metabissulfite was from BDH Chemicals Ltd.

Sequencing-grade modified trypsin (porcine) was from Promega (Portugal). R-

Cyano-4-hydroxycinnamic acid was purchased from Applied Biosystems

(Germany). Silver nitrate, sodium carbonate, and formaldehyde were purchased

from Merck (Germany).

Grape Seed Fraction (GSF) Isolation. Condensed tannins were

extracted from Vitis vinifera grape seed extract. This extract was fractionated

through a TSK Toyopearl HW-40(s) gel column (100 mm x 10 mmi.d., with 0.8

mL.min-1 methanol as eluent), yielding two fractions according to the method

described in the literature.52 The first fraction was obtained after elution with

99.8% (v/v) methanol during 5 h (240 mL) and the second one after elution with

methanol/5% (v/v) acetic acid during the next 14 h (670 mL). Both fractions were

mixed with deionized water, and the solvent was eliminated using a rotary

evaporator under reduced pressure at 30°C and then freeze-dried. The



95

procyanidin composition of fractions was determined by direct analysis by ESI-

MS (Finnigan DECA XP PLUS). The first fraction contains mainly catechins (m/z

= 290), procyanidin dimers, and their galloyl derivatives, and the second fraction

contains procyanidin dimers galloylated, procyanidin trimers, and their galloyl

derivatives and procyanidin tetramers. The latter has a mean MW of 936 and a

polymerization degree average of 3.2. Only the second fraction named grape

seed fraction (GSF) was used because it is composed of more polymerized and

galloylated procyanidins, which are known to be more reactive toward

proteins.38,53

Saliva Collection, Treatment, and Analysis. Whole human saliva was

always collected freshly at 2 pm from a healthy, nonsmoking female volunteer.

The saliva samples were taken under unstimulated conditions and after at least 1

h without ingestion of food or beverages. Collection time was standardized in

order to reduce concentration variability connected to circadian rhythms of

secretion.30 As the objective of this work was to establish a relationship between

different families of PRPs (basic, acidic, and glycosylated PRPs) saliva was

supplied only by one volunteer in order to simplify the proteomic analysis and

chromatogram division.

TFA solution (10% aqueous TFA) was immediately added to 900 µL of collected

saliva (1:90 v/v), and the solution was centrifuged at 8000g for 5 min. After

centrifugation, the supernatant (acidic saliva, AS) was separated from the

precipitate, and 90 µL was immediately analyzed on a HPLC-DAD Elite Lachrom

system (L-2130) equipped with a Vydac C8 column, with 5 µm particle diameter

(column dimensions 150 x 2.1 mm); detection was carried out at 214 to 280 nm,

using a diode array detector (L-2455). The HPLC solvents were (eluent A) 0.2%

aqueous TFA and (eluent B) 0.2% TFA in ACN/water 80/20 (v/v). The gradient

applied was linear from 10 to 40% (eluent B) in 60 min, at a flow rate of 0.30

mL/min. After this program, the column was washed with 100% eluent B for 20

min in order to elute S-type cystatins and other late-eluting proteins. After

washing, the column was stabilized in the initial conditions.

Twelve selected salivary fractions containing different families of salivary proteins

were obtained during the HPLC analysis by separate collection of the eluent

deriving from the diode array detector.


SDS-PAGE. The 12 selected salivary fractions were dried in a SpeedVac

and dissolved in 25 µL of 1x electrophoresis sample buffer (50 mM Tris-HCl, pH

6.8, 12% v/v glycerol, 4% SDS, 2.5% v/v β-mercaptoethanol, and 0.01%

bromophenol blue) and heated at 60°C for 1 h with shaking. The 12 samples

were analyzed by SDS-PAGE in a tris-tricine buffer system according to the

method of Schägger54 using 16% acrylamide resolving gel. The stacking gel was

5% acrilamide. The cathode buffer was 0.1 M Tris, 0.1 M tricine, and 0.1% SDS,

and the anode buffer was 0.2 M Tris-HCl, pH 8.9. Electrophoresis was performed

on a Bio-Rad MiniProtean Cell electrophoresis apparatus (Bio-Rad). After

electrophoresis, the gels were stained with Imperial Protein Stain, a Coomassie

R-250 dye-based reagent, or silver stained. The staining with Imperial Protein

Stain was done according to the supplier's instructions. The destaining step was

done by washing the gels with water until the bands were visible.

Molecularweights were estimated by comparison with the migration rates of

standard proteins. The silver staining procedure was done according to

O’Connell and Stults.55

Periodic Acid Schiff’s (PAS) Staining. The PAS staining was done

according to Zacharius.56 After electrophoresis, the gels were fixed overnight

(40% ethanol and 7% acetic acid) and then immersed in a solution of 1% periodic

acid for 60 min. The gels were washed with water and incubated in the dark with

Schiff’s reagent for 60 min. The gels were then washed three times (0.58%

potassium metabissulfite and 3% acetic acid).

Tryptic Digestion. The protocol used for tryptic digestion was according

to Vitorino et al.57 After electrophoresis, the bands of interest were excised from

the gel and transferred to a rack. The gel pieces were washed twice with 25 mM

ammonium bicarbonate/50% ACN, one time with 100% ACN, and after the

washes, the gel pieces were dried in a SpeedVac (Thermo Savant). Twenty

microliters of 10 µg/mL trypsin in 50 mM ammonium bicarbonate was added to

the dried residue, and the samples were incubated overnight at 37°C. After the

incubation, the extraction of tryptic peptides was performed by the addition of

10% formic acid/50% ACN three times, followed by lyophylization in a SpeedVac

(Thermo Savant). Tryptic peptides were resuspended in 10 µL of a 50%

ACN/0.1% formic acid solution.



97

Mass Spectrometry Analysis. The AS and the 12 HPLC salivary

fractions were analyzed by LC-ESI-MS and MALDI-TOF/TOF, respectively. The

AS was analyzed by LC-ESI-MS with the HPLC analysis performed on a liquid

chromatograph (Hewlett-Packard 1100 series) equipped with the same column

referred previously. The solvents and the HPLC gradient used were the same as

those reported above for the HPLC analysis. Double online detection was done in

a photodiode spectrophotometer and by mass spectrometry. The mass detector

was a Finnigan LCQ Deca (Finnigan Corporation, San Jose, CA) equipped with

an API source, using electrospray ionization (ESI) interface. Both the auxiliary

and the sheath gases were a mixture of nitrogen and helium. The capillary

voltage was 15 V and the capillary temperature 325°C. Spectra were recorded in

positive ion mode between m/z 250 and 2000 Da. The 12 HPLC salivary fractions

were analyzed by MALDI-TOF/TOF, using a 4800 MALDI-TOF/TOF analyzer

(Applied Biosystems, Foster City, CA) in the linear mode to obtain the molecular

weight of larger species and in the reflectron mode to obtain the peptide

sequence of small species. In linear mode, all samples were mixed (1:1) with a

matrix solution (3 mg/mL) of α-cyano-4-hydroxycinnamic acid matrix prepared in

50% ACN/0.1% TFA. Aliquots of samples (0.35 µL) were spotted onto the MALDI

sample target plate, and spectra were obtained in the mass range between 1500

and 70000 Da with ca. 1000 laser shots.

Top Down Analysis. Characterization of smaller species present in each

fraction was performed in the positive ion reflector mode using the above matrix

composition. Each fraction was applied in triplicate and MS spectra were

obtained in the mass range between 800 and 4500 Da with ca. 1200 laser shots.

A fragmentation voltage of 2 kV was used for MS/MS analysis. Automated

acquisition of MS and MS/MS data in the batch mode employed an interpretation

method with the following settings: number of shots per spot =10; minimum S/N

filter =50 to select peaks for MS/MS analyses, chromatogram peak width =3, and

fraction resolution of precursor exclusion window =200 fwhm.

Peptide Mass Fingerprint (PMF). SDS-PAGE bands were digested with

trypsin, and the generated tryptic peptides were analyzed. Peptide mass spectra

were obtained on a MALDI-TOF/-TOF mass spectrometer (4800 Analyzer;

Applied Biosystems, Foster CA) in conditions similar to those in the top-down

analysis in the positive ion reflector mode automated acquisition of MS and


MS/MS data. In this case, the interpretation method excludes the trypsin

autolysis peaks.

Data Analysis. The spectra were processed and analyzed by the Global

Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which

uses internal Mascot software (v.2.1.0.4, Matrix Science Ltd., U.K.) for

protein/peptide identification based on peptide mass fingerprints and MS/MS

data. The search was performed against the SwissProt protein database (march

2009, 428650 entries) for Homo sapiens. A MS tolerance of 30 ppm was found

for precursor ions and 0.3 Da for fragment ions, as well as two missed cleavages.

In the case of top-down, no enzyme was selected, and in the case of PMF,

trypsin was selected. Protein identifications were considered as reliable when the

MASCOT score was >70 (the MASCOT score was calculated as – 10 x log P,

where P is the probability that the observed match is a random event). This is the

lowest score indicated by the program as significant (P < 0.05) and indicated by

the probability of incorrect protein identification. In order to estimate the false

discovery rate (FDR) and considering the repetitive PRP motif, a random decoy

database was created for all SwissProt and internal database entries resulting in

10% of FDR (false positive peptides/(false positive peptides + total peptides)) x

100. Unique peptides retrieved from the FDR search were considered.

Protein-Tannin Interaction. The AS sample was analyzed by HPLC-

DAD before and after the interaction with increasing concentrations of GSF. The

control condition was a mixture of AS (150 µL) and acetate buffer 0.1 M, pH 5.0,

and 12% ethanol (50 µL) (final volume 200 µL). For the experiments with GSF,

the necessary volume (10 to 39 µL) of a GSF stock solution (2.66 mM) was

added to AS (150 µL) to obtain the desired final concentration, plus the volume of

acetate buffer to make the final volume 200 µL. GSF was tested in the following

final concentrations: 0.133, 0.306, 0.399, 0.505, 0.599, 1.300 mM, and each

concentration was an independent experiment. After shaking, the mixture reacted

at room temperature (20°C) for 5 min and then was centrifuged (8000 g, 5 min).

The supernatant was injected into the HPLC-DAD. These experiments were also

made in large scale with two different concentrations of GSF 0.133 and 0.232

mM to do semipreparative HPLC-DAD. The 12 fractions were collected by

recovering of the eluent deriving from the diode array detector. The 12 fractions

were dried in a SpeedVac and analyzed by SDS-PAGE.



99

� RESULTS AND DISCUSSION

In order to observe in vitro the effect of condensed tannins in quantitative and

qualitative changes of the HPLC profile of human saliva, the first major task is the

identification of the proteins corresponding to the HPLC profile. The HPLC

method and identification described herein were based on the work developed by

Messana et al.30

Several HPLC analytical problems may appear related to the quantity of mucins

and other high molecular weight glycoproteins present in saliva. Indeed, these

proteins can block and contribute to the degeneration of HPLC columns and poor

reproducibility. In order to overcome these problems, saliva samples were mixed

immediately after collection with aqueous TFA (final concentration 0.1%). This

acidic treatment causes the precipitation of several high molecular weight SP

(such as α-amylases, mucins, carbonic anhydrase, and lactoferrin) contributing to

a decrease of the viscosity and also preserves sample protein composition since

TFA partially inhibits intrinsic protease activity.30,58 Peptides and proteins such as

histatins, basic and acidic proline-rich proteins (PRPs), statherin, cystatins, and

defensins are soluble in acidic saliva (AS) solution and may be directly analyzed

by RP-HPLC. The use of TFA provides a satisfactory compromise between high

ion-pairing strength for chromatographic separation and protein ionization for ESI

analysis. The HPLC chromatogram of this AS solution at 214 nm is presented in

Fig.1.1A, and the profile was similar to the one previously described in the

literature by Messana et al.30

On the basis of the identification of the different salivary proteins, the HPLC

chromatogram of the AS solution was roughly divided into 12 peptide salivary

fractions.

Identification of Salivary Proteins. In order to identify the main SP of

each HPLC fraction, the AS solution was analyzed by several techniques such as

RP-HPLC-ESI-MS, MALDI-TOF/TOF, and SDS-PAGE-MALDI-TOF/TOF. The

total ion current (TIC) chromatogram profile obtained by HPLC-ESI-MS (Fig.1.1B)

is similar to the chromatogram obtained at 214 nm (Fig.1.1A).

These identifications were first achieved by LC-MS analysis and deconvolution of

the averaged ESI mass spectra. An example of the deconvolution process is

presented in Fig.1.2 for the peak eluted at 40 min in Fig.1.1B.


A

B

Fig.1.1(A) Typical RP-HPLC profile detected at 214 nm of the acidic saliva (AS) solution of whole human saliva. The pointed lines and numbers show the ranges and names assigned to each HPLC fraction, with the outline of the main proteins identified for each HPLC fraction (Table 1). At the bottom, there is the distribution of the different families of salivary proteins along the chromatogram. *, fragments of proteins; His, histatin; Stat, sthaterin; (di-, mono-, n-)-phosp, (di-, mono-, non-)-phosphorylated; bPRP, basic proline-rich protein; gPRPs, glycosylated proline-rich proteins, aPRPs, acidic proline-rich proteins. (B) Total ion current (TIC) profile of AS solution collected by the ion-trap mass spectrometer (ESI-MS).

15 20 25 30 35 40 45 50 55 60 650

5

10

15

20

25

x 1041 2 3 5 6 7 10 124 8 9 11

bPRPs gPRPs aPRPs Statherin

IB-8

bP-

J, IB

-8c,

His

3*,

bPR

P2*,

bPR

P3*

IB-9

, IB

-4, H

is 7

, 8 a

nd 9

, His

3*

IB-6

, His

3*

His

5, H

is 3

*, P

-B*

bPR

P3

bPR

P3, I

I-2, I

B-1

His

3

PRP3

, PR

P1

His

1

Sta

t (di

-, m

ono-

, n-p

hosp

), P

eptid

e P-

B

IB-6

, IB

-4, I

B-9

, His

3*

t (min)

Ab

s (m

Au

)

Legend:* - fragments of proteinsHis - histatinbPRP - basic Proline-rich ProteinaPRP - acidic Proline-rich ProteingPRP - glycosylated Proline-rich ProteinStat - Statherindi-, mono-, n-phop - di-, mono-, n-phosphorilated

15 20 25 30 35 40 45 50 55 600

20

40

60

80

100

t (min)

Re

lativ

e A

bund

ance



101

Fig.1.2 Deconvolution process for fraction 10. (A) ESI mass spectrum obtained by the average of 77 mass spectra collected in the 38.49-40.39 min range during the HPLC separation reported in the TIC profile (Fig.1.1B). (B) The bottom panel reports the deconvolution of the upper ESI-MS (A).

In general, the deconvolution of the average TIC spectra of each area allowed

the identification of 27 peptides shown in Table 1.1

Table 1.1. Experimental Masses (Da) Detected in the Twelve Chromatographic Fractions by RP-HPLC-ESI-MS, and MALDI-TOF/TOFa

HPLC fraction Peptide Theoric

mass Exp. ESI Exp. MALDI*

1 IB-8b 4371 4369.9 4369.1

bPRP2 (379-407,379-408,379-412)

- - 2817.3,2874.1,3271.1

bPRP4(261-305)

- - 4336.7

bPRP1(236-279)

- 4392.9 4392.5

2 His 3(33-43,24-36,20-36)

- - 1434.5,1750.8,2161.1

bPRP2(379-412,368-407,368-408)

- - 3271.3,3963.6,4020.6

bPRP3(268-288,219-261)

- - 2027.8,4396.5

P-J 5945 5942.9 5949.3

IB-8c 5843 5841.5 5847.7

bPRP1(94-139,235-261)

- - 2692.3,4376.5

IB-8b 4371 4369.6 4369.2

bPRP4(263-307)

- - 4398.5

His 8 1562 1561.7 1562.7

His 10 1719 - 1718.8

3 IB-9 6023 6023.3 6029.9

IB-4 5590 5589.6 5593.5

His 7 1718 - 1718.8

His 3(42-51,30-43,20-36,20-37,20-39)

- - 1264.5,1846.9,2161.1,2298.0,2522.1

His 8 1562 1561.7 1562.7

His 9 1875 - 1874.9

4 His 3(24-33,20-33,30-43)

- - 1356.7,1766.8,1846.9

IB-5 6950 6949.1 6957.4

IB-9 6023 6022.7 6028.2

IB-4 5590 - 5593.1

5 His 3(20-33,37-51,29-43,26-43,24-

- - 1766.9,1918.8,2009.9,2341.1,2405.2,2815.3

IB-6 11517 11515.6 -

Peptide P-B (57-67)

- - 1161.5

HPLC fraction Peptide Theoric

mass Exp. ESI Exp. MALDI*

6 His 5 3036 3036.6 3035.3

His 3(24-43, 34-51, 24-44)

- 2625.0 2625.1, 2312.8, 2781.2

Peptide P-B(55-67)

- - 1315.6

His 6 3192 3192.0 3191.4

7 bPRP3 - - -

Unknown - 23467.0 -

His 3(55-69, 33-51)

- 1920.8, 2459.9

Peptide P-B(55-67, 55-69)

- 1315.7, 1469.7

8 bPRP3 - - -

Unknown - 23467.0 -

II-2 (phos.) 7608 7607.1 7613.7

IB-1 9590 9590.7 -

His 3(33-51)

- - 2459.9

Peptide P-B(68-79)

- - 1200.6

9 His 3 4061 4061.0 4061.0

10 PRP1 (di-phos) 15514 15512.0 -

PRP3 (di-phos) 11161 11160.0 -

Cys S 14347.0 -

Cys SN - -

11 His 1 4926 4927.3 4932.1

His 1(37-57, 34-57, 33-57, 32-57)

- 2617.9,3012.1,3159.2,3287.3

His 2(31-57)

3443 3441.4 3443.6

12 Statherin (di-phos) 5378 5378.7 5381.5

Statherin (mono-phos) 5299 - 5299.9

Statherin (n-phos) 5219 - 5216.9

Peptide P-B 5789 5791.2 5796.3 a The experimental masses obtained by ESI-MS were compared to the theoretical masses of human salivary proteins reported in international data banks,and the experimental masses obtained by MALDI-TOF/TOF were identified as referred to in the Materials and Methods section. The superscriptednumbers are the residue numbers for the peptides identified for each protein; the proteins in bold are the main proteins for each fraction. Exp. ESI,experimental masses obtained in ESI analysis; * masses obtained by MS/MS and linear MALDI. Swiss-prot code: IB-8b, PRP1 and PRP3 (P02810); IB-8c and bPRP2 (P02812); bPRP1, P-J, IB-4, IB-6 and II-2 (P04280); bPRP3 (Q04118); bPRP4 and IB-5 (P10163); IB-9 (P02811); IB-1 (P04281); peptide P-B (P02814); His 3, 5, 6, 7, 8, 9 and 10 (P15516); His 1 (P15515); Cys S (P010136); Cys SN (P01037); statherin (P02808).



103

Table 1.1 reports all the proteins/peptides identified in the 12 HPLC fractions of

the AS solution. It also indicates the experimental masses detected by RP-HPLC-

ESI-MS, MS/MS, and MALDI-TOF (linear or after trypsin digestion). Using these

different techniques, we were able to identify several peptides/small proteins.

From the results presented in Table 1.1, it can be observed that the different

techniques are complementary to identifying the major SP, probably because the

peptides/proteins ionized differently according to the technique used.

The assignment of experimental masses to peptides/proteins was performed

against the SwissProt protein database for Homo sapiens and also taking into

account the previous identifications and experimental masses of these SP by

ESI-MS and MALDI-TOF approaches by other groups, using similar experimental

conditions.30,59

In order to confirm the assignment of some peptides performed by ESI-MS data

and to increase the information of each HPLC fraction, the 12 chromatographic

fractions displayed in Fig.1.1A were isolated individually by HPLC and analyzed

by different approaches: (a) sequencing of peptides with MW below 5000 Da by

direct MALDI-TOF MS/MS of each fraction, (b) SDS-PAGE of each fraction using

different staining procedures, (c) identification of peptides/proteins with MW

above 5000 Da by trypsin digestion of the bands after the SDS-PAGE analysis,

and (d) linear MALDI-TOF analysis of each fraction. This strategy has been used

by expert groups in this area.30,59

It is also important to refer that for glycosylated PRPs (gPRPs) the identification

is even more difficult. The lack of the “total” molecular weight of these proteins

including the sugar moiety makes the direct identification by ESI more

complicated. Nevertheless, the digestion with trypsin and analysis by MALDI-TOF

as well as Schiff’s staining of the SDS-PAGE gels (see below) make it possible to

identify a protein (bPRP3) belonging to the gPRPs class in HPLC fractions 7 and

8.

It is interesting to notice that the deconvolution of the ESI average spectra for

HPLC fractions 7 and 8 gives a protein with a mass of 23467 Da, which could not

be identified since there is no report in the databases or elsewhere in the

literature. Messana et al. 30 have also identified a SP with this mass, but they

were not able to identify this protein. However, they suggested that this unknown

protein may be a putative basic PRP candidate since that protein was resistant to

trypsin cleavage, which is a main characteristic of this kind of proteins.


The relative quantification of the proteins identified in each fraction based on the

intensities of the peaks is inaccurate due to differences in ionization ability of the

different proteins but could be a good approximative approach.

For the peptides identified by ESI-MS/MS, the extracted ion current (XIC)

strategy was applied, which compares the ion currents originated by the

peptides. The deconvolution process considers the major ions of ESI spectra

giving the relative quantity of each protein identified. On the basis of that

approach, the major proteins of each HPLC fraction were identified and are

indicated with bold text in Table 1.1.

From the results presented, it is possible to observe that several proteins were

found, namely, IB-1, IB-8b, IB-4, IB-9, IB-5, IB-6, bPRP3, II-2, PRP1, PRP3,

histatin 5, 7, 8, and several forms of statherin; small fragments were assigned as

histatin 3, bPRP1, bPRP2, and bPRP3 fragments. These observations had been

previously reported by Vitorino et al.5

In summary, the chromatogram is roughly divided into four regions corresponding

to the different families of SP. The first zone (1 to 6) comprises proteins that

belong to the classes of bPRPs and His. The second region (7 and 8) comprises

mainly a gPRP, the bPRP3. The third region (10) has as main proteins the

aPRPs. The last region (12) has phosphorylated and nonphosphorylated forms of

statherin.

SDS-PAGE. In order to obtain additional information and to do trypsin

digestion of the SP of each HPLC fraction, SDS-PAGE of each fraction was

carried out, and the results are presented in Fig.1.3.

Fig.1.3 SDS-PAGE of the 12 HPLC fractions isolated from the HPLC after the injection of the AS solution. The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa as marked on the left side. The gels were stained by with Imperial Protein Stain, a Coomassie R-250 dye-based reagent.



105

Although SP can be separated and analyzed by SDS-PAGE, there are some

particular aspects that have to be considered, especially regarding PRPs. PRPs

stain poorly with conventional Coomassie Blue and even with silver staining

procedures. However, when Coomassie Blue R-250 is used and organic solvents

are omitted from the destain solution, PRPs stain pink-violet; regarding silver

staining, some improved staining can be achieved by a modified silver staining

procedure. Nevertheless, for PRPs the sensitivity is lower than that for other

proteins.60 From the results presented in Fig.1.3, it is possible to observe that for

fractions 1 (F1) and 12 (F12) there were no bands detected, even after testing

different sample concentrations and different staining methods with these

fractions.

It is also necessary to be cautious with the apparent molecular weight of this kind

of proteins on SDS-PAGE gels; several reports have described that PRPs do not

migrate in SDS-PAGE at the expected molecular weight.61,62 The high proline

content presumably increases the rigidity of the protein, which makes the protein

migrate slower in SDS-PAGE than globular proteins with the same molecular

weight (MW).

Unusual mobility on SDS-PAGE is one of the characteristics of intrinsically

unstructured proteins. Because of their unusual amino acid composition, PRPs

bind less SDS than usual, and their apparent MW on SDS-PAGE gel is often 1.2-

1.8 times higher (MW factor) than the real one.61

For fraction 2 (F2), amajor band smaller than 6.5 kDa is visible, which could

correspond to several peptides previously identified by ESI-MS and MALDI-TOF-

MS for this fraction, peptides deriving from histatin 3, bPRP2, and bPRP3 with a

molecular weight between 1.4 and 4.4 kDa. Besides these, peptides P-J and IB-

8c with a molecular weight of 5.9 and 5.8 kDa, respectively, were also identified

for this fraction. These peptides could correspond to a small smear just below the

6.5 kDa marker.

For fraction 3, it is possible to observe a significant smear at the bottom of the gel

and a high molecular weight band. The smear at the bottom of the gel spreads

below the 6.5 kDa. The upper part of the smear could correspond to IB-9 and IB-

4 proteins. These proteins have molecular weights of 6.0 and 5.5 kDa,

respectively, and they could migrate in the area of the 6.5-7.0 kDa (in this case,

the MW factor is 1.2). However, the other identified peptides, histatins 7, 8, 9,

and fragments of histatin 3, have lower molecular weight around 2.1 kDa, and


therefore, they will migrate at the bottom of the gel. Regarding the band at the top

of the gel, its identification was not possible.

For fraction 4 (F4), the results presented are quite similar to F3. There is a

significant smear at the bottom of the gel. At the top of the smear, there is

probably IB-5, IB-9, and IB-4 with molecular weights of 6.9, 6.0, and 5.6 kDa,

respectively. These proteins could migrate in the area of 6.7 to 8.0 kDa (MW

factor of 1.2).

For fraction 5 (F5), it is possible to observe a pink-violet band in the region of 20

kDa. This band could be IB-6 protein, which was identified in this fraction by

deconvolution of the average ESI. Indeed, the real MW is 11.5 kDa, but as

referred to above, it could migrate with an apparent MW of 11.5 x 1.8 (MW factor)

= 20.7 kDa. This identification is further supported by the pink-violet color

(characteristic of PRPs) displayed by this band. This fraction presents one smear

at the bottom of the gel. At the bottom of this smear could be the several

fragments of histatin 3 previously identified (MW around 2.0 kDa), but there were

no identified peptides/proteins around 6.5 kDa. Therefore, the upper part of the

smear, as well as the band at 66 kDa, was not identified.

Fraction 6 (F6) presents two important bands. The analysis of the intense low

molecular weight band revealed the presence of histatin 5 (3.0 kDa) and several

fragments of histatin 3 at 2.5 kDa. The other band at the region of 45 kDa is

probably one protein that appears in the ESI analysis with 23.4 kDa (will migrate

with an apparent MW of 42 kDa). This protein seems also to be eluted in fraction

7 collected from the HPLC. Messana et al.30 have also verified the existence of

that protein, and they suggested that it could correspond to basic PRPs.

For fractions 7 (F7) and 8 (F8), there is a main band at the top of the gel with an

apparent MW of around 66 kDa. The analysis of this band by MALDI-TOF after

trypsin digestion allowed the identification of bPRP3 protein, a glycosylated

bPRP. The presence of sugars in protein structure was confirmed by periodic

acid schiff (PAS) staining, which is a basic procedure for the analysis of

glycoproteins (Fig.1.4). Basically, this procedure stains the sugar moieties of

glycoproteins yielding magenta bands with a colorless background.

Only these two fractions gave positive results to the PAS staining, which

indicates that glycosylated proteins are eluted only in those two fractions

(Fig.1.4).



107

Fig.1.4 SDS-PAGE of each HPLC fraction isolated from the HPLC after the injection of the AS solution. The molecular weight markers were substituted by lines and the molecular mass is expressed in kDa. The gels were stained by the periodic acid Schiff procedure in order to visualize glycoproteins.

Although two other proteins, namely, II-2 and IB-1, have been identified in F8 by

ESI deconvolution, they do not appear in the SDS-PAGE gel probably because

their quantity is below the detection limit of the Coomassie stain.

For fraction 9 (F9), only histatin 3 (4.0 kDa) was identified in the SDS-PAGE gel.

Fraction 10 (F10) presents one band with an apparent MW of 24-29 kDa. The

previous analysis of this fraction by ESI-MS indicated the presence of two

aPRPs, PRP1 and PRP3 (15.5 and 11.2 kDa, respectively). Considering the

factor 1.8 into the apparent MW, we determined that these proteins would appear

in the SDS-PAGE gel in the zone of 20-27 kDa, which is in agreement with the

results obtained.

The band observed at the bottom of the SDS-PAGE gel of fraction 11 (F11) could

be attributed to the identified histatin 1 (4.9 kDa).

For fraction 12 (F12), different isoforms of statherin were identified, namely, di

(5.4 kDa)-, mono (5.3 kDa)-, and nonphosphorylated (5.2 kDa) forms. The

peptide P-B (5.8 kDa) was also identified, but surprisingly no bands were

detected in the SDS-PAGE gel stained with a Coomassie based dye. However,

the silver staining revealed the presence of a smear in the 6.5 kDa region;

probably this smear corresponds to the several isoforms of statherin and to the

peptide P-B since that these proteins have approximately the same molecular

weight.

Interaction between Salivary Proteins and Condense d Tannins. The

experiments in this study were all performed in buffer with 12% ethanol to mimic

a model wine and at pH of 5.0 as has already been referred to as a pH at which

salivary proteins strongly interact with condensed tannins63 and correspond to an

intermediary pH between wine pH (3.4) and saliva pH (7.0). Indeed, salivary pH

drops with the ingestion of acidic drinks, andthe degree of acidity in saliva

depends on the sampled volume, buffering capacity, and mode of drinking.64,65 In


order to compare the reactivity of the identified proteins with condensed tannins,

different concentrations of grape seed fraction (GSF) were mixed with acidic

saliva (AS) solution, and after centrifugation, the supernatant was analyzed by

HPLC. The profiles of AS solution before and after the interaction with GSF are

shown in Fig.1.5.

Fig.1.5 RP-HPLC profile detected at 214 nm of the AS solution before (AS control) and after the interaction with increasing concentrations of grape seed fraction (GSF).

The results presented in Fig.1.5 show that the HPLC profile of the AS solution is

interestingly affected by the interaction with GSF. Effectively, GSF interacts in a

different way with the different groups of proteins. While the areas of HPLC

fractions 10 and 12 declines significantly with the lowest GSF concentration, the

other fractions' areas remain relatively constant. It is important to mention that the

mixture of AS solution with GSF always resulted in the formation of insoluble

precipitates, which increased along with GSF concentration.

The decrease of percentage area for each HPLC fraction after the AS solution

interaction with increasing concentrations of GSF is summarized in Fig.1.6.

From the results presented in Fig.1.6, it is possible to observe that fractions 10,

11, and 12 interact more importantly with GSF (Fig.1.6C). For the lowest GSF

concentration (0.133 mM) assayed, the area of these fractions was much

reduced to 20% for fractions 10 and 12 and to 40% for fraction 11. With the

increase in GSF concentration (0.505 mM), fractions 5 and 6 were also greatly

reduced to 30% (Fig.1.6B). However, regarding the fractions 1 to 4 and 9, only

higher GSF concentrations significantly reduced their areas (Fig.1.6A e C). The

results were gouped by SP families (Fig.1.6D), and it is possible to observe that

the major SP families that interact with GSF were aPRPs and statherin. For the



109

SP families gPRPs and bPRPs, their interaction depends on the concentration of

GSF. For the lowest concentration of GSF, the family that interacts less with GSF

is gPRPs. However, increasing the concentration of GSF leads to a more

significant interaction with gPRPs. Among the three main families of SP, for the

highest GSF concentration, bPRPs are the ones that interact less with GSF.

A B

C D

Fig.1.6 Percentages of area decrease of each HPLC fraction after the interaction of AS solution with increasing concentrations of GSF (A, B, and C). Percentages of area decrease for each family of salivary proteins (D).

In order to obtain additional information about which SP of each HPLC fraction is

interacting with tannins, SDS-PAGE of AS before and after the interaction with

GSF was carried out. The resulting precipitate was also analyzed by SDS-PAGE,

which is widely used for the analysis of human fluids containing proteins as well

as for the study of tannin protein interactions.41 In fact, gel electrophoresis is a

useful tool for assessing tannin binding proteins in human saliva because the

proteins dissociate from the insoluble tannin/protein complexes in the presence

of SDS and can be visualized and identified in the gel.

0.00 0.25 0.50 0.75 1.00 1.25 1.500

20

40

60

80

100

1

2

3

4

% area

HPLC pool:

GSF (mM)

0.00 0.25 0.50 0.75 1.00 1.25 1.500

20

40

60

80

100 5

6

7

8

% area HPLC pool:

GSF (mM)

0.00 0.25 0.50 0.75 1.00 1.25 1.500

20

40

60

80

100

9

10

11

12

% area

HPLC pool:

GSF (mM)

0.00 0.25 0.50 0.75 1.00 1.25 1.500

20

40

60

80

100

bPRP

gPRP

aPRP

statherin

% area

Salivary protein family:

His 1

GSF (mM)


Fig.1.7 presents the results of the SDS-PAGE of AS before and after the

interaction with 0.133 and 0.232 mM of GSF.

The results presented in Fig.1.7 clearly show that the proteins existing in the

control AS precipitate by GSF. It is also perceptible that increasing GSF

concentration also increases the amount of SP that appears in the respective

precipitate.

Fig.1.7 SDS-PAGE of the AS solution before and after the interaction with two concentrations of grape seed fraction (GSF) (0.133 mM and 0.232 mM). The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa.

The HPLC fractions that previously showed a significant interaction with GSF

(Fig.1.5 and Fig.1.6), namely, fractions 5 to 12, were also analyzed by SDS-

PAGE after the interaction with GSF. The same saliva sample was incubated with

two different concentrations of GSF (0.133 and 0.232 mM) and centrifuged, and

the supernatant was analyzed by HPLC. After HPLC collection, the fractions

were dried in a SpeedVac, dissolved in the same control volume, and analyzed

by SDS-PAGE. The results are presented in Fig.1.8. The gels for F5 and F12 are

absent because it was not possible to see any band in the control assay.

Fig.1.8 SDS-PAGE of HPLC fractions 6 to 11 isolated from the HPLC after the injection of the AS solution before (C) and after the interaction with two concentrations of grape seed fraction (0.133 and 0.232 mM). The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa.



111

From the results presented, it is possible to see that for F6 the band that appears

at 45 kDa, probably corresponding to a bPRP as previously referred, and

disappears with the increase in GSF concentration. Similar behavior was

observed for the same protein that also appears in F7.

The intensity of the band of bPRP3 (near the 66 kDa) in F7 decreases slightly

with increasing GSF concentration. However, the effect of GSF in this

protein/band is more visible in F8. In general, these results are in agreement with

the ones obtained in the HPLC analysis (Fig.1.5); for F7 and F8, only with GSF

concentration above 0.5 mM is the profile greatly reduced. For the lowest GSF

concentrations, the HPLC profile of these fractions is not very affected.

The band corresponding to aPRPs in F10 disappear after 0.232 mM GSF

concentration. This result is in agreement with the results obtained from the

HPLC analysis (Fig.1.5F). Furthermore, the band's intensity corresponding to

histatin 3 and 1 in F9 and F11, respectively, is reduced importantly with the

increase in GSF concentration. Overall, the main family of SP that disappears

considerably after GSF incubation is the aPRPs (PRP1 and PRP3) present in

HPLC fraction 10.

These results seem to indicate that statherin and especially acidic PRPs (aPRPs)

have a high relative affinity toward condensed tannin complexation compared to

that of the other SP, in a competitive assay at pH 5.0. The acidic proteins PRP1

and PRP3 have 150 and 106 aminoacid residues, respectively. The first 106

residue sequence from PRP1 corresponds to PRP3. The acidic character of

these proteins is confined roughly to the first 30 amino acids at the N-terminal

due to the presence of many aspartic and glutamic acid residues. The remaining

part is basic and, similarly to basic PRPs, shows repeated sequences of proline

and glutamine.22

In general, PRPs have open randomly coiled structures. These open structures

allow the exposure of peptide carbonyl groups to hydrogen bonding as well as

the exposure of proline residues to act as binding sites for tannin by hydrophobic

interaction of the aromatic portion of tannin with the pyrrolidine structure of

proline. Nevertheless, in the case of aPRPs, previous studies on calcium binding

to aPRPs pointed to an interaction between the N- and C-terminal regions of the

proteins (possibly by electrostatic forces) indicating that the structures are not so

open when compared to those of other PRPs.66,67 However, at pH 5.0 as studied

herein, which is close to the isoelectric point (pI) of PRP1 and PRP3 (4.63 and

4.14, respectively), several acidic amino acids in the N- terminal part are not


expected to be charged, reducing the interaction with C-terminal and opening the

structure of aPRPs.42 Consequently, proline residues are readily accessible to

promote hydrophobic interaction with tannins. However, the presence of an

important number of carboxyl groups in aspartic and glutamic amino acid

residues in the N-terminal part may contribute importantly to strengthen the

interaction with tannins through hydrogen bonds.

Concerning the basic PRPs (bPRPs), these results generally show a significant

decrease of their area but only for the highest GSF concentrations (0.599 and 1.3

mM) and when almost all the other proteins have been depleted. These results

seem to indicate that the bPRPs have a low relative affinity toward condensed

tannin complexation compared to that of the other SP, in a competitive assay at

pH 5.0. Not totally in agreement with this, Lu and Bennick35 have measured the

amount of condensed tannins (crude quebracho tannin) and tannic acid

precipitated by pure bPRPs (IB-1, IB-4, IB-8b, and IB-6), aPRPs (PIF-s), and

gPRPs (gPRPs) at pH 7.4. These authors have observed that each bPRP

precipitated a higher quantity of tannins compared to that of the other PRPs.

Also, they showed that there were only small differences in the tannin-

precipitating ability of various bPRPs of different sizes or sequences, indicating

that, although there is considerable phenotypic variation of PRPs, it is not likely to

cause marked individual variation in tannin-binding ability. However, it is difficult

to compare the results obtained by these authors with the ones obtained herein

because first of all, the tannins used are completely different from ours, being a

complex mixture and not properly characterized. Also, these authors measured

the quantity of tannin precipitated, which depends not only on the PRP affinity but

also on the stoichiometry of the complexes. However, the pH used by these

authors (pH 7.4) and herein (pH 5.0) are substantially different, which will

probably differently affect the interaction for the reasons mentioned above.

The interaction of GSF with phosphorylated forms of statherin (HPLC fraction 12)

is also important. The results yielded by the HPLC analysis show a significant

interaction with statherin being one of the main SPs that interacts with condensed

tannins in the conditions described here. According to our knowledge, only Nayak

and Carpenter68 showed that statherin is precipitated by polyphenols, namely, tea

polyphenols. Statherin is abundant in tyrosine residues and is phosphorylated at

Ser2 and Ser3. Several variants have been identified and are derived both by

alternative splicing and posttranslational modifications. The presence of protic



113

groups (phosphates) with acidic character may favor the hydrogen bond

interactions which probably explain statherin's high affinity to tannins.

Regarding the glycoproteins, bPRP3 did not show a high interaction at lowest

GSF concentration. Nevertheless, increasing GSF concentration leads to a

significant decrease of that protein. For GSF concentration of 1.3 mM, the area of

HPLC fractions 7 and 8 has been decreased about 60%, while other HPLC

fractions (bPRPs 1 to 6) only decreased about 40%. As previously mentioned,

bPRP3 is a glycosylated protein that contains about 50% carbohydrate, which is

composed of highly fucosylated N-linked saccharides.69 Glycosylated PRPs

(gPRPs), which have been implicated in the oral epithelium lubrication,70 were

shown here to resist precipitation compared to that of aPRPs, histatins, and

statherin. Sarni-Machado et al.46 have also observed that low concentrations of

condensed tannins (mainly (+)-catechin, (-)-epicatechin, and epicatechin gallate)

first precipitate lower molecular weight SP and only for higher concentrations

precipitate glycosylated PRPs. More recently, the same authors14 have observed

that the glycosylation of human PRPs favors the formation of soluble complexes

and reduces tannin precipitation with regard to tannin amounts. This is in

agreement with Pascal et al.15 who have stated that protein glycosylation (gPRP,

similar to II-1 herein) prevented PRP condensed tannin precipitation when

compared to that of a nonglycosylated PRP (IB-5). These authors have

concluded that gPRPs are effective in binding tannins but that these interactions

do not necessarily result in precipitation. In conclusion, although gPRPs are not

readily precipitated by condensed tannins compared with other SP, they can form

complexes with tannins modifying the rheological properties of saliva such as

viscosity and consequently astringency.15

The GSF also interacts importantly with histatin 1 (HPLC fraction 11), as shown

both by HPLC analysis and by SDS-PAGE. The results also showed an

interaction of tannins with histatins 3 and 5 (HPLC fractions 6 and 9), although

not as significant as the one with histatin 1. Contrary to these results, Naurato

and co-workers71 have found that His 1 bound only about half the amount of

condensed tannin (crude quebracho tannin and epigallocatechin gallate) than

histatins 3 and 5, and there was no difference between these two latter in their

ability to precipitate condensed tannins. Moreover, they found that histatins 3 and

5 share the same condensed tannin-binding region, with more tannin binding to

the C-terminal region. However, all the experimental conditions and methodology


used were quite different, namely, pH (7.4), buffer (isotonic barbital buffer), and

temperature of interaction (37°C).

Regarding the histatin (His) group, their interaction with tannins is well known.42,71

They comprise a group of structurally related, small histidine-rich proteins found

only in the saliva of humans and some monkeys. Twelve His, named His 1 to 12,

have been isolated from human saliva and their primary structures determined.21

The most prominent members are His 1, 3, and 5, and they account for 85-90%

of this family. Histidine is the prominent amino acid, accounting for about 25% of

all residues and, together with basic amino acids, makes up 30% to 75% of total

amino acids. In contrast to PRPs, they contain no proline residues except for a

single residue in His 1. It is interesting that, among the identified histatins, it was

His 1 that was the most effective in binding polyphenols.

In conclusion, regarding the interaction between tannins and different families of

SP, the published data at present is somehow controversial. Some authors have

stated that all salivary PRP families have similar affinities toward different

condensed tannins at a molecular level by means of a competition assay, while

others stated that basic PRPs are the main family of SP that interact with

condensed tannins. However, the experimental conditions described in the

literature are often different, and the results should be analyzed carefully.

The results present herein provide important insights concerning the influence of

the different families of SP in the development of astringency. In fact, when all

the main families of SP are present in a competitive assay, like in the oral cavity,

they demonstrate that tannins interact first with aPRPs and statherin and also

interact significantly with histatins and gPRPs. For future experiments, it would be

interesting to evaluate the interaction between condensed tannins and saliva

from different donors and also to evaluate the interaction with hydrolysable

tannins.

� AUTHOR INFORMATION

Corresponding Author

*Department of Chemistry, Faculty of Sciences, University of Porto, Rua do

Campo Alegre 687, 4169-007 Porto, Portugal. Phone: +351 220402558. Fax:

+351 220402659. E-mail: [email protected].



115

Funding Sources

This work was supported by Fundação para a Ciência e Tecnologia by one Ph.D.

grant [SFRH/BD/41946/2007] and one project grant [PTDC/AGR-

ALI/67579/2006].

� ACKNOWLEDGMENT

We thank Professor Massimo Castagnola for his help, knowledge, and

advice in the interpretation of ESI-MS spectra of salivary proteins.

� ABBREVIATIONS USED

PRPs, proline-rich proteins; bPRPs, basic proline-rich proteins; aPRPs, acidic

proline-rich proteins; gPRPs, glycosylated proline-rich proteins; SP, salivary

proteins; GSF, grape seed fraction; AS, acidic saliva; DLS, dynamic light

scattering; PMF, peptide mass fingerprint; TFA, trifluoracetic acid;MW, molecular

weight; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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Capítulo 2

Effect of Condensed Tannins Addition on the Astringency of Red Wines

Soares, S., Sousa, A., Mateus, N., de Freitas, V.

Chem. Senses 2011, 37, 191-198



125

Effect of Condensed Tannins Addition on the Astring ency of Red Wines

Susana Soares, André Sousa, Nuno Mateus and Victor de Freitas

Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of

Sciences, University of Porto, 4169-007 Porto, Portugal

Correspondence to be sent to: Victor de Freitas, Department of Chemistry,

Faculty of Sciences, University of Porto, Rua do Campo Alegre 687, 4169-007

Porto, Portugal. e-mail: [email protected]

Accepted August 11, 2011

Astringency has been defined as a group of sensations involving dryness,

tightening, and shrinking of the oral surface. It has been accepted that

astringency is due to the tannin-induced interaction and/or precipitation of the

salivary proline-rich proteins (PRPs) in the oral cavity, as a result of the ingestion

of food products rich in tannins, for example, red wine. The sensory evaluation of

astringency is difficult, and the existence of fast and reliable methods to its study

in vitro is scarce. So, in this work, the astringency of red wine supplemented with

oligomeric procyanidins (condensed tannins), and the salivary proteins (SP)

involved in its development were evaluated by high-performance liquid

chromatography analysis of human saliva after its interaction with red wine and

by sensorial evaluation. The results show that for low concentration of tannins,

the decrease of acidic PRPs and statherin is correlated with astringency intensity,

with these families having a high relative complexation and precipitation toward

condensed tannins comparatively to the other SP. However, for higher

concentrations of tannins, the relative astringency between wines seems to

correlate’s to the glycosylated PRPs changes. This work shows for the first time

that the several families of SP could be involved in different stages of the

astringency development.

KEYWORDS: human saliva, oligomeric procyanidins, proline-rich proteins, sensory analysis


� INTRODUCTION

The astringency perception on the human palate has been defined as a complex

group of sensations involving dryness, tightening and shrinking of the oral

surface, and puckering sensations of the oral cavity (ASFTTO Materials 1989).

Astringency sensation results from the ingestion of some food products rich in

tannins, namely tea and red wine. Since 1954, when Bate-Smith (1954) proposed

that astringency results from the interaction of tannins with salivary proteins (SP)

in the mouth, it has been generally accepted and supported by recent literature

(De Freitas and Mateus 2001; Kallithraka et al. 2001; Mateus et al. 2004;

Hofmann et al. 2006; Preys et al. 2006) that astringency is due to the tannin-

induced interaction and/or precipitation of salivary proline-rich proteins (PRPs) in

the oral cavity. Although many research groups and literature support this

mechanism for astringency development, astringency is a very complex sensory

experience, and actually, the possible mechanisms for its development are

controversially discussed by the scientific community. Schwarz and Hofmann

(2008) proposed that astringency sensory perception is related to the ‘‘nonbound

free’’ astringent stimulus present in saliva and also suggested the involvement of

laminin receptor in its development; others suggested that modifications of the

viscous and lubrication properties of saliva are important, either by the

precipitation of several proteins (Rossetti et al. 2008) and increased friction

(Green 1993) or by interaction with glycosylated PRPs (gPRPs) modifying their

rheological properties (Pascal et al. 2008).

SP have been grouped into 6 structurally related major classes namely, histatins,

basic PRPs (bPRPs), acidic PRPs (aPRPs), gPRPs, statherin, and cystatins

(Oppenheim et al. 1971, 1988; Bennick 1982; Shomers et al. 1982; Hay et al.

1988; Schlesinger et al. 1989; Helmerhorst and Oppenheim 2007). All these

peptides, except bPRPs, have well-defined important biological functions in

saliva, including maintenance of ionic calcium concentration (aPRP and

statherin), antimicrobial action (histatins and cystatins), or protection of oral

tissues against degradation by proteolytic activity (cystatins). For bPRPs, it has

been proposed (Mehansho et al. 1983, 1985; Lu and Bennick 1998) that one of

their functions is to bind tannins, preventing their toxic effects in the

gastrointestinal tract.

One well-known beverage rich in tannins is red wine. Red wine quality takes into

account the fine balance between traditional parameters, such as acidity, sugar,

color, bitterness, and astringency, some of which are related to anthocyanins and



127

tannins. Anthocyanins and tannins are polyphenolic compounds responsible for

the color and astringency attributes, respectively.

The composition of wines in tannins from grapes (condensed tannins) changes

drastically between wines depending on variety, wine-making procedures, region,

and among other factors. These aspects influence importantly the wines

sensorial characteristics, mainly the flavor. So, it is of fundamental importance to

understand the effect of tannins on wine astringency, in order to not compromise

the overall wine quality. The sensory evaluation of astringency is difficult, time

consuming, and expensive. Moreover, it is often wrongly associated with

bitterness, being sometimes difficult for a panel to distinguish different

astringency attributes (Peleg et al. 1999), and the existence of fast and reliable

methods to study the astringency in vitro is scarce. So, in this work, the

astringency of red wine supplemented with oligomeric procyanidins (OPC,

condensed tannins isolated from grape seeds), and the SP involved in its

development were evaluated by chromatographic analysis of human saliva after

its interaction with red wine and also by sensorial evaluation by a trained panel.

This approach also allowed to study the effect of the red wine matrix in the

reactivity of condensed tannins toward human SP.


Reagents. All reagents used were of analytical grade or better.

Acetonitrile (ACN) and hydrochloric acid were purchased from Panreac Quimica;

acetic acid (HOAc) was purchased from Carlo Erba Reagents; Folin-Ciocalteu,

sodium acetate, and trifluoroacetic acid (TFA) were purchased from Fluka

Biochemica; ethanol was purchased from AGA, Álcool e Géneros Alimentares,

SA.; ethyl acetate was purchased from Valente e Ribeiro, Lda; chloroform was

purchased from Pronalab, José M. Vaz Pereira, Lda; sodium hydroxide was

purchased from LaboratórioMaialab, Lda; sodium carbonate was purchased from

Sigma-Aldrich; and tartaric acid was purchased from Aldrich.

Isolation of OPC from grape seeds. OPC (condensed tannins) were

extracted from Vitis vinifera grape seeds with an ethanol/water/chloroform

solution (1:1:2, v/v/v), and the chloroform phase, containing chlorophylls and

lipids, was rejected. Then, the hydroalcoholic phase was extracted with ethyl

acetate. The organic solvent was removed using a rotary evaporator (30°C)

yielding a residue (OPC) that corresponds to catechin monomers and


procyanidinoligomers (Darné and Madero 1979; De Freitas et al. 1998). This

residue was characterized by high-performance liquid chromatography (HPLC)

regarding the composition in procyanidins, accordingly to De Freitas and Glories

(1999). Briefly, 2 Lichrospher (C18) ODS (250 x 4.6 mm id) columns placed in

line were used for all analysis. The chromatograms were monitored at 280 nm

using an ultraviolet (UV) detector.The elution system consisted of 2 solvents, A:

2.5% HOAc and B: 80% ACN + 20% A. The gradient applied was linear from 7%

to 20% (eluent B) in 90 min at a flow rate of 1.0 mL.min–1. The procyanidins

identified and quantified were: (+)-catechin (56.0 mg.g–1), (–)-epicatechin (50.0

mg.g–1), dimers B1–B3 (270.0 mg.g–1), B2 (138.0 mg.g–1), B4 (38.0 mg.g–1), B5

(26.0 mg.g–1), B2-gallate (151.0 mg.g–1), and epicatechin gallate (16.0 mg.g–1).

Total of catechins and procyanindins correspond to 745.0 mg.g–1 and remaining

255.0 correspond to others high molecular weight (MW) OPC (255.0 mg.g–1).

Red wine supplementation with grape seed OPC. A red wine (V.

vinifera, Touriga nacional, and T. Franca cv.) from the Douro demarked region

was provided by Lavradores de Feitoria, S. A. This wine has been determined to

have 0.993 ± 0.006 g catechin equivalents.L–1 of condensed tannins, determined

based on the Folin-Ciocalteu method described by Singleton and Rossi (1965).

Red wine was also characterized by HPLC regarding the composition in

procyanidins, accordingly to De Freitas and Glories (1999), as described above.

The procyanidins identified and quantified were: (+)-catechin (2.51 ± 0.11 mg.L–

1), (–)-epicatechin (1.57 ± 0.02 mg.L–1), dimers B1–B3 (109.63 ± 10.37 mg.L–1),

B2 (50.40 ± 5.86 mg.L–1), B4 (3.38 ± 0.70 mg.L–1), B5 (20.95 ± 0.63 mg.L–1), B6

(10.33 ± 0.63 mg.L–1), B2-gallate (42.64 ± 2.35 mg.L–1), and epicatechin gallate

(2.35 ± 0.21 mg.L–1). The total quantity of procyanidins is 243.76 mg.L–1. Different

red wines with different concentration in OPC were prepared by adding

increasing quantities of OPC extract to the selected wine (control wine): 0.5, 1.0,

1.5, 2.0, 2.5, and 3.0 g.L–1. The resulting wines were bottled in triplicate

(triplicates of 50 mL) and kept in the dark.

Human saliva collection. Saliva was collected from 6 healthy

nonsmoking volunteers, and 2 mL of saliva from each volunteer were used to

make a saliva pool (whole saliva, WS). Collection time was standardized at 2 PM

in order to reduce concentration variability connected to circadian rhythms of

secretion (Messana et al. 2004). The saliva pool was mixed with 10% TFA (final

concentration 0.1%), mixed and centrifuged at 8000g for 5 min. After the



129

centrifugation, the supernatant (acidic saliva, AS) was separated from the

precipitate and used for the following experiments.

Protein–tannin interaction. The proteins in AS sample were analyzed by

HPLC before and after the interaction with increasing volumes of red wines

enriched with different concentration of OPC. The control condition was a mixture

of AS (150 µL) and acetate buffer 0.1 M, 12% ethanol, and pH 5.0 (50 µL) in

order to maintain the final volume constant (200 µL) between the several tested

volumes of red wine. The control condition was done in buffer with 12% ethanol

to simulate a model wine and at pH 5.0 because it is the average pH between

wine (3.4) and saliva (7.0). The first experiments were made with increasing

volumes (10, 20, 30, 40, and 50 µL) of red wine supplemented with 1.5 g.L–1 of

OPC, and acetate buffer was added to make the final volume 200 µL. Each

volume tested of red wine was an independent experiment. After shaking, the

mixture reacted at room temperature for 5 min and then was centrifuged (8000g,

5 min). The reaction time (5 min) was established as the minimal time to have

stable interactions and good reproducible results. The supernatant was analyzed

by HPLC. The precipitate resultant from some experiments was resolubilized in

110 µL of eluent A used for the HPLC analysis (see below) and analyzed by

HPLC. The same experiments were done for the other red wines supplemented

with different concentrations of OPC (control, 0.5, 1.0, 2.0, 2.5, and 3.0 g.L–1),

using 10 or 20 µL of wines in the reaction with saliva.

Negative control experiments were made with OPC dissolved in acetate buffer.

OPC solutions were made in increasing concentrations (the same concentrations

referred above), and 20 µL of these solutions were used to react with saliva in the

same experimental conditions described above.

HPLC analysis. Ninety microliters of each solution was injected on an

HPLC Lachrom system (L-7100) equipped with a Vydac C8 column, with 5 µm

particle diameter (column dimensions 150 x 2.1 mm); detection was carried out at

214 nm, using a UV-Vis detector (L-7420). The HPLC solvents were (eluent A)

0.2% aqueous TFA and (eluent B) 0.2% TFA in ACN/water 80/20 (v/v). The

gradient applied was linear from 10% to 40% (eluent B) in 60 min, at a flow rate

of 0.30 mL.min–1. After this program, the column was washed with 100% eluent B

for 20 min in order to elute S-type cystatins and other late-eluting proteins. After

washing, the column was stabilized with the initial conditions (Messana et al.

2004; Soares et al. 2011).


Tannin-specific activity. The tannin-specific activity (TSA) toward WS

and AS was determined by nephelometry as described by De Freitas and Mateus

(2001). This method is based on the characteristic property of tannins to interact

and precipitate proteins. The red wines supplemented with condensed tannins

were diluted 50 times with filtered (0.45 µm) model solution (12% ethanol, 5.0

g.L–1 tartaric acid, pH 3.20). About, 4.0 mL of this solution were transferred to a

test tube and mixed with 50 µL of WS or AS. The test tube was kept in the dark

for 30 min, and after this time, the maximum turbidity was measured in a

turbidimeter HACH 2100 N adapted for cells of 100 x 12 mm. The TSA is

expressed in turbidity units NTU/mL of wine and is determined by the following

expression, where 0.08 corresponds to the dilution factor of wine:

Turbidity �NTU/mL� = Turbidity ��

0.08

Sensory evaluation. Red wines (control and supplemented wines with

OPC) were rated for astringency by a 6-member trained sensory panel. The

wines were presented to the panel members in glass cups (at room temperature,

20°C) in random order. The panel members were asked to rate the intensity of

the perceived astringency for each sample in each series on a 1–7 score scale.

Water was used for mouth rinsing between consecutive samples.

Data and statistical analysis. Values are expressed as the arithmetic

means ± standard deviation. Statistical significance of the difference between the

TSA of WS and AS was evaluated by t-test unparametric. Differences were

considered to be statistically significant when P < 0.05. Statistical significance of

the difference between the perceived astringency by the sensory panel was

evaluated by one-way analysis of variance, followed by the Bonferroni test.

Differences were considered to be statistically significant when P < 0.1.

� RESULTS

The reactivity of red wines supplemented with condensed tannins (OPC) toward

SP was evaluated by chromatographic analysis of human saliva after its

interaction with red wine and also by sensorial analysis.

The initial acidic treatment of saliva with TFA is used to precipitate several high

MWSP (such as α-amylases, mucins, carbonic anhydrase, and lactoferrin) and to

preserve sample protein composition because TFA partially inhibits intrinsic

protease activity (Messana et al. 2004). However, peptides and proteins like



131

histatins, bPRPs and aPRPs, statherin, cystatins, and defensins are soluble in

AS solution and may be directly analyzed by Reverse Phase HPLC, as

previously described (Messana et al. 2004; Soares et al. 2011).

The HPLC chromatogram of this AS solution at 214 nm is presented in Fig.2.1,

and the profile was similar to the one previously described in the literature

(Messana et al. 2004; Soares et al. 2011). The top of the figure shows the

distribution of the different families of SP along the chromatogram that were

established previously by proteomic approaches, namely ESI-MS and MALDI-

TOF/TOF (Messana et al. 2004; Soares et al. 2011).

Fig.2.1 Typical Reverse Phase HPLC profile detected at 214 nm of the AS solution of human saliva. The dotted lines and numbers show the ranges and the main SP family assigned to each HPLC peptide region.

The HPLC chromatogram of the AS solution is roughly divided into 4 salivary

peptide family regions: The first Region 1 comprises proteins that belong to the

classes of bPRPs and histatins. The bPRPs identified in this region include IB-8b,

IB-8c, IB-9, IB-4, and P-J; and the histatins include histatins 3, 5, 7, 8, and 9.

Region 2 comprises mainly a gPRPs, the bPRP3. Region 3 corresponds entirely

to aPRPs, namely PRP1 and PRP3, and the last Region 4 has phosphorylated

and nonphosphorylated forms of statherin and peptide P-B (Soares et al. 2011).

The peak between regions 2 and 3 has not been previously assigned to any

family of SP, and in this work, it has shown a weak reactivity toward OPC. So,

this peak was not considered in the results analysis and discussion.

A red wine enriched with increasing concentrations of OPC was used in order to

compare the reactivity of the several SP families toward wine tannins directly in a

wine matrix. The wines were mixed with AS, and the insoluble aggregates were

removed by centrifugation. The supernatant was analyzed by HPLC.

15 20 25 30 35 40 45 50 55 60 650

2

4

6

8

101 3 4

bPRPs gPRPs aPRPs statherin

2

t (min)

Ab

s (A

u)


The first experiments were done with the red wine supplemented with 1.5 g.L–1 of

OPC, which is the middle concentration of OPC used. These experiments were

done with increasing volumes of red wine from 10–50 µL in order to study if the

interaction with the different families of SP was affected by the wine volume.

Some examples of the HPLC profiles of AS solution before and after the

interaction with different volumes of this red wine arepresented in Fig.2.2A.The

percentage of area decreases for each HPLC region after the AS solution

interaction with increasing volumes of red wine supplemented with 1.5 g.L–1 of

OPC are summarized in Fig.2.2B. It was also perceptible that the amount of

precipitate increased with the volume of red wine added to AS.

A

B

Fig.2.2 (A) HPLC profile detected at 214 nm of the AS solution before (control) and after the interaction with increasing volumes of red wine supplemented with 1.5 g.L–1 of OPC (condensed tannins). (B) Percentages of area decrease of each HPLC salivary peptide region after the interaction of AS solution with increasing volumes of red wine supplemented with 1.5 g.L–1 OPC. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.

20 25 30 35 40 45 50 55 60 65 700

2

4

6

8

10

12

t (min)

Ab

s (A

u)

0 (control)

10 µµµµL

20 µµµµL

30 µµµµL

Volume of red wine supplementedwith 1.5 g.L -1:

0 10 20 30 40 500

20

40

60

80

100

Red wine supplementedwith 1.5 g.L -1of OPC (µµµµL)

Are

a (

%)

1

2

3

HPLC peptide regions:

bPRPs

gPRPs

aPRPs

4 statherin



133

The results displayed in Fig.2.2 show that the HPLC profile of the AS solution

and, therefore, the area of each different family of proteins are interestingly

affected in a different way by the interaction with increasing volumes of red wine

HPLC regions 3 (aPRPs) and 4 (statherin) decline significantly (to 50% and 20%,

respectively) with the lowest red wine volume added (10 µL), the area of the

other regions remains relatively constant (Fig.2.2B). Indeed, only the highest

volume added (50 µL) decrease the bPRPs to about 40% and gPRPs to 30%.

Volumes of 10 and 20 µL were chosen for further experiments with the other

wines supplemented with different concentrations of OPC. These volumes were

chosen because they correspond to the lowest volumes that affected differently

the several regions of the chromatogram and allowed comparing the reactivity of

several wines toward different families of SP. Indeed, for volumes of wine of 30

µL and higher, statherin and aPRPs precipitated almost completely (Fig.2.2B).

Fig.2.3 presents the HPLC profile of AS solution before and after the interaction

with 10 µL of red wine supplemented with increasing concentrations of OPC (1.0,

2.0, and 3.0 g.L–1). Only some chromatograms representative of the overall

tendency are presented.

Fig.2.3 Reverse Phase HPLC profile detected at 214 nm of the AS solution before (control) and after the interaction with 10 µL of each red wine supplemented with different concentrations of OPC.

From the results presented in Fig.2.3, it is possible to observe that for the same

volume of wine (10 µL) added to AS solution, the resulting decrease of SP is

markedly different. The addition of 10 µL of red wine supplemented with 3.0 g.L–1

of OPC, results in an important decrease of almost all regions. On the other

hand, for the red wine supplemented with 1.0 g.L–1 of OPC, only the areas of

aPRPs and statherin decrease importantly. These experiments were done with 2

15 20 25 30 35 40 45 50 55 60 650

2

4

6

8

101 3 42

t (min)

Abs

(Au

)

0 (control)

1.0 g.L -1 3.0 g.L -1

2.0 g.L -1

Red wine supplemented with OPC:


different volumes of wine (10 and 20 µL), and the decrease of percentage area is

summarized in Fig.2.4.

A – 10 µL B – 20 µL

Fig.2.4 Percentages of area decrease of each HPLC peptide regions after the interaction of AS solution with (A) 10 and (B) 20 µL of red wine supplemented with increasing concentrations of OPC. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.

The results presented in Fig.2.4 clearly indicate that the interaction of SP present

in the AS solution with red wine is affected by the volume of red wine used and

also by the quantity of condensed tannins present in the red wines. While the

aPRPs and statherin are significantly affected for the lowest volume used

(Fig.2.4A) and with the increasing in the concentration of condensed tannins in

wines, bPRPs are practically not precipitated. Increasing the volume of red wine

to 20 µL leads to a significant decrease of gPRPs and a slight decrease of

bPRPs.

The area of statherin is reduced below 20% with addition of control red wine

(without OPC supplementation). This means that the tannins present in the

control red wine are enough to complex and precipitate statherin in the

concentration that it is present in saliva, and the presence of higher quantities of

condensed tannins leads to a total depletion of this protein.

For aPRPs, the behavior is similar: 20 µL of control red wine is enough to reduce

its area to 20%. However, for the experiments with 10 µL, it is possible to

observe that the area of aPRPs is reduced importantly for wine enriched in OPC.

For gPRPs, only the experiments with 20 µL showed a significant decrease of

their area. Indeed, increasing the volume to 20 µL and increasing the

0.0 0.5 1.0 1.5 2.0 2.5 3.00

20

40

60

80

100

Red wine suplementedwith OPC (g.L -1)

Are

a (

%)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

20

40

60

80

100

Red wine suplementedwith OPC (g.L -1)

Are

a (

%)

1

2

3

HPLC region:

bPRPs

gPRPs

aPRPs

4 statherin



135

concentration of condensed tannins lead to a decrease of their area up to 20%.

For bPRPs, neither increasing the volume nor increasing the concentration of

condensed tannins, at least at the concentrations used, reduce significantly its

area.

In order to study the contribution of OPC added to supplemented wines in the SP

precipitation, blank experiments were done in which the interaction with SP was

made using OPC solutions made in acetate buffer (pH = 5.0), with the same

range of concentrations used previously (Fig.2.5).

Fig.2.5 Percentages of area decrease of each HPLC peptide regions after the interaction of AS solution with 20 µL of OPC solutions in acetate buffer matrix. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.

From these results, it is possible to observe that aPRP and statherin families

decrease significantly along with the concentration of OPC as it was observed in

red wines (Fig.2.4B, 20 µL), whereas gPRP are practically not affected by OPC in

blank experiments. The concentration of OPC (catechin and dimers) in original

wines (244 mg.L–1, see the experimental section) probably do not explain the

high precipitation observed in wines (Fig.2.4B, 20 µL). Indeed, in Fig.2.5, for the

concentration around 244 mg.L–1, only a small percentage of SP precipitates.

This means that in original wine, already exists other components (e.g., tannin-

like structures) that interact strongly with SP. However, the slight increase of

tannins concentration in wines with OPC addition has an important contribution to

discriminate wines concerning their ability to precipitate SP.

It is important to refer that the mixture of AS solution withred wine always resulted

in the formation of insoluble precipitates that perceptibly increased along with the

added volume of wine and also with the concentration in OPC.

0.0 0.5 1.0 1.5 2.0 2.5 3.00

20

40

60

80

100

120

Acidic buffer with OPC (g.L -1)

Are

a (

%)

bPRP

gPRP

aPRP

Statherin

HPLC peptide regions:


The HPLC analysis of the precipitates resulting from the interaction of 10 or 20

µL of red wine supplemented with 0.5, 2.0, and 3.0 g.L–1 of OPC with the AS are

shown in Fig.2.6.

Fig.2.6 Reverse Phase HPLC profile detected at 214 nm of the precipitates resultant from the interaction of 10 or 20 µL of red wine supplemented with 0.5, 2.0, and 3.0 g.L–1 of OPC with the AS.

It is possible to observe that several SP families are effectively precipitated by

wine tannins. Indeed, in the HPLC chromatogram of the solubilized precipitate,

aPRPs (region 3) and statherin (region 4) appear in the precipitate formed by the

addition of 10 µL of red wine supplemented with 0.5 g.L–1 of condensed tannins.

Increasing the concentration in condensed tannins (red wine supplemented with

2.0 g.L–1 of OPC) also increases the percentage of those proteins in the

precipitate. The difference between the percentages of those proteins in the

precipitates obtained with 10 µL of wines supplemented with 0.5 and 2.0 g.L–1 of

OPC is not very high because the lowest concentration of condensed tannins

leads to depletion of almost all these 2 families of proteins.

For the highest volume of the more concentrated wine (20 µL of red wine

supplemented with 3.0 g.L–1 of OPC), it is possible to observe the appearance of

other proteins belonging to gPRPs in the precipitate. However, regarding bPRPs,

small peaks start to be detected in the insoluble aggregates resulting from the

interaction with 20 µL of red wine supplemented with 3.0 g.L–1 of OPC. The

appearance of these groups of proteins in the precipitate is in agreement with

their disappearance in the respective supernatant described previously. In

general, the first proteins to be precipitated by condensed tannins are aPRPs and

statherin, followed by gPRPs.

Overall, these results seem to indicate that statherin and aPRPs have a high

relative affinity toward condensed tannins complexation and precipitation

comparatively to the other SP, in a competitive assay in a wine matrix. On the

2

15 20 25 30 35 40 45 50 55 60 65 700

4

8

12

161 3 4

t (min)

Abs

(A

u)

10 µµµµL red wine supplemented with 2.0 g.L -1 of OPC

20 µµµµL red wine supplemented with 3.0 g.L -1 of OPC

10 µµµµL red wine supplement w ith 0.5 g.L -1 of OPC

Precipitates:



137

other hand, these results seem to indicate that bPRPs, when present in a wine

matrix, have a low relative tannin affinity.

In order to study if the proteins present in WS and that are precipitated with TFA

are important for the interaction with condensed tannin in wines, the TSA of WS

and AS toward the several enriched red wines was also measured (Table 2.1).

Table 2.1. Tannin specific activity (TSA) of 50 µL of whole (WS) and acidic saliva (AS) toward red wines supplemented with several concentrations of oligomeric procyanidins (OPC). For the same concentration of OPC, the results of WS and AS are significantly different (P<0.05), except for the 2.0 g.L-1 concentration (athese results are statistically equal). Astringency rating from the sensorial evaluation. The orders with different letters are significantly different (P<0.1).

Concentration of OPC / g.L -1 0 0.5 1.0 1.5 2.0 2.5 3.0

TSA / NTU.mL -1

Whole saliva (WS) 66.6 ± 0.7 83.8 ± 7.3 90.5 ± 1.4 95.0 ± 3.1 86.6 ± 1.5a 103.7 ± 3.2 114.2 ± 4.4

Acidic saliva (AS) 77.8 ± 2.9 98.8 ± 6.5 103.8 ± 0.3 119.4 ± 7.1 97.5 ± 10.7a 129.7 ± 11.7 141.3 ± 9.4

Astringency rating 1.2 ± 0.4b 2.3 ± 1.4b,c 3.0 ± 0.0 c,d 4.0 ± 0.0 c,d,e 4.8 ± 1.6 e ,f 6.0 ± 0.0 f,g 6.7 ± 0.8 g

From the results presented in Table 2.1, it is possible to observe that the

aggregation is higher with AS comparatively to WS. However, these differences

are more significant for the higher supplemented red wines. Proteins present in

WS, such as α-amylase and mucins, that were removed with TFA, seem to affect

negatively the formation of aggregates. These results suggest that the AS, which

has been analyzed by HPLC, contains the most important proteins that react with

condensed tannins and that may contribute to astringency sensation.

As expected, the TSA increased regularly with the concentration of OPC added

to wine. This behavior concurs with the perceived astringency of those wines

(Table 2.1). The analysis of the SP families of this AS showed that the different

families could have different potentialities in developing astringency. From

Fig.2.4, it is possible to conclude that for low wine volume and therefore lower

concentration of tannins (10 µL of wine), the decrease of aPRPs is correlated

with astringency intensity. However, for higher concentrations of tannins (20 µL of

wine), the relative astringency between wines seems to be correlated to the

gPRPs changes.

� CONCLUSION

Astringency is a very complex sensation with different descriptives very hard to

define by sensorial analysis. Those descriptives could be related with tannins’

structure, concentration, wine medium, and class of SP precipitated.


The results presented show that human AS could be used for an in vitro

evaluation of the astringency of a sample because both HPLC and TSA results

using AS are in agreement with the results obtained by the sensory panel.

Moreover, this work shows for the first time that increasing the volume of red

wine, as well as OPC concentration in red wine matrix, affects differently the

several families of SP, with aPRPs and statherin being the most affected either

for low volumes as for low concentrations of OPC. Nevertheless, for higher

volumes of red wine and higher concentration of OPC, gPRPs are also

significantly affected and precipitated. So, it seems that the several SP families

have relative discriminatory functions in rating the wine astringency depending on

the concentrations of condensed tannins and the volume of wines. In summary,

the several families of SP could be involved in different stages of the

development of astringency.

The future work stands in studying the interaction of different human saliva

proteins with different classes of tannins.

� FUNDING

This work was supported by Fundação para a Ciência e Tecnologia by one phD

grant [SFRH/BD/41946/2007] and one project grant [PTDC/AGR-

ALI/67579/2006].

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Capítulo 3

Interaction of Different Classes of Salivary Proteins with Food Tannins

Soares, S., Mateus, N., de Freitas, V.

Food Res. Int. 2012, 49, 807-813



145

Interaction of different classes of salivary protei ns with food tannins

Susana Soares, Nuno Mateus and Victor de Freitas*

Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

Tannins are polyphenolic compounds commonly present in vegetables, fruits and

derived products that interact with proteins, namely salivary proline-rich proteins

(PRPs). This interaction forms (in)soluble aggregates that are supposed to be at

the origin of the astringency sensation but that also influences tannins

bioavailability. Therefore, this interaction was studied herein by HPLC and

dynamic light scattering (DLS) using procyanidin trimer and PGG

(pentagalloylglucose), to obtain information about the relative affinity of the

interaction, solubility (both by HPLC) and size of the complexes formed (by DLS).

The results show that mainly acidic PRPs (aPRPs) and statherin interact with

both tannins, forming a significant quantity of complexes (either insoluble or

soluble), while bPRPs (basic PRPs) interact poorly with procyanidin trimer and

gPRPs (glycosylated PRPs) only complex with PGG. In general, PGG formed a

high amount of insoluble complexes with salivary proteins while procyanidin

trimer formed soluble complexes (except for statherin). For PGG, the maximum

aggregation and complexes size (~200 nm) corresponds to the stoichiometric

concentration (232 µM). For procyanidin trimer, the concentrations used did not

allow to reach the maximum of aggregation. These results highlight the influence

and mechanisms that different tannins and salivary proteins could present in their

interaction, and consequently in the development of the astringency sensation.

KEYWORDS: proline-rich proteins, condensed tannins, hydrolyzable tannins, soluble complexe

� INTRODUCTION

Tannins are a complex group of polyphenolic compounds that can be found in

vegetal foodstuffs, particularly in fruits, cereal grains and beverages (red wine,

tea and beer). These compounds are structurally divided in condensed (polymers

of catechin) and hydrolysable tannins (esters of glucose with gallic acid). During

foodstuff consumption, these polyphenols interact with salivary proteins,

especially with proline-rich proteins (PRPs), forming insoluble aggregates that

are supposed to be at the origin of astringency sensation (Kallithraka, Bakker et


al. 1998; Lu and Bennick 1998; Bacon and Rhodes 2000; Soares, Vitorino et al.

2011). Astringency has been defined has a dryness of the oral surface, puckering

and tightening sensations of the oral mucosa typically experienced during the

ingestion of tannin-rich food, in particular red wine (ASTM 1989). It is considered

to be a tactile, diffuse and poorly localized sensation.

Although there are a few theories about the origin of astringency sensation, the

most widely accepted one relies on the interaction between tannins and salivary

proteins. In general, the nature of tannin/protein interactions can be described as

covalent or non-covalent based on whether the molecules are irreversible bound

to each other or not, and which could result in the formation of soluble or

insoluble complexes. Non-covalent interactions are thought to involve the cross-

linking of separate protein molecules by the tannin, which acts as a polydentate

ligand on the protein surface involving hydrophobic and hydrogen bonds (Asano,

Shinagawa et al. 1982; Siebert, Troukhanova et al. 1996; de Freitas and Mateus

2001; Bennick 2002; Cala, Pinaud et al. 2010; Canon, Giuliani et al. 2010).

From tannin/protein interaction it is also known that there is an optimum

tannin/protein ratio for which maximum precipitation occurs (Fig.3.1A). In fact,

Hagerman and Robbins (1987) verified this fact with the addition of increasing

concentrations of BSA to a fixed amount of tannin. For higher or lower

tannin/protein ratios the quantity of insoluble precipitates was lower (hyperbolic

behavior, Fig.3.1A). The mechanism to explain these experimental observations

was proposed later by Siebert and co-workers (Siebert, Troukhanova et al. 1996)

(Fig.3.1B).

Fig.3.1 A. Adapted from Hagerman and Robbins (1987). Titration of a plant extract containing tannin with protein (BSA): region A, excess tannin; region B, equivalence point; region C, excess protein. B. Adapted from Siebert and co-workers (Siebert, Troukhanova et al. 1996). Model for protein/polyphenol interaction that explains the results observed in Fig. 3.1A. Polyphenols are depicted as having two ends that can bind to protein. Proteins are depicted as having a fixed number (three) of polyphenol binding sites.

If a protein is considered as having a fixed number of sites to which a tannin can

bind and a tannin is thought as having two (or more) ends that can bind to

protein, then the situation where the total concentration of tannin’s ends is



147

roughly equal to the number of binding sites in the protein will result in a large

network, corresponding to large colloidal particles and maximum precipitation. On

the other hand, if there is a large excess of protein to tannin, each tannin

molecule is able to find binding sites in two proteins to attach to. However, it is

unlikely that there will be sufficient additional tannin molecules to bridge many of

these “protein dimers” together. This results in smaller particles and less haze.

The same is true if there is a large excess of tannin to protein, because nearly all

the sites in proteins would be occupied. This would make difficult for tannin

attached at one end to find an available site on another protein to bridge to. The

result would again be smaller particles and less precipitation.

As referred previously, astringency is thought to result from tannin/salivary

proteins interaction. The main salivary proteins have been grouped into six

structurally related major classes, namely histatins, basic PRPs (bPRPs), acidic

PRPs (aPRPs), glycosylated PRPs (gPRPs), statherin, and cystatins (Humphrey

and Williamson 2001; Huq, Cross et al. 2007). The differences between the

several families of PRPs depend on their charge and presence or absence of

carbohydrate. All these proteins have important biological functions in saliva

associated with calcium binding to enamel, maintenance of ionic calcium

concentration (PRPs and statherin), antimicrobial action (histatins and cystatins),

or protection of oral tissues against degradation by proteolytic activity (cystatins)

(Hay, Bennick et al. 1988; Oppenheim, Xu et al. 1988; Schlesinger, Hay et al.

1989; Kauffman, Keller et al. 1993; Helmerhorst and Oppenheim 2007).

Despite the knowledge about tannin/protein interactions, there is no much

information about the interaction of different families of salivary proteins with

different classes of tannins. Therefore, this work aimed to study the interaction

between different families of salivary proteins with condensed and hydrolysable

tannins, to obtain information about the relative affinity of the interaction, solubility

and size of the complexes formed.


Reagents. All reagents used were of analytical grade.

Procyanidin trimer C2 (condensed tannin) synthesis. Procyanidin

trimer (catechin-(4-8)-catechin-(4-8)-catechin) was synthesized according to the

procedure described in literature (Bras, Goncalves et al. 2010). Briefly, (+)-

taxifolin (Extrasynthèse) and (+)-catechin (ratio 1:3) were dissolved in ethanol


and the mixture was treated with sodium borohydride (in ethanol). The pH was

then lowered to 4.5 by addition of CH3COOH/H2O 50% (v/v), and the mixture

stood under argon atmosphere for 30 min. The reaction mixture was extracted

with ethyl acetate. After evaporation of the solvent, water was added and the

mixture was passed through C18 gel, washed with water, and recovered with

methanol. After evaporation of methanol, the fraction was passed through a TSK

Toyopearl HW-40(s) gel column (300 mm × 10 mm i.d., 0.8 mL.min-1, coupled to

a UV-Vis detector, using methanol as eluent). Several fractions were recovered

and analyzed by ESI-MS (Finnigan DECA XP PLUS) yielding procyanidins

dimers (B3 and B6) and trimer (catechin-(4-8)-catechin-(4-8)-catechin). The

structure was elucidated by HPLC-MS and NMR analysis (Tarascou, Barathieu et

al. 2006; Absalon, Fabre et al. 2011).

β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose (PGG) (h ydrolysable

tannin) synthesis. PGG was synthesized from tannic acid according to Chen

and Hagerman (2004). Tannic acid (5.0 g) was dissolved (“methanolyzed”) in

70% methanol in acetate buffer (0.1 M, pH 5.0) at 65 °C for 15 h. After this time,

the pH of the mixture was immediately adjusted to 6.0 with NaOH. Methanol was

then removed from the mixture by evaporation under reduced pressure at <30

°C, with addition of water to maintain the volume constant. The resulting solution

was extracted with 3 volumes of diethyl ether and 3 volumes of ethyl acetate. The

diethyl ether extracts were rejected while the ethyl acetate extracts were

combined and evaporated, with addition of water to maintain the volume. The

resulting suspension was centrifuged, and the precipitate was redissolved by

heating in 2% methanol solution. PGG precipitated as the solution cooled to room

temperature and was collected by centrifugation. PGG was washed twice with an

ice-cold 2% methanol solution and once with ice-cold distilled water. The final

material was lyophilized to yield a white powder with an overall mass yield of

23%. The purity of the obtained PGG was assessed by HPLC analysis and 1H

NMR spectroscopy (Fernandes, Fernandes et al. 2009).

Saliva Collection. Saliva was collected from six healthy non-smoking

volunteers and 2 mL of saliva from each volunteer were used to make a saliva

pool (whole saliva). Collection time was standardized at 2 p.m. in order to reduce

concentration variability connected to circadian rhythms of secretion (Messana,

Cabras et al. 2004). The saliva pool was mixed with 10% TFA (final concentration

0.1%) to precipitate several high molecular weight salivary proteins (such as α-



149

amylases, mucins, carbonic anhydrase and lactoferrin) and to preserve sample

protein composition, since TFA partially inhibits intrinsic protease activity. After

centrifugation (8000g for 5 min), supernatant (acidic saliva, AS) was separated

from the precipitate and used for the following experiments.

Protein and Tannin Interaction. The AS sample was analyzed by HPLC

before and after interaction with increasing concentrations of PGG or procyanidin

trimer. The control condition was a mixture of AS (150 µL) and 0.1 M acetate

buffer (pH 5.0, 12% ethanol) (50 µL) (final volume 200 µL). For the experiments

with tannins, the necessary volume (8 to 60 µL) of tannin stock solution (2.66

mM) was added to AS (150 µL) to obtain the desired final concentration, plus the

volume of acetate buffer to make the final volume 200 µL. Tannins were tested in

the following final concentrations: 53.0, 133.0, 232.0, 299.0 and 399.0 µM, and

each concentration was an independent experiment. After shaking, the mixture

reacted at room temperature (20 ⁰C) for 5 min and was then centrifuged (8000g,

5 min). Supernatant was recovered for subsequent HPLC-DAD and DLS analysis

(Fig.3.2).

Fig.3.2 Scheme of the experimental approach used to study protein/tannin interaction indicating the supposed kind of complexes formed and removed in each step.

HPLC analysis. Half of the supernatant was directly injected into the

HPLC-DAD and the other half was filtered (cellulose acetate 0.22 µm) and only

then injected into the HPLC-DAD. 90 µL of each solution were injected on a

HPLC Lachrom system (Merck Hitachi, L-7100) equipped with a Vydac C8

column, with 5 µm particle diameter (column dimensions 150 x 2.1 mm);

detection was carried out at 214 nm, using a UV-Vis detector (L-7420). The

HPLC solvents were 0.2% aqueous TFA (eluent A) and 0.2% TFA in ACN/water

80/20 (v/v) (eluent B). The gradient applied was linear from 10 to 40% (eluent B)

in 60 min, at a flow rate of 0.30 mL.min-1. After this program the column was

washed with 100% eluent B for 20 min in order to elute S-type cystatins and

other late-eluting proteins. After washing, the column was stabilized with the

initial conditions (Messana, Cabras et al. 2004; Soares, Vitorino et al. 2011).


Dynamic Light Scattering (DLS) Measurements. The size of the

aggregates present in the supernatant was determined by DLS. Half of the

supernatant was directly analyzed by DLS and the other half was filtered

(cellulose acetate 0.22 µm) and only then analyzed by DLS (Fig.3.2). The size of

procyanidin trimer/salivary proteins and PGG/salivary proteins aggregates in

filtered and non-filtered solutions was measured using the Zetasizer Nano ZS

Malvern instrument. This technique measures the time-dependent fluctuations in

the intensity of scattered light that occur because particles undergo Brownian

motion. The analysis of these intensity fluctuations enables determination of the

diffusion coefficients of particles, which are converted into a size distribution. The

sample solution was illuminated by a 633 nm laser, and the intensity of light

scattered at an angle of 173 was measured by an avalanche photodiode. This

analysis provides information concerning particle size (obtained by average size

parameter) and polydispersity. In general, Z average size results were

considered valid when the polydispersity of solutions was below 0.5.

Statistical Analysis. All assays were performed in n = 3 repetitions. The

mean values and standard deviations were evaluated using analysis of variance

(ANOVA); all statistical data were processed using GraphPad Prism version 5.0

for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).

� RESULTS

This work aimed to provide new insights about the influence of structural factors

of polyphenols and salivary proteins on the development of astringency

sensation. The interaction between different families of salivary proteins with

condensed and hydrolysable tannins was studied in order to have some new

information about the relative affinity of the interaction, solubility and size of the

complexes formed. This information was achieved by HPLC analysis of human

saliva before and after interaction with the referred tannins and by DLS

measurements of the complexes formed.

Salivary proteins

The initial acidic treatment of human saliva with TFA is used to precipitate several

high molecular weight salivary proteins (such as α-amylases, mucins, carbonic

anhydrase and lactoferrin) and to preserve sample protein composition, since

TFA partially inhibits intrinsic protease activity (Messana, Cabras et al. 2004).

However, peptides and proteins like histatins, basic, acidic and glycosylated



151

PRPs, statherin, cystatins, and defensins are soluble in acidic saliva (AS)

solution and were thus directly analyzed by RP-HPLC, as previously described

(Messana, Cabras et al. 2004; Soares, Vitorino et al. 2011). The HPLC

chromatogram of this AS solution (at 214 nm) is displayed in Fig.3.3. The top of

the first HPLC chromatogram shows the distribution of different families of

salivary proteins along the chromatogram that were established previously by

proteomic approaches, namely ESI-MS and MALDI-TOF/TOF (Messana, Cabras

et al. 2004; Soares, Vitorino et al. 2011).

Fig.3.3 RP-HPLC profile detected at 214 nm of the AS solution before (control saliva) and after the interaction with 133 and 399 µM of A, PGG and B, procyanidin trimer. All these solutions were filtered (0.22 µm) prior to HPLC analysis.

The HPLC chromatogram of the AS solution is roughly divided into four salivary

proteins family regions: the first region comprises proteins that belong to bPRPs

and histatins classes. The bPRPs identified in this region include IB-8b, IB-8c, IB-

9, IB-4 and P-J and the histatins include histatins 3, 5, 7, 8 and 9. Second region

comprises mainly a gPRP, bPRP3. The next region corresponds entirely to


aPRPs, namely PRP1 and PRP3, and last region contains phosphorylated and

non-phosphorylated forms of statherin and peptide P-B (Soares, Vitorino et al.

2011).

Interaction of salivary proteins with tannins

The experiments in this study were all performed in buffer with 12% ethanol to

mimic a model wine and at pH 5.0. At this pH, salivary proteins have showed to

strongly interact with tannins (de Freitas and Mateus 2002) and it corresponds to

an intermediary pH between wine pH (3.4) and saliva pH (7.0). Indeed, salivary

pH drops with the ingestion of acidic drinks, and the degree of acidity in saliva

depends on the sampled volume, buffering capacity, and mode of drinking

(Johansson, Lingström et al. 2004; Brand, Tjoe Fat et al. 2009). In order to

compare the reactivity of salivary proteins with tannins, different concentrations of

procyanidin trimer (condensed tannins) and PGG (hydrolysable tannins) were

mixed with AS solution, and after removal of insoluble complexes by

centrifugation, half of the supernatant was directly analyzed by HPLC and the

other half was filtered (0.2 µm) prior to the analysis by HPLC. Some HPLC

profiles of AS solution after interaction with both tannins (filtered) are shown in

Fig.3.3.

In Fig.3.3, the area of each peak represents the amount of protein that did not

precipitate with tannins and eventually the amount of protein involved in

formation of soluble complexes with tannins that were dissociated by HPLC

conditions analysis.

The HPLC profile of the AS solution is interestingly affected by interaction with

both tannins. Effectively, each tannin interacts differently with each group of

proteins. In general, PGG was much more efficient than procyanidin trimer in

precipitating all salivary proteins, especially for higher concentrations. For

instance, for the same tannin concentration (133 µM) both tannins affected

differently several families of salivary proteins. In fact, while both tannins

precipitated much more efficiently aPRPs and statherin proteins, practically no

bPRPs and gPRPs were precipitated. However, for 399 µM concentration, PGG

practically precipitated all salivary proteins including bPRPs and gPRPs, while

procyanidin trimer only precipitated significantly aPRPs and statherin. So, both

protein and tannin structures are crucial features that govern tannin/protein

interactions.



153

Formation of soluble and insoluble salivary protein/tannin

complexes

Protein/tannin complexes can be soluble and insoluble, depending among other

things on protein and tannin structure, concentration and protein/tannin ratio. The

presence of insoluble salivary protein/tannin complexes was visually perceived

and easily removed by centrifugation. However, the formation of soluble

complexes is more difficult to assess and was estimated herein by filtration

comparing filtered and non-filtered supernatants (Fig.3.4) and by DLS

measurements. Insoluble complexes were removed previously by centrifugation.

The HPLC chromatogram of the filtered and non-filtered AS solutions, without

tannins, were exactly the same (data not shown), indicating that the filtration

process does not retain proteins from AS.

Fig.3.4 RP-HPLC profiles detected at 214 nm of the AS solution after the interaction with 399 µM of procyanidin trimer and 133 µM of PGG. These solutions were non-filtered and filtered (0.22 µm) prior to HPLC analysis.

From Fig.3.4 it is possible to observe that filtration induced a decrease of some

chromatographic peaks of salivary proteins, in particular for aPRPs and statherin

proteins. That difference could be explained by retention of salivary protein/tannin

soluble complexes by the filter. In the case of the non-filtered solutions, the

observed higher area of some chromatographic peaks may be justified by

dissociation of these soluble complexes by the HPLC solvents into their

precursors (tannin and protein). This is particular evident in the PGG

experiments, were the chromatographic peak of PGG appear only in the non-

filtered solution, which may result from dissociation of soluble salivary

proteins/PGG complexes during HPLC analysis. In fact, the efficiency of HPLC

solvents in dissociate salivary protein/tannin complexes was tested with the

insoluble complexes formed and initially removed by centrifugation. These


experiments revealed that the HPLC solvents dissolve, at least partially, the

formed precipitates because the salivary proteins have appeared in the

chromatogram after dissolution (data not shown).

The variations in the chromatographic peaks of each family of salivary proteins

after interaction with both tannins before and after filtration are presented in

Fig.3.5. The results are expressed as percentage of the area of these proteins

relatively to the control saliva (without tannin). The results were divided by family

of salivary proteins. bPRPs peak areas were not considered in non-filtered

solutions because of the co-elution with PGG peak (shown in Fig.3.4) which

difficult its analysis.

Fig.3.5 The graphics represent the variation of the chromatographic peaks area of the several salivary proteins studied with the increase in tannins concentration. The results are expressed as percentage of the area of each protein relatively to the control saliva (100%) analyzed by HPLC, before and after solutions filtration (0.22 µm). Statherin graphic was used as an example indicating the percentage of protein involved in the formation of soluble and insoluble complexes. These results represent the average of three independent experiments.

From the results presented in Fig.3.5 it is possible to observe systematically a

reduction of proteins’ area comparing non-filtered and filtered solutions. Once

again, it is possible to observe that PGG is much more efficient than procyanidin

trimer in reducing almost all salivary proteins, especially for higher

concentrations.



155

The observed differences between non-filtered and filtered solutions point to the

existence of soluble complexes that are retained by the filter and that could

explain the proteins’ area reduction upon filtration, as explained previously.

The differences between non-filtered and filtered solutions were also visible in the

size experiments measurements by DLS. The size of salivary protein/tannin

(soluble) complexes present in the centrifuged solution of AS and tannins that

was analyzed by HPLC, was determined by DLS and the results are presented in

Fig.3.6. This DLS analysis comprises all the salivary proteins together present in

AS solution and not the different families of salivary proteins separately, which

are only possible to separate from AS by HPLC.

Fig.3.6 Influence of tannin concentration on the average size of salivary soluble proteins/tannins complexes present in solution. These results represent the average of three independent experiments.

The presented results show unequivocally the existence of soluble salivary

protein/tannin complexes that are retained by the filter (0.22 µm). Although these

complexes present a lower size than the cut-off of the filter used in the

experiment, they are almost totally retained by filtration. This fact could be

explained by the presence of some higher complexes that are firstly retained by

the filter and that could act as adjuvants in the filtration of smaller complexes.

For procyanidin trimer, soluble complexes size increase along with its

concentration (240 nm for the highest concentration). However, the behavior for

PGG is not entirely similar. The size of the soluble complexes that result from

salivary proteins interaction with PGG first increased with PGG concentration

until it reaches a maximum (200 nm) and then it was reduced.


� DISCUSSION

In order to highlight the differences between non-filtered and filtered solutions

and facilitate discussion, the percentages of proteins involved in the formation of

soluble and non-soluble complexes for the highest tannin concentration are

shown in Table 3.1.

As schematically presented in Fig.3.5 for statherin, the percentage of insoluble

complexes was obtained subtracting the area of the chromatographic peaks of

each family obtained in the HPLC analysis of the non-filtered solution to the

respective area obtained in the control saliva (AS without tannin). The

percentage of soluble complexes was calculated in the same way but subtracting

the data obtained in the HPLC analysis of the non-filtered solution to the

respective area obtained in the filtered solution.

Table 3.1 Percentage of salivary proteins involved in the formation of soluble and insoluble complexes formed by the interaction with 399 µM of each tannin. These results represent the average of three independent experiments.

Tannin Complexes Salivary protein families (%)


PGG Insoluble - 59.3 ± 1.7 65.6 ± 4.5 70.1 ± 1.7

Soluble - 35.5 ± 3.6 30.9 ± 4.0 28.1 ± 4.5

Procyanidin trimer Insoluble ~0 ~0 19.3 ± 2.6 72.1 ± 1.5

Soluble 14.3 ± 3.1 ~0 29.0 ± 12.4 24.9 ± 0.3

The results show that most of the salivary protein families’ studied interact with

both tannins, specially aPRPs and statherin. In fact, it is possible to observe that

these two families are involved in the formation of a significant quantity of

complexes (either insoluble or soluble) with both tannins, while bPRPs and

gPRPs interact poorly and practically do not complex with procyanidin trimer in

these conditions.

On the other hand, the amount of salivary proteins involved in the formation of

soluble and insoluble complexes is in general higher for PGG with the exception

of statherin for which the amount of complexes is practically the same for both

tannins. This shows that statherin is the only salivary protein that presents no

selectivity towards tannin structures.

In general, PGG interacted with all salivary proteins forming high amount of

insoluble complexes that precipitate. Oppositely, procyanidin trimer tends to form

soluble complexes with the exception of statherin.



157

Comparing the different families of salivary proteins, the difference in the

percentage of proteins involved in the formation of complexes, especially the

insoluble ones, seem to be more affected by the protein structure for the

procyanidin trimer in comparison with PGG. In fact, for PGG it seems that protein

structure only affects the efficiency of the interaction. PGG seems to be more

reactive towards salivary proteins while procyanidin trimer is more selective. It is

also interesting to observe that gPRPs only complex with PGG.

Regarding the size measurements interpretation, it is interesting to notice that

PGG behavior is in agreement with the protein/tannin interaction model most

widely accepted and presented in Fig.3.1, in which the maximum aggregation

and complexes size corresponds to the stoichiometric concentration (in PGG

case 232 µM). For lower and higher PGG concentration the soluble complexes

formed have a lower size because there are fewer and higher PGG molecules

respectively, to occupy proteins binding sites, forming smaller complexes.

Regarding the procyanidin trimer, the used concentrations did not allow to reach

the stoichiometric maximum of aggregation for this tannin, although the size

seemed to begin to stabilize for the higher concentrations.

In order to interpret the size measurements in the light of the results observed for

the HPLC experiments, it is important to notice that the salivary proteins that are

probably involved in the formation of soluble complexes with the highest size

(~200 nm) at 232 µM of PGG (Fig.3.5) were mainly aPRPs and statherin.

� CONCLUSION

In summary, these results suggest that the kind (soluble or insoluble) and size of

complexes formed depends not only on tannin class/structure but also on protein

structure and on protein:tannin ratio. PGG originates mainly insoluble complexes

with salivary proteins (mainly composed by aPRPs or statherin proteins) with the

soluble complexes having a maximum size about 200 nm at near 200 µM, while

procyanidin trimer originates mainly soluble complexes (in particular for PRPs)

but also insoluble complexes (mainly composed by statherin) with maximum size

higher than 240 nm at a concentration superior to 400 µM.

These results highlight the diverse influence and mechanisms that different

tannins and salivary proteins could present in the development of the astringency

sensation.


� AKNOWLEDGMENTS

The authors thank the financial support by Fundação para a Ciência e

Tecnologia by one phD grant [SFRH/BD/41946/2007] and one project grant

[PTDC/AGR-ALI/67579/2006].

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Journal of Proteome Research, 3, 792-800.

Oppenheim, F. G., Xu, T., McMillian, F. M., Levitz, S. M., Diamond, R. D., Offner,

G. D., & Troxler, R. F. (1988). Histatins, a novel family of histidine-rich proteins in


fungistatic effects on Candida albicans. Journal of Biological Chemistry, 263,

7472-7477.

Schlesinger, D. H., Hay, D. I., & Levine, M. J. (1989). Complete primary structure

of statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva

of the monkey, Macaca arctoides. International Journal of Peptide and Protein

Research, 34, 374-380.

Siebert, K. J., Troukhanova, N. V., & Lynn, P. Y. (1996). Nature of polyphenol-

protein interactions. Journal of Agricultural and Food Chemistry, 44, 80-85.

Soares, S., Vitorino, R., Osorio, H., Fernandes, A., Venancio, A., Mateus, N.,

Amado, F., & de Freitas, V. (2011). Reactivity of human salivary proteins families

toward food polyphenols. Journal of Agricultural and Food Chemistry, 59, 5535-

5547.

Tarascou, I., Barathieu, K., Andre, Y., Pianet, I., & Dufourc, E. J. (2006). An

Improved Synthesis of Procyanidin Dimers: Regio- and Stereocontrol of the

Interflavan Bond. European Journal of Organic Chemistry, 2006, 5367-5377.

Capítulo 4

Mechanistic Approach by Which Polysaccharides Inhibit α-Amylase/Procyanidin Aggregation

Soares, S., Gonçalves, R. M., Fernandes, I., Mateus, N., de Freitas, V.

J. Agric. Food Chem. 2009, 57, 4352–4358



163

Mechanistic Approach by Which Polysaccharides Inhib it α-Amylase/Procyanidin Aggregation

Susana Soares, Rui M. Gonçalves, Iva Fernandes, Nuno Mateus and Victor de

Freitas


The present work studies the inhibition of aggregation of α-amylase and

procyanidin fractions by different polysaccharides (arabic gum, β-cyclodextrin,

and pectins). Several analytical approaches, namely, fluorescence quenching,

nephelometry, and dynamic light scattering (DLS), were used. In general,

nephelometry showed that the presence of the polysaccharides in solution

reduced the formation of insoluble aggregates. The fluorescence quenching

measurements showed two effects: arabic gum and β-cyclodextrin reduce the

quenching effect of procyanidin fractions on α-amylase fluorescence, whereas

pectins do not affect the quenching of α-amylase fluorescence by procyanidin

fractions. DLS measurements have revealed that the polysaccharides studied

induce a decrease in aggregates size, which probably is due to the formation of

smaller aggregates resulting from the disruption and reorganization of the

procyanidin fractions/α-amylase aggregates. Overall, the results obtained for

arabic gum and β-cyclodextrin strongly suggest that the main mechanism by

which these two compounds inhibit protein/polyphenol aggregation is by

molecular association between these polysaccharides and polyphenols,

competing with protein aggregation. In the case of pectins, the results obtained

provide evidence that the main mechanism by which they reduce

protein/polyphenol aggregation is by forming a protein/polyphenol/polysaccharide

complex, enhancing its solubility in aqueous medium.

KEYWORDS: Aggregation; α-amylase; fluorescence; polysaccharides; tannin

� INTRODUCTION

Tannins are phenolic compounds, yielded from the secondary metabolism of

higher plants, being found worldwide in many different families of plants (1).


Bate-Smith and Swain defined the plant tannins as water-soluble phenolic

compounds with molecular masses between 300 and 3000 Da, displaying the

usual phenolic reactions [e.g., blue color with iron(III) chloride] and precipitating

alkaloids, gelatins, and other proteins (2, 3). However, this definition does not

include all tannins. Indeed, more recently, molecules with a molecular mass of up

to 20000 Da have been isolated and should also be classified as tannins on the

basis of their phenolic reactions. High concentrations of tannins can be found in

nearly every part of the plant, such as in wood, leaves, fruit, roots, and seed.

Usually, tannins are divided in two major classes: condensed (proanthocyanidins)

and hydrolyzable tannins. The first ones are polymers of catechin, and the latter

are gallic or ellagic esters of glucose. It is assumed that these compounds have a

biological role in the plant related to protection against infection and herbivores

(1).

From a nutritional point of view, the interaction between tannins and enzymes,

such as α-amylase or trypsin, has been shown to have harmful effects, with the

inhibition of these enzymes and decrease in body weight gain (4-6).

On the other hand, the interaction of α-amylase and other salivary proteins such

as proline-rich proteins (PRPs) and histatins with tannins is thought to be

responsible for the astringency sensation. This event is thought to result from the

formation of protein/tannin insoluble aggregates that precipitate, reducing the

palate lubrication and causing an unpleasant sensation of roughness, dryness,

and constriction (7, 8). Astringency is often perceived as a negative attribute as in

dairy products, nuts, and juices (9). However, in some beverages such as tea,

beer, and red wine, astringency could be perceived as a positive quality factor, if

not too intense.

The affinities of salivary proteins to complex tannins depend on many factors and

mainly on their chemical structures (10, 11). Previous works have shown that a

PRP (IB8c) binds to condensed tannins much more effectively than α-amylase

(12). This can be explained not only by their primary structure but also by the

three-dimensional structure of these proteins. In fact, whereas α-amylase is a

globular protein, PRPs are extended randomly coiled proteins, which offer more

sites to interact with tannins. However, R-amylase seems to bemore specific and

selective than PRPs in the aggregation with samples containing different

amounts of proanthocyanidins (13). α-Amylase from porcine pancreas (hereafter

PPA) is a single polypeptide chain of 496 residues of which 17 are tryptophan

residues, which are responsible for its intrinsic fluorescence (14). Mammalian α-



165

amylases show a high degree of homology, so it is considered that in a general

way porcine and human α-amylases are very similar (15, 16).

The presence of polysaccharides in solution is also an important factor that

affects the interaction between tannins and proteins. Several studies have

demonstrated the ability of some neutral and anionic polysaccharides to disrupt

the binding of polyphenols to proteins (17-19). For instance, loss of astringency is

one of the principal changes that occur during the ripening of many edible fruits,

which has been related to the increase of soluble pectins during maturation. Two

mechanisms have been proposed to explain this phenomenon: (I)

polysaccharides form a ternary complex protein/polyphenol/polysaccharide,

which enhances solubility in an aqueous medium; (II) there is a molecular

association in solution between polysaccharides and polyphenols, competing for

protein aggregation (17, 19).

The effect of polysaccharides on polyphenol complexation with proteins has been

studied in solution by techniques such as enzyme activity, NMR, and

nephelometry (17, 19, 20). Whereas NMR gives important data about the

interaction between tannin and protein at a molecular level, nephelometry

measures the formation of high molecular weight structures at a macromolecular

level, taking into account all physical-chemical driving forces involved in the

aggregates’ formation. Over recent years, the fluorescence quenching technique

has been employed, with success, to study the specificity of the protein/tannin

interaction at a molecular level (21-23). Changes on PPA structure resulting from

the interaction with tannins were evaluated by the measurement of intrinsic

fluorescence intensity of protein tryptophan residues. Fluorescence

measurements give information about the molecular environment in the vicinity of

the chromophore molecule. The decrease of protein intrinsic fluorescence

intensity is called quenching, which can occur by different mechanisms, namely,

collisional quenching, when the excited-state fluorophore is deactivated upon

contact with some other molecule in solution (the quencher), or static quenching,

whereby fluorophores form nonfluorescent complexes with quenchers (24).

The aim of this work is to link the fluorescence quenching, nephelometry, and

dynamic light scattering (DLS) techniques to provide insights, at a molecular

level, of the process whereby polysaccharides (arabic gum, β-cyclodextrin, and

pectins with different methoxylation degrees) inhibit α-amylase aggregation with

grape seed procyanidins. α-Amylase/tannin aggregates are thought to influence

food astringency and, on the other hand, inhibit enzyme activity.



Reagents. α-Amylase from porcine pancreas (≥98%) was purchased from

Sigma. Arabic gum was purchased from Aldrich. β-Cyclodextrin (≥99%, HPLC)

was purchased from Fluka. Pectins (from citrus peel and standardized by the

addition of sucrose) with different methoxylation degrees (MD of 27, 58-63, and

72%) were kindly supplied by CPKelco Co.

Grape Seed Tannin Isolation. Condensed tannins were extracted as

described in the literature (25, 26). Briefly, condensed tannins were extracted

from Vitis vinifera grape seeds with an ethanol/water/chloroform solution (1:1:2,

v/v/v), and the chloroform phase, containing chlorophylls and lipids, was rejected.

The resulting hydroalcoholic phase was extracted with ethyl acetate. The organic

solvent was removed using a rotary evaporator (30°C), and the resulting residue,

corresponding to catechin monomers and oligomeric procyanidins, was

fractionated through a TSK Toyopearl gel column TSK Toyopearl HW-40(s) gel

column (100 mm X 10 mm i.d., with 0.8 mL.min-1 methanol as eluent), yielding

four fractions. Fractions I and II were obtained after elution with 99.8% (v/v)

methanol during 1 and 5 h, respectively (cumulative time); fraction III was

obtained after elution with methanol/5%(v/v) acetic acid during the next 14 h; and

fraction IV was obtained after elution with methanol/10% (v/v) acetic acid during

the next 8 h. All of the fractions were mixed with deionized water; the solvent was

eliminated using a rotary evaporator under reduced pressure at 30°C and then

freeze-dried. The composition of procyanidins in each fraction was determined by

direct analysis by ESI-mass spectrometry (Finnigan DECA XP PLUS) as

described in the literature (26). Fraction I contains catechin, galloyl derivatives,

procyanidin dimer, the galloyl derivative, and procyanidin trimer; fraction II

contains procyanidin trimer and tetramer and its galloyl derivatives (mean MW =

950); fraction III contains procyanidin pentamer and the galloyl derivative (mean

MW = 1512); fraction IV contains procyanidin pentamer digalloyl, procyanidin

tetramer tetragalloyl, procyanidin hexamer galloyl, procyanidin heptamer, and the

galloyl derivative (mean MW = 2052). The mean molecular weight was

determined on the basis of the relative abundance of each flavanol in the

fractions.

Fluorescence Quenching and Nephelometry Measuremen ts. The

quenching effect between α-amylase and procyanidin fractions with different

molecular weights was assayed in the presence of increasing concentrations of



167

different polysaccharides (arabic gum, β-cyclodextrin, and pectins with different

methoxylation degrees; 27, 58-63, and 72%) present in foods or added during

food processing (27-31). A Perkin-Elmer LS 45 fluorometer was used for

fluorescence quenching and nephelometry measurements. For the fluorescence

quenching assays the excitation wavelength was set to 282 nm and the emission

spectrum was recorded from 300 to 450 nm. Both slits were 10 nm. For

nephelometry analysis, the fluorometer was used as a 90° light scattering

photometer; for that, both excitation and emission wavelengths selected were the

same (400 nm). At this wavelength, protein, tannins, and polysaccharides do not

absorb the incident light (32). Considering the possibility of fluorescence

resonance energy transfer (FRET) between the protein and the procyanidin

fractions, the absorption spectra of both were analyzed (data not shown):

procyanidin fractions have an absorption maximum at 270 nm and decrease

significantly until near 310 nm. The protein’s emission spectrum starts at 320 nm,

and at this λ the polyphenols practically do not absorb. On the other hand,

procyanidin fractions have their emission maximum at 330 nm, whereas the

protein’s absorption spectrumis between 200 and 290 nm. Thus, it does not

seem possible that energy transference between these two molecules may

occur.

The experiments were performed in 100 mM acetate buffer with 12%

ethanol/water (v/v) as solvent (pH 5.0), and stock solutions of α-amylase (100

µM), procyanidin fractions (1 mM), and polysaccharides were prepared in this

buffer. A pH of 5.0 was chosen because it is already known that α-amylase

interactions with polyphenol are high at this pH, and it is close to mouth pH (7.0)

(33).

Because the reactivity of procyanidins toward α-amylase depends on their MW,

the concentration of each procyanidin fraction corresponds to the maximum

aggregation for a fixed concentration of α-amylase (1 µM). Fraction I interacted

weakly with α-amylase and was rejected in this study. The concentrations used

(fraction II = 120 µM; fraction III = 50 µM; fraction IV = 17 µM) were obtained by a

nephelometry curve, corresponding to the aggregation with increasing

concentration of procyanidins (data not shown). The α-amylase concentration

was chosen to be in the linear range of concentration with fluorescence.

In several microtubes, a volume of procyanidin fraction stock solution was mixed

with different volumes (to make a final volume of 250 µL) of acetate buffer. After

this, different volumes of the polysaccharide stock solution were added, and the


tubes were mixed. The mixture was allowed to stand for 30 min, and the blank

was measured (only for nephelometry) before the addition of 4 µL of the α-

amylase stock solution. After the addition of α-amylase, the mixture was mixed

and allowed to react for 1 h. After this, the microtube was mixed and the emission

spectra and intensity of aggregates were measured in the fluorometer cell.

The effect of polysaccharide on the protein’s fluorescence intensity was

previously analyzed. For this, a mixture of α-amylase (1 µM) with the highest

concentration of each polysaccharide (0.6 g.L-1 for arabic gum, 4.4 g.L-1 for

cyclodextrin, and 80.0 mg.L-1 for all pectins) was prepared, and after 1 h, the

protein’s fluorescence intensity was measured.

Because the procyanidin fractions possess intrinsic fluorescence at λex (282 nm),

their spectrum was measured and subtracted in all fluorescence experiments.

The effect of polysaccharides in the procyanidin fraction emission spectra was

also evaluated; it was observed that polysaccharides did not affect the

procyanidin fractions spectra.

All assays were performed in triplicate.

Dynamic Light Scattering (DLS) Measurements. The size of

aggregates present in solution was determined by DLS. The size of procyanidin

fractions/PPA aggregates and the effect of the carbohydrates were measured

using the Zetasizer Nano ZS Malvern instrument. This technique measures the

time-dependent fluctuations in the intensity of scattered light that occur because

particles undergo Brownian motion. The analysis of these intensity fluctuations

enables the determination of the diffusion coefficients of particles, which are

converted into a size distribution. The sample solution was illuminated by a 633

nm laser, and the intensity of light scattered at an angle of 173 was measured by

an avalanche photodiode. This analysis provides information concerning particle

size (obtained by the parameter intensity) and polydispersity.

These experiments were performed in triplicate.

Statistical Analysis. All assays were performed in n = 3 repetitions. The

mean values and standard deviations were evaluated using analysis of variance

(ANOVA); all statistical data were processed using GraphPad Prism version 5.0

for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).



169

� RESULTS AND DISCUSSION

Fluorescence quenching of α-amylase/tannin complexes in the presence of

increasing concentrations of polysaccharides was followed simultaneously with

the measure of α-amylase/tannin aggregates by nephelometry. As the

nephelometry measurements of aggregation are affected by several factors,

mainly by the size of aggregates, this aspect was also evaluated by DLS

technique.

Effect of Arabic Gum on PPA/Procyanidin Fraction Co mplexation.

Fig.4.1 shows the fluorescence emission spectrum (at λex = 282 nm) obtained for

PPA and procyanidin fraction IV (mean MW = 2052) solution in the absence and

increasing concentrations of arabic gum (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 g.L-1). Similar

spectra were obtained with procyanidin fractions II and III (data not shown). As

the concentration of arabic gum rises, the intensity of fluorescence increases.

Fig.4.1 Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) and procyanidin fraction IV (17 µM; mean MW = 2052) solution, in the absence (full line) and presence of increasing concentrations of Arabic gum (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 g.L–1). The experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).

The effect of different polysaccharides on the protein’s fluorescence intensity was

assayed to determine if the observed variations in fluorescence intensity were

only due to modifications of procyanidin interaction with α-amylase (Fig.4.2A).

300 350 400 4500

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λλλλ (nm)

Inte

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arabic gum

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A.

B. Procyanidins fractions alone Procyanidins fractio ns in presence of polysaccharides

Fig.4.2 A. Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) (solid spectra) and in the presence of the highest polysaccharide concentration [arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g.L-1, - - -; and pectins 80 mg.L-1 (27% MD, ; 58-63% MD, ; and 72% MD, ]. B. Several fluorescence emission spectra of procyanidin fractions alone and in the presence of the highest polysaccharide concentration (arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g L-1, ; and pectin 58-63% MD 80 mg.L-1, ). The experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).

In general, the different polysaccharides studied did not affect significantly the α-

amylase intrinsic fluorescence. On the other hand, procyanidins have been

shown to have much lower fluorescence intensity than the one observed for

proteins at the λex (Fig.4.2B). Nevertheless, procyanidin fraction spectra were

measured and subtracted from the emission spectra of α-amylase. Therefore, the

results obtained in the fluorescence quenching assays are only due to

modifications in the interaction between protein and the quencher (procyanidin).

This means that the quenching effect of procyanidins on PPA fluorescence

decreases in the presence of arabic gum. However, the increase in fluorescence

does not reach the protein’s intrinsic fluorescence itself (∼200 of intensity, in the

same experimental conditions without procyanidins and arabic gum) even for

higher concentrations of polysaccharide (data not shown). The increasing

percentages of intrinsic fluorescence by the presence of arabic gum for all tested

300 340 380 420 4600

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171

procyanidin fractions are shown in Fig.4.3 (upper data). All procyanidin fractions

followed the same behavior.

Overall, arabic gum was shown to be more efficient in reducing the quenching

effect of PPA for the more highly polymerized fraction (IV).

Fig.4.3 Influence of increasing concentrations of arabic gum in (upper data) variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates measured by nephelometry. Mean MWs of fractions II, III, and IV are 950, 1512, and 2052, respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).

For the same solution, the formation of aggregates (macromolecules) between α-

amylase and procyanidins, which are supposed to be in part insoluble, was also

followed by nephelometry (Fig.4.3, lower data). For all of the procyanidin

fractions tested, a decrease in aggregation with the increase in arabic gum

concentration was observed. This decreasing intensity of aggregation seems to

be greater for the highly polymerized fractions. These results are in agreement

with previous works (19, 34).

It is interesting to observe that the aggregation decreases concomitantly with the

increase of fluorescence. The competition between arabic gum and α-amylase

for procyanidins leads to the decrease of the quenching effect and, consequently,

to less aggregation.

Effect of β-Cyclodextrin on PPA/Procyanidin Fraction Complexat ion.

The influence of β-cyclodextrin concentration in procyanidin/PPA intrinsic

fluorescence is similar to that observed with arabic gum (Fig.4.1). The variation of

intrinsic fluorescence and aggregation is shown in Fig.4.4.

0.0 0.1 0.2 0.3 0.4 0.5 0.60

20

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180

200

Fraction II

Fraction III

Fraction IV

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%


Fig.4.4 Influence of increasing concentrations of β-cyclodextrin in (upper data) variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution and (lower data) percentage of procyanidin/PPA (1 µM) insoluble

aggregates measured by nephelometry. Mean MWs of procyanidin fractions II, III, and IV are 950, 1512, and 2052,

respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).

As observed for arabic gum, fluorescence in the presence of β-cyclodextrin

increases concomitantly with the decrease in percentage of aggregation. With

regard to the quenching effect, the lower MW fraction (II) is the most affected,

whereas the higher MW fraction (IV) is the less affected, oppositely to arabic

gum, where fraction IV was the most affected fraction.

For arabic gum and β-cyclodextrin assays, the interaction between procyanidins

(quenchers) with PPA is restrained by the presence of these polysaccharides,

reducing the quenching effect and consequently reducing the aggregation, as

confirmed by nephelometry. Altogether, these results provide strong evidence for

a molecular association in solution between polysaccharides and procyanidins,

competing for protein aggregation and inhibiting the formation of aggregates.

Comparison of these two polysaccharides shows that arabic gum is by far the

most effective in restraining the interaction between PPA and procyanidins,

because the amount required was about 10-fold smaller than that of β-

cyclodextrin. This is probably due to the polysaccharide structures, because β-

cyclodextrin has a cone-shaped structure conferring conformational restrictions

for the interaction with procyanidins. Effectively, β-cyclodextrin is a cyclic

oligosaccharide, composed of seven 1→4-linked α-D-glucopyranoside units. The

interior of the structure is less hydrophilic than the surface, being able to host

hydrophobic molecules in its interior (30, 31). However, some studies (35, 36)

have shown that only small molecules, such as (+)-catechin, can be included

inside the cyclodextrin cavity, which is not the case with procyanidin oligomers.

On the other hand, arabic gum is a heteropolysaccharide composed by a

polysaccharide and hydroxyproline-rich protein moieties able to ensure

hydrophobic interactions with procyanidins (27, 28). This polysaccharide moiety

0.0 1.0 2.0 3.0 4.0 5.00

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173

has also a slight acidic character, resulting from the presence of only 20% of

glucuronic acids. Its more acidic character compared to β-cyclodextrin may help

to establish electrostatic and cooperative hydrogen bonds that strengthen the

interaction with procyanidins. This may explain the higher efficiency of arabic

gum in restraining the aggregation between procyanidin fractions and PPA.

These kinds of electrostatic and ionic interactionswith proteins and tannins have

previously been shown with pectins from wines (32, 37). The restraining effect of

both polysaccharides in the aggregation between procyanidin fractions and α-

amylase is lower for fraction II than for the other two fractions. The effect of

polysaccharides in aggregation seems to be affected differently with the increase

in procyanidin MW. This effect was greater for fractions IV and III for arabic gum

and α-cyclodextrin, respectively.

Effect of Pectin with Different MDs on PPA/Procyani din Fraction

Complexation. The variation in intrinsic fluorescence of α-amylase/procyanidin

with different concentrations of pectins is shown in Fig.4.5 (upper data).

A B

C

Fig.4.5 Percentage of procyanidin/PPA (1 µM) insoluble aggregates in the presence of increasing concentrations of different pectins measured by nephelometry. Mean MWs of procyanidin fractions II (a), III (b), and IV (c) are 950, 1512, and 2052, respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).

0.0 20.0 40.0 60.0 80.00

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Pectin 27% (mg.L -1)

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Oppositely to arabic gum and β-cyclodextrin, the fluorescence remains more or

less constant with the increase of pectin concentration. In these cases, the

quenching effect of α-amylase by procyanidins is not affected by the presence of

pectins: the molecular environment in the vicinity of the chromophore (tryptophan

residues) seems to remain unchanged.

The nephelometry analysis showed a systematic decrease in aggregation with

the increase in pectin concentration, similar to that with arabic gum and β-

cyclodextrin. Once again, this effect was lower for the procyanidin fraction with

lower MW (II).

It is interesting to observe that although the effect of procyanidins in α-amylase

fluorescence is not affected by the presence of pectins, their aggregation ability is

importantly affected. Altogether, these results demonstrate that there is probably

a molecular association between polysaccharides, procyanidin fraction, and

protein in solution, resulting in a ternary complex and thereby forming more

soluble aggregates in aqueous medium. Thus, the fluorescence quenching of

PPA by procyanidin fractions is not reduced, but the formation of less soluble

aggregates is still inhibited.

Pectins were the most effective polysaccharides that inhibited procyanidins/PPA

aggregation, because the amount required was about a 10-fold lower

concentration than that of arabic gum. This is probably due to their more acidic

character and higher flexibility, when compared to the other polysaccharides

tested. In fact, pectins are a very complex mixture of different polysaccharide

molecules with a large range of molecular weights that vary with origin and

extraction conditions. In general, they are composed by partially methylated poly-

R-(1→4)-D-galacturonic acid residues (38). Pectins with high MD have reduced

polarity, whereas pectins with low MD have a few hydrophobic groups.

However, no correlation between the pectins’ MD and the restraining effect on

procyanidin fractions/PPA aggregation was observed. Apparently, differences in

the ratio of hydrophobic/hydrophilic groups between pectins did not seem to be

enough to induce important different behaviors. Overall, among all the studied

polysaccharides, pectins have a higher ionic character capable of establishing

hydrophilic interactions.

Size Measurement of Aggregates. The measurement of the size of

aggregates present in solution resulting from the interaction between the three

procyanidin fractions (II, III, and IV) and PPA in the absence and presence of

carbohydrates was performed by DLS. Table 4.1 shows the average size (Z) of



175

aggregates and also the polydispersity of solutions. In general, the polydispersity

of solutions is below 0.5, which means that the Z and polydispersity will give

values that can be used for comparative purposes.

Table 4.1 Average size of aggregates (Z) present in procyanidin fractions and PPA (1 µM) solutions (100 mM acetate buffer with 12% ethanol/water (v/v) pH 5.0) and polydispersity in absence and presence of carbohydrates, measured by dynamic light scattering. Fraction II Fraction III Fraction IV Carbohydrate Z (nm) polydispersity Z (nm) polydispersity Z (nm) polydispersity

none 646 ± 38 0.221 485 ± 49 0.121 465 ± 28 0.346 β-cyclodextrin (4.5 g.L-1) 646 ± 39 0.833 445 ± 26 0.279 416 ± 25 0.021 arabic gum (0.6 g.L-1) 123 ± 7 0.525 156 ± 2 0.246 107 ± 6 0.500 Pectin (80 mg.L-1): MD 27% 249 ± 15 0.495 177 ± 11 0.248 173 ± 10 0.477 MD 58-63% 289 ± 17 0.464 196 ± 12 0.287 164 ± 10 0.425 MD 72% 300 ± 18 0.707 178 ± 11 0.307 219 ± 13 0.477

Despite the decrease of procyanidin fraction concentration relative to

themolecular weight, the aggregates present in solution have approximately the

same size, particularly for procyanidin fractions III and IV (magnitude of 500 nm).

This could be explained by the fact that the concentrations of procyanidin

fractions used correspond to their maximum aggregation with PPA (stoichiometric

concentrations), for which the increase in tannin concentration does not change

aggregation (see Materials and Methods).

In general, the size of aggregates decreases in the presence of carbohydrates.

Exceptionally, the one obtained for fraction II in the presence of β-cyclodextrin did

not change. In that case, a large width of the particle size distribution was also

observed (high polydispersity), indicating a very heterogeneous population of

aggregates.

In the presence of pectin and especially of arabic gum there is a significant

decrease in the size of aggregates, much higher than for β-cyclodextrin. These

carbohydrates have induced a reorganization of the aggregates leading to the

formation of smaller structures that reduce the scattered light, which is in

agreement with the observed decrease of nephelometric measurements.

Therefore, as previously mentioned, the disrupting effect of polysaccharides

toward protein/tannin aggregation has been proposed to result from two

mechanisms (Fig.4.6): (i) the ability of polysaccharides to form a ternary complex

protein/polyphenol/polysaccharide, thereby enhancing its solubility in aqueous

medium, resulting in less insoluble aggregates; and (ii) the molecular association

in solution between polysaccharides and polyphenols competing with protein

aggregation.


Fig.4.6 Possible mechanisms (i and ii) involved in the inhibition of the aggregation of tannins and proteins by polysaccharides. P, protein; T, tannin; C, polysaccharide/carbohydrate.

From the results obtained herein, it can be proposed that the mechanisms i and

ii, by which polysaccharides restrain the protein/polyphenol aggregation, are

governed by the polysaccharide structure. The results obtained for arabic gum

and β-cyclodextrin strongly suggest that the main mechanism by which these two

compounds inhibit protein/polyphenol aggregation is mechanism ii (Fig.4.6).

Arabic gum and β-cyclodextrin interact with procyanidin fractions, with more or

less strength depending on procyanidin MW and also on the polysaccharide

structure, and inhibit procyanidin interaction with PPA. Effectively, the quenching

effect on α-amylase by procyanidins is decreased by the action of these

polysaccharides promoting an increase in the observed intrinsic fluorescence

intensity of α-amylase. Simultaneously, the aggregation decreases concomitantly

with the increase of polysaccharide concentration.

The interaction between polysaccharides and polyphenols may result from

cooperative hydrogen bonding, between the hydroxyl groups of polysaccharide

and phenolics, and from hydrophobic interactions. The higher effect of arabic

gum, compared to β-cyclodextrin, is probably due to its more acidic character,

resulting from the presence of glucuronic acids able to establish more hydrogen

bonds with procyanidins.

In the case of pectins, the results obtained provide substantial evidence for

demonstrating that the main mechanism by which these compounds inhibit

protein/polyphenol aggregation is mechanism i. Pectins seem to interact with

procyanidin fractions and with PPA to form a ternary complex

protein/polyphenol/polysaccharide, thereby enhancing its solubility in aqueous

medium, resulting in smaller andmore soluble aggregates. Effectively, opposite to

Stechoimetric

concentrations

polyphenol protein

+ Largest network of

protein-tannin

insoluble complexes

+

i)

ii

Protein/tannin/polysaccharide

ternary complex

Soluble complex of

tannin/polysaccharide

polysaccharide

Protein with a few

tannin molecules



177

arabic gum and β-cyclodextrin, the environment of the tryptophan residues of α-

amylase is not affected by the presence of pectins, so the intrinsic fluorescence

is not changed while solubilization occurs. This could mean that procyanidins still

interact with α-amylase even in the presence of pectins.

Using DLS, it was observed that carbohydrates induce a decrease in aggregates

size. The decrease in nephelometry intensity seems to be due to the formation of

smaller aggregates, which results from the disruption and reorganization of the

aggregates.

Overall, the combination of different analytical techniques – fluorescence

quenching, nephelometry, and DLS – allowed important insights into the two

mechanisms by which different polysaccharides are thought to inhibit

protein/tannin aggregation. This development may help further investigations

regarding the influence of carbohydrates in food taste.


PRP, proline-rich protein; PPA, porcine pancreas α-amylase; MD, methoxylation

degree; DLS, dynamic light scattering; FRET, fluorescence resonance energy

transfer.

� ACNOWLEDGMENT

We thank Prof. Graham Jones from the University of Adelaide (Australia) for

scientific advice and grammatical corrections.

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Biochimie 2003, 85, 109–121.

Capítulo 5

Carbohydrates Inhibit Salivary Proteins Precipitation by Condensed Tannins

Soares, S., Mateus, N., de Freitas, V.

J. Agric. Food Chem. 2012, 60, 3966-3972



185

Carbohydrates Inhibit Salivary Proteins Precipitati on by Condensed Tannins

Susana Soares, Nuno Mateus and Victor de Freitas*


Condensed tannins are a group of polyphenols that are associated with the

astringency sensation, as they readily interact and precipitate salivary proteins.

As this interaction is affected by carbohydrates, the aim of this work was to study

the effect of some carbohydrates used in the food industry [arabic gum (AG),

pectin, and poligalacturonic acid (PGA)] on the salivary proteins/grape seed

procyanidins interaction. This was assessed monitoring the salivary proteins that

remain soluble in the presence of condensed tannins with the addition of

carbohydrates (HPLC) and analysis of the respective precipitates (SDS-PAGE).

The results show that pectin was the most efficient in inhibiting protein/tannin

precipitation, followed by AG and PGA. The results suggest that pectin and PGA

exert their effect by formation of a ternary complex

protein/polyphenol/carbohydrate, while AG competes with proteins for tannin

binding (competition mechanism). The results also point out that both hydrophilic

and hydrophobic interactions are important for the carbohydrate effects.

KEYWORDS: proline-rich proteins, grape seed tannins, pectin, arabic gum, polygalacturonic acid

� INTRODUCTION

Condensed tannins are a complex group of polyphenolic polymers of catechin

(polyphenol compounds) that can be found in vegetal foodstuffs, particularly in

fruits, cereal grains, and beverages (red wine, tea, and beer). During foodstuff

consumption, these polyphenols interact with salivary proteins forming insoluble

aggregates that are supposed to be at the origin of the astringency sensation.1-4

Astringency has been defined as a dryness of the oral surface, puckering and

tightening sensations of the oral mucosa typically experienced during the

ingestion of tannin-rich food, in particular red wine.5 It is considered to be a

tactile, diffuse, and poorly localized sensation. In fact, this sensation is often the


last one to be detected as it can take 15 s or more for the perception to fully

develop.6

Although there are a few theories about the origin of the astringency sensation,

the most widely accepted one relies on the interaction between tannins and

salivary proteins. In general, these tannin.protein interactions are thought to

involve the cross-linking of separate protein molecules by the tannin, which acts

as a polydentate ligand on the protein surface involving hydrophobic and

hydrogen bonds.7-12

Salivary proteins include very structurally diverse proteins such as á-amylase,

albumin, lysozyme, proline-rich proteins (PRPs), histatins, cystatins, and

statherin. The main salivary proteins have been grouped into six structurally

related major classes, namely, histatins, basic proline-rich proteins (bPRPs),

acidic proline-rich proteins (aPRPs), glycosylated proline-rich proteins (gPRPs),

statherin, and cystatins.13,14 These proteins have important biological functions in

saliva associated with calcium binding to enamel, maintenance of ionic calcium

concentration (PRPs and statherin), antimicrobial action (histatins and cystatins),

or protection of oral tissues against degradation by proteolytic activity

(cystatins).15-19

Regarding tannin-protein interactions in vitro, these are reported to be affected by

several factors, in particular ionic strength, pH of the medium, percentage of

ethanol, temperature, and presence of carbohydrates.20-24

The first report of the inhibitory effect of carbohydrates in those interactions

concerned the proposed mechanisms for the astringency loss during fruit

ripening: as the cellular structure softens during fruit ripening, there is an

increase in watersoluble pectin fragments that could prevent the formation of

aggregates between fruit tannins and salivary proteins in the mouth, leading to a

modified astringency response.25-27 Other studies showed that the astringency of

tannins, as well as their interaction with proteins, is reduced by the addition of

carbohydrates.23,28,29

Two mechanisms have been proposed to explain this inhibitory effect of

carbohydrates: (I) carbohydrates form ternary complex protein-polyphenol-

carbohydrates, which enhance solubility in an aqueous medium; (II) there is a

molecular association in solution between carbohydrates and polyphenols hence

competing for protein aggregation.25,27

The effect of carbohydrates in protein-tannin interactions has a great impact in

the perception and choice of foodstuffs. Carbohydrates are frequently used in



187

food industry as food colloids (gums) and are also naturally present in several

food products, thereby affecting their astringent features.

Despite the knowledge about the effect of carbohydrates on protein-tannin

interactions, there is still little information when considering this effect on the

interaction of tannins with salivary proteins. In fact, there is a lack of information

in which way the structure of the different salivary proteins and the different

carbohydrates affect their action. Most of the literature on this subject has mainly

reported studies involving other proteins, namely, model proteins such as bovine

serum albumin (BSA) and α-amylase.

Therefore, this work aimed to study the effect of several carbohydrates used in

the food industry as food colloids [Arabic gum (AG), pectin, and polygalacturonic

acid (PGA)] on the interaction between salivary proteins and grape seed fraction

(GSF) (condensed tannins).


Reagents. All reagents used were of analytical grade. AG (purity not

provided) was purchased from Aldrich. Pectin (purity 79.5%) and PGA (purity

80%) (both from citrus peel) were purchased from Sigma.

GSF Isolation. Procyanidins were extracted from grape seeds (Vitis

vinifera) with an ethanol/water/chloroform solution (1:1:2, v/v/v). The resulting

solution was centrifuged, and the chloroform phase, containing chlorophylls,

lipids, and other undesirable compounds, was rejected. The hydroalcoholic

phase was then extracted with ethyl acetate, and the organic phase was

evaporated using a rotary evaporator (30°C). The resulting residue

corresponding essentially to oligomeric procyanidins was fractionated through a

TSK Toyopearl HW-40(s) gel column (100 mm × 10 mm i.d., with 0.8 mL.min−1

methanol as eluent), yielding two fractions according to the method described in

the literature.30 The first fraction was obtained after elution with 99.8% (v/v)

methanol during 5 h (240 mL), and the second was eluted with methanol/5% (v/v)

acetic acid during the next 14 h (670 mL). Both fractions were mixed with

deionized water, and the organic solvent was eliminated using a rotary

evaporator under reduced pressure at 30°C and then freeze-dried. The

procyanidin composition of fractions was determined by direct analysis by

electrospray ionization mass spectrometry (ESI-MS) (Finnigan DECA XP PLUS)

as described in the literature.31 The first fraction contains mainly catechins,


procyanidin dimers, and their galloyl derivatives, and the second fraction contains

essentially procyanidin dimers galloylated, procyanidin trimers and their galloyl

derivatives, and procyanidin tetramers. For the second fraction, the average full

mass spectra was obtained, and the mean degree of polymerization (mDP) was

estimated from the ratio between the sum of the relative abundance of each

compound multiplied by its number of elementary catechin units and the sum of

the relative abundances for all compounds in the fraction. The fraction has a

mean MW of 936 and a polymerization degree average of 3.2. Only the second

fraction named GSF was used herein, as it was shown to be the most reactive

toward proteins.

Saliva Collection. Saliva was collected from six healthy nonsmoking

volunteers, and 2 mL of saliva from each volunteer was used to make a saliva

pool (whole saliva). The collection time was standardized at 2 p.m. to reduce

concentration variability connected to circadian rhythms of secretion.32 The saliva

pool was mixed with 10% trifluoroacetic acid (TFA) (final concentration, 0.1%) to

precipitate several high molecular weight salivary proteins (such as α-amylase,

mucins, carbonic anhydrase, and lactoferrin) and to preserve sample protein

composition, since TFA partially inhibits intrinsic protease activity. After the

centrifugation (8000g for 5 min), the supernatant (acidic saliva, AS) was

separated from the precipitate and used for the following experiments.

Pectin Purification. Pectin was purified by precipitation with ethanol.

Briefly, after dissolution in a small amount of water, ethanol was added to

achieve a 70% (v/v) CH3CH2OH/H2O concentration. The precipitated

carbohydrate was recovered by filtration under vacuum (1 µm filters) and dried.

Pectin was analyzed by colorimetric methods and gas chromatography to

determine sugar composition and esterification degrees.33,34 It was found to be

composed by 85% galacturonic acid, 10% galactose, and 5% other sugars. The

degrees of methylation and acetylation were determined to be 14 and 1%,

respectively.

Protein and Tannin Interaction. The AS sample was analyzed by high-

performance liquid chromatography (HPLC) before and after the interaction with

increasing concentrations of GSF. These experiments were made to obtain the

minimal GSF concentration that precipitates almost totally the salivary proteins.

The control condition was a mixture of AS (150 µL) and water (50 µL) (final

volume, 200 µL). Different volumes of a GSF stock solution (30.0 mM) prepared



189

in water were added to AS (150 µL) to obtain the desired final concentrations,

150, 300, or 750 µM. The final volume was adjusted to 200 µL with pure water.

The mixture was shaken and kept for 5 min at room temperature (±20 °C) and

then centrifuged (8000g, 5 min). The supernatant was injected into the HPLC.

After these first experiments, the GSF concentration chosen for the experiments

with the carbohydrates was 300 µM.

Effect of Carbohydrates on Protein/Tannin Interacti on. For the

experiments with carbohydrates, stock solutions of AG (25.0 g.L–1), PGA (30.0

g.L–1), and pectin (10.0 g.L–1) were prepared in water, and different volumes of

these stock solutions were added to GSF solution to obtain different final

concentrations. The final volume (50 µL) was adjusted with water to obtain the

desired carbohydrates final concentration (between 2.4 and 30.0 g.L–1,

depending on carbohydrates). The mixture was shaken and kept at room

temperature (±20 °C) for 30 min. After that, 150 µL of AS was added to the

previous mixture (final volume, 200 µL), which was shaken and kept for 5 min at

room temperature (±20 °C). The mixture was then centrifuged (8000g, 5 min), the

supernatant was injected into the HPLC, and the precipitate was analyzed by

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH

of the supernatant was measured prior to analysis, and it was 3.1 [which is near

to some beverages pH such as red wine (pH 3.5)].

HPLC Analysis. Ninety microliters of each solution was injected on a

HPLC Lachrom system (L-7100) (Merck Hitachi) equipped with a Vydac C8

column (Grace Davison Discovery Sciences), with 5 µm particle diameter

(column dimensions 150 mm × 2.1 mm); detection was carried out at 214 nm,

using a UV−vis detector (L-7420). The HPLC solvents were 0.2% aqueous TFA

(eluent A) and 0.2% TFA in acetonitrile (ACN)/water 80/20 (v/v) (eluent B). The

gradient applied was linear from 10 to 40% (eluent B) in 60 min, at a flow rate of

0.30 mL.min–1. After this program, the column was washed with 100% eluent B

for 20 min to elute S type cystatins and other late eluting proteins. After washing,

the column was stabilized with the initial conditions.1,32

SDS-PAGE. The precipitates that resulted from the interaction between

AS and GSF in absence and presence of the highest carbohydrates

concentration, as well as the AS control solution, were analyzed by SDS-PAGE.

The precipitates were resolubilized in 200 µL of electrophoresis sample buffer (50

mM Tris-HCl, pH 6.8, 12% v/v glycerol, 4% SDS, 2.5% v/v β-mercaptoethanol,


and 0.01% bromophenol blue) to have the same initial volume used in the

previous experiments during precipitation. Eighty microliters of electrophoresis

sample buffer was added to 80 µL of these solutions. The control solution was

composed by 80 µL of the AS solution and 80 µL of a twice-concentrated

electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8, 24% v/v glycerol, 8%

SDS, 5% v/v β-mercaptoethanol, and 0.02% bromophenol blue). The samples

were heated at 60°C for 1 h with shaking and then analyzed by SDS-PAGE in a

tris-tricine buffer system according to the method of Schägger using 16%

acrylamide resolving gel. The stacking gel was 5% acrylamide. The cathode

buffer was 0.1 M Tris, 0.1 M tricine, and 0.1% SDS. The anode buffer was 0.2 M

Tris-HCl, pH 8.9. Electrophoresis was performed on a Bio-Rad MiniProtean Cell

electrophoresis apparatus (Bio-Rad) at constant voltage (150 V). After

electrophoresis, the gels were stained with Imperial Protein Stain (Thermo

Scientific), a Coomassie R-250 dye-based reagent. The staining with Imperial

Protein Stain was done according to the supplier's instructions. The destaining

step was done by washing the gels with water until the bands were visible.

Molecular weights (Sigma) were estimated by comparison with the migration

rates of standard proteins (β-galactosidase from Escherichia coli, 116000 Da;

phosphorylase b from rabbit muscle, 97000 Da; albumin bovine serum, 66000

Da; glutamic dehydrogenase from bovine liver, 55000 Da; ovalbumin from

chicken egg, 45000 Da; glyceraldehyde-3-phosphate dehydrogenase from rabbit

muscle, 36000 Da; carbonic anhydrase from bovine erythrocytes, 29000 Da;

trypsinogen from bovine pancreas, 24000 Da; trypsin inhibitor from soybean,

20000 Da; á-lactalbumin from bovine milk, 14200 Da; and aprotinin from bovine

lung, 6500 Da).

Half Maximal Effective Concentration (EC 50) Calculation and

Statistical Analysis. EC50 values were calculated considering the area variation

of chromatogram peaks of each protein in the presence of carbohydrates. The

software GraphPad Prism 5 (GraphPad Software, Inc.) was used to calculate

EC50 and statistical analysis. Statistical significance of the difference between the

several calculated EC50 was evaluated by one-way analysis of variance, followed

by the Bonferroni test. Differences were considered to be statistically significant

when P<0.05. All of the experiments were performed in n = 3 repetitions.



191

� RESULTS

The carbohydrates AG, pectin, and PGA are commonly present in food and are

also used in the food industry as additives. The influence of these three different

ionic carbohydrates on the interaction between salivary proteins and grape seed

condensed tannins was assessed by HPLC analysis and SDS-PAGE. The

disrupting effect of these compounds on protein−tannin aggregates was

assessed by monitoring the increase of the salivary proteins that remain soluble

in the presence of condensed tannins with the addition of each of these

carbohydrates.

Salivary Proteins. The initial acidic treatment of human saliva with TFA is

used to precipitate several high molecular weight salivary proteins (such as α-

amylases, mucins, carbonic anhydrase, and lactoferrin) and to preserve sample

protein composition, since TFA partially inhibits intrinsic protease activity.32

However, peptides and proteins like histatins, basic, acidic, and glycosylated

PRPs, statherin, cystatins, and defensins are soluble in AS solution and may be

directly analyzed by RP-HPLC, as previously described.1,32

The HPLC chromatogram of this AS solution at 214 nm is presented in Fig.5.1.

The top of the figure shows the distribution of the different families of salivary

proteins along the chromatogram that were established previously by proteomic

approaches, namely, ESI-MS and matrix-assisted laser desorption/ionization

time-of-flight/time-of-flight (MALDI-TOF/TOF).1,32

Fig.5.1Typical RP-HPLC profile detected at 214 nm of the AS solution of human saliva in the absence (— ) and presence (- - -) of 300 µM GSF. The vertical dotted lines show the ranges and the main salivary proteins family assigned to each HPLC peptide region: bPRPs, gPRPs, and aPRPs.

The HPLC chromatogram of the AS solution is roughly divided into four salivary

proteins family regions: the first region comprises proteins that belong to the

15 20 25 30 35 40 45 50 55 60 650

2

4

6


t (min)

Abs

(A

u)


classes of bPRPs and histatins. The bPRPs identified in this region include IB-8b,

IB-8c, IB-9, IB-4, and P-J, and the histatins include histatins 3, 5, 7, 8, and 9. The

second region comprises mainly one gPRPs, the bPRP3. The next region

corresponds entirely to aPRPs, namely, PRP1 and PRP3, and the last region has

phosphorylated and nonphosphorylated forms of statherin and peptide PB.1

Interaction of Salivary Proteins with Procyanidin Fraction from

Grape Seeds (GSF). The first experiments were made to assess the minimal

procyanidin concentration that leads to a significant precipitation of salivary

proteins and then to study if the carbohydrates were able to inhibit that

precipitation. It was important to establish this concentration since an insufficient

quantity of procyanidins would lead to no significant precipitation of most of the

salivary proteins, and an excess of GSF would lead to free molecules of GSF,

thereby influencing the further effect of carbohydrates. Bearing this, three

increasing GSF concentrations were tested (150, 300, and 750 µM).

From these experiments, the GSF concentration chosen for the experiments with

carbohydrates was 300 µM, since it corresponded to the GSF concentration that

precipitated almost all of the salivary proteins present in the AS (Fig.5.1). In fact,

while for 150 µM GSF only aPRPs and statherin were significantly affected, the

750 µM concentration led to the precipitation of all of the proteins but also to an

excess of free molecules of GSF (data not shown).

It can be seen from Fig.5.1 that the addition of 300 µM GSF reduced significantly

the amount of gPRPs and practically depleted aPRPs and statherin. On the other

hand, bPRPs were not significantly affected. Therefore, the effect of

carbohydrates on the interaction between salivary proteins and tannins was

focused on gPRPs, aPRPs, and statherin proteins.

Effect of Carbohydrates on the Interaction between Salivary Proteins

and GSF. The experimental approach described herein intends to mimic the

phenomenon that occurs in the mouth during food mastication, where tannins

and carbohydrates simultaneously contact with salivary proteins. So, a solution of

tannins−carbohydrates was prepared, to which the AS solution was subsequently

added. The control experimental condition was made only with AS and 300 µM of

GSF. Fig.5.2 shows part of the chromatogram corresponding only to the families

of salivary proteins that were effectively affected by the GSF in the absence or

presence of the different carbohydrates.



193

Fig.5.2 Part of the chromatograms of the AS solution after the interaction with GSF (300 µM) in the absence (AS solution + 300 µM GSF) and presence of the several tested carbohydrates (pectin, 5.0 g.L–1; AG, 10.0 g.L–1; and PGA, 20.0 g.L–1).

From the presented results, it can be observed that the carbohydrates in solution

enhance the chromatographic peaks corresponding to salivary proteins, in

especially for aPRPs and statherin. Fig.5.3 shows variation of the

chromatographic peaks area of the several salivary proteins studied with the

increase in the carbohydrate concentration, expressed in percentage of the area

of these proteins relatively to the respective area in control saliva (AS without

tannin).

A B

C

Fig.5.3 Influence of carbohydrate concentration on salivary proteins precipitation by condensed tannins (GSF, 300 µM). (A) AG, (B) PGA, and (C) pectin. These results represent the average of three independent experiments.

30 35 40 45 50 55 60 65 700

2

4

6

8

10

t (min)

Abs

(A

u)AS + GSF

AS + GSF + Pectin

AS + GSF + PGA

AS + GSF + AG

gPRPs aPRPs statherin

0.0 5.0 10.0 15.0 20.0 25.00

20

40

60

80

100

AG (g.L -1)

Are

a (

%)

of c

hrom

ato

gra

phic

pea

ks o

f sa

liva

ry p

rote

ins

0.0 10.0 20.0 30.00

20

40

60

80

100

PGA (g.L -1)

Are

a (

%)

of c

hro

ma

togr

aph

icp

ea

ks o

f sa

liva

ry p

rote

ins

0.0 2.0 4.0 6.0 8.0 10.00

20

40

60

80

100

Pectin (g.L -1)

Are

a (

%)

of c

hro

ma

togr

aph

icp

ea

ks o

f sa

liva

ry p

rote

ins

gPRP

aPRP

statherin


In general, it is possible to observe that salivary proteins in solution increase

concomitantly with the carbohydrates concentration. The observed changes may

be interpreted as the inhibition of salivary proteins precipitation by tannins in the

presence of increasing concentrations of carbohydrates.

Pectin was by far the most effective carbohydrate in preventing precipitation of

salivary proteins by condensed tannins. While pectin prevents almost total

precipitation of salivary proteins at the concentration of 10.0 g.L−1, AG and PGA

only showed similar effects for concentrations higher than 20.0 g.L−1.

Besides the HPLC analysis of the supernatant, it was also important to analyze

the pellets that resulted from the AS interaction with GSF in the absence and

presence of the different carbohydrates. So, to analyze these pellets, all

precipitates were resolubilized by heating (60°C) in 200 µL of electrophoresis

sample buffer and further analysis by SDS-PAGE. The proteins bands were

subsequently analyzed by densitometry, and the results are presented in Fig.5.4.

Fig.5.4 SDS-PAGE of the pellets that resulted from the interaction between AS and GSF in the absence (C) and presence of the several carbohydrates (pectin, 10.0 g.L−1; AG, 25.0 g.L−1; and PGA, 25.0 g.L−1). The molecular weight markers were substituted by lines, and the molecular mass marked on the left side is expressed in kDa. The gels were stained with Imperial Protein Stain, a Coomassie R-250 dye-based reagent. The table shows the ratio between the densitometry values obtained for total salivary proteins present in AS (control AS) and in each experiment. The values with equal letters are not significantly different (P < 0.05).

The results obtained by SDS-PAGE analysis of the referred pellets, as well as by

the densitometry analysis performed to the gel, clearly demonstrate that the

presence of these carbohydrates, in particular pectin and AG, decreases the

salivary proteins that are precipitated by tannins (Fig.5.4). The maximum of

salivary proteins precipitation results from the interaction between AS and GSF in

the absence of AG and pectin (lane C). The experiment with AS and GSF

resulted in a value of 0.46, while in the presence of pectin or AG, the obtained

value is significantly lower (0.16 and 0.11,respectively). Regarding the PGA

effect, looking at the SDS-PAGE results, it is also possible to observe that it also

Ratio Control AS 1.00 ± 0.07

C (AS + GSF) 0.46 ± 0.00a Pectin 0.16 ± 0.02b

AG 0.11 ± 0.02b PGA 0.43 ± 0.08a



195

leads to a decrease of salivary proteins precipitation but in a fewer extent than

the other carbohydrates. SDS-PAGE analyses are probably not comparable with

HPLC results because there are other proteins (cystatins and other late eluting

proteins) not analyzed by HPLC that could be precipitated by tannins and so

appear in the SDS-PAGE gel. In this way, SDS-PAGE results only reflect the

efficiency of the carbohydrates in inhibiting tannin−protein interaction.

To compare properly the effectiveness of carbohydrates to inhibit salivary

proteins precipitation by tannins, the half maximal effective concentration (EC50)

was calculated from the HPLC results (Table 5.1).

Table 5.1 Half maximal effective concentration (EC50) in inhibition of tannin/salivary protein precipitation by three carbohydrates: AG, PGA and pectin. The values with equal letters are not significantly different (P<0.05).

EC50 (g.L -1) Carbohydrate gPRP aPRP statherin

AG 4.78 ± 0.39a 10.10 ± 0.16 5.68 ± 0.65a PGA 19.47 ± 0.50 15.12 ± 0.58 f 14.34 ± 1.61f

Pectin 4.75 ± 0.38d 4.83 ± 0.10d 4.77 ± 0.18d

Pectin has been shown to have the lowest EC50 value (around 4−5 g.L−1) being

the most effective in the inhibition of salivary proteins precipitation by tannins,

while PGA was shown to be the less effective carbohydrate in inhibiting

tannin−salivary proteins precipitation with an EC50 between 14 and 19 g.L−1.

Oppositely to AG, the effect of the other carbohydrates is quite similar for the

three groups of salivary proteins, especially for pectin, which indicates that the

influence at this latter is independent of the salivary protein structure. For AG, its

effect is more pronounced for gPRPs and statherin rather than for aPRPs. In fact,

the EC50 obtained for AG and aPRP is nearly twice of the one obtained for the

other salivary proteins. These results seem to suggest that the mechanism by

which AG exerts its effect is probably different and more selective toward salivary

protein structure as compared to pectin and PGA.

It had already been shown that AG inhibits the interaction between a protein (α-

amylase) and condensed tannins by a different mechanism than pectin.23 While

pectin was described to have the ability to form a ternary complex protein-

polyphenol-carbohydrate (Fig.5.5, i), thereby enhancing its solubility in aqueous

medium, resulting in less insoluble aggregates, AG has been described to have

the ability to inhibit protein−tannin interaction by competing with tannins by

protein aggregation (Fig.5.5, ii). Although this previous work was performed with

a different protein (α-amylase),23 a similar behavior could explain the high


efficiency and lack of selectivity observed herein for pectin and PGA. In the

present case, as tannins complex with both proteins and carbohydrates (pectin or

PGA), there is no competition mechanism and thus less selectivity.

Fig.5.5 Possible mechanism (i and ii) involved in the inhibition of the aggregation of tannins and proteins by carbohydrates. P, protein; T, tannin; and C, carbohydrate.40

PGA and pectin are quite similar polymers of α-(1→4)-linked D-galacturonic acid

units that differ essentially on the degree of methoxylation of their carboxyl

groups.35 This structural similarity probably explains the identical inhibition

mechanism described, leading to the formation of ternary complexes. However,

pectin is more esterified than PGA, which reduces its polarity and consequently

its readiness to hydrophilic interactions. On the other hand, esterification favors

its ability to establish van der Waals interactions. Thus, pectin would be more

prone to hydrophobic interactions than PGA, which favors the interaction with

condensed tannins (that tend to be more hydrophobic molecules). This particular

feature seems to be important to increase the ability of pectin to inhibit salivary

protein−tannin precipitation despite its low selectivity.

Regarding AG, its structure is quite different from the structures of pectin and

PGA, which could explain the different mechanism of inhibition of this

carbohydrate. A competition mechanism is once again proposed herein for AG

(Fig.5.5, ii). The effectiveness of this process is expected to depend on the

relative affinities of tannin toward AG and salivary proteins. If tannins have a

higher affinity to AG than to salivary proteins, AG would be able to prevent

salivary proteins-tannins precipitation. AG is a heteropolysaccharide composed

of a polysaccharide and hydroxyproline-rich protein moieties able to ensure

hydrophobic interactions with procyanidins. The polysaccharide moiety has also

a slightly acidic character, resulting from the presence of 20% glucuronic acids.

The acidic character may help to establish electrostatic and hydrogen bonds that



197

could help to strengthen the interaction with condensed tannins. On the other

hand, the presence of the protein-proline moiety could also contribute to an

increase in the efficiency of AG to complex strongly with tannins, competing for

the interaction with salivary proteins. This is readily apparent from the obtained

results as AG was found to be the second more efficient carbohydrate in a range

of concentrations generally much lower than the ones for PGA.

Altogether, these results seem to point out that both hydrophilic and hydrophobic

interactions are important for the carbohydrate effects where the hydrophilic

interactions (hydrogen bonds) seem to contribute to the selectivity of the

inhibition, while the hydrophobic ones seem to contribute to the efficiency. This

was especially clear for pectin that was shown to be the most efficient in

inhibiting salivary proteins precipitation by condensed tannins toward the aPRP,

gPRP, and statherin, despite its low selectivity.

Besides the central role of the carbohydrate structure, these results also point to

the importance of the protein structure for the effect of those carbohydrates.

PRPs are classified in different classes (acidic, basic, and glycosylated) based on

small and specific structural differences, in particular richness in acidic or basic

residues and the presence of sugar molecules in their structure.36-38 The acidic

character of aPRPs is confined roughly to the first 30 amino acids, due to the

presence of many aspartic and glutamic acid residues. The remaining part is

basic and, similarly to bPRPs, shows repeated sequences of proline and

glutamine often separated by glycine residues. Regarding the gPRP, there is little

information about the glycosylation pattern of these proteins. Statherin is

abundant in tyrosine residues and is phosphorylated at Ser-2 and Ser-3. This

latter protein also has a highly acidic amino-terminal hexapeptide (first six

residues) that is needed to inhibit calcium phosphate crystal growth. These

structural aspects of salivary proteins are important for their competition with

carbohydrates toward tannins in the competition mechanism previously referred.

In fact, the efficiency of the carbohydrate to inhibit protein-tannin aggregation

depends not only on the carbohydrate structure but also on the affinity between

the salivary proteins and the tannins. This is evident for AG that has been shown

to be less efficient (high EC50) in inhibiting aPRPs-tannin aggregation as

compared to other salivary proteins. In this case, AG has been shown to be more

effective in inhibiting gPRPs-tannins and statherin-tannins precipitation (EC50 =

4.78 and 5.68 g.L–1, respectively) than aPRPs-tannin precipitation (EC50 = 10.10

g.L–1). In fact, aPRPs have been described recently to interact more strongly with


condensed tannins than the other salivary protein families.1 On the other hand,

gPRPs are known to not interact strongly with tannins as compared to other

salivary proteins because of the presence of the sugar moiety in their structure,39

which probably explains the higher inefficacy of AG to inhibit gPRP-tannin

precipitation.

Overall, the obtained results show that carbohydrates commonly used in the food

industry are able to inhibit the interaction and precipitation of salivary proteins

with tannins and thus influence the perceived astringency of some food products.

The extent and the mechanism by which this inhibition occurs are related with the

carbohydrate structure and in the last instance also to the protein structure.



*Tel: +351 220402558. Fax: +351 220402659. E-mail: [email protected].

Funding

This work was supported by Fundação para a Ciência e Tecnologia by one Ph.D.

grant (SFRH/BD/41946/2007) and one project grant (PTDC/AGR-

ALI/67579/2006).

Notes

The authors declare no competing financial interest.


ACN, acetonitrile; aPRPs, acidic proline-rich proteins; AS, acidic saliva; AG,

arabic gum; bPRPs, basic proline-rich proteins; ESI-MS, electrospray ionization

mass spectrometry; gPRPs, glycosylated proline-rich proteins; GSF, grape seed

fraction; HPLC, high-performance liquid chromatography; MALDI-TOF/TOF,

matrix-assisted laser desorption/ionization time-of-flight/time-of-flight; PGA,

polygalacturonic acid; PRPs, proline-rich proteins; SDS-PAGE, sodium dodecyl

sulfate−polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid

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Capítulo 6

Different Phenolic Compounds Activate Distinct Subsets of Human Bitter Taste Receptors

Soares, S., Kohl, S., Thalmann, S., Mateus, N., Meyerhof, W., de Freitas, V.

Submetido ao J. Agric. Food Chem.



205

Different Phenolic Compounds Activate Distinct Subs ets of Human Bitter Taste Receptors

Susana Soares, † Susann Khol, ‡ Sophie Thalmann, ‡ Nuno Mateus, † Wolfgang

Meyerhof‡,* and Victor de Freitas†,*

†Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

‡ DIFE, German Institute of Human Nutrition, Potsdam-Rehbrücke, 14558 Nuthetal, Germany

Bitterness is a major sensory attribute of several common foods and beverages

rich in polyphenol compounds. These compounds are reported as very important

for health as chemopreventive compounds but they are also known to taste bitter.

So, in this work, we analyzed the activation of the human bitter taste receptors,

TAS2Rs, by six polyphenol compounds. The compounds chosen are present in a

wide range of plant-derived foods and beverages, namely red wine, beer, tea and

chocolate. Pentagalloylglucose (PGG) is a hydrolysable tannin, (-)-epicatechin is

a precursor of condensed tannins, procyanidin dimer B3 and trimer C2 belong to

the condensed tannins, and malvidin-3-glucoside and cyanidin-3-glucoside are

anthocyanins. The results show that the different compounds activate different

subsets of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors, TAS2R4,

TAS2R5 and TAS2R39 whereas only two receptors, TAS2R5 and TAS2R39,

responded to PGG. In contrast, malvidin-3-glucoside and procyanidin trimer

stimulated only one receptor, TAS2R7 and TAS2R5, respectively. Notably,

tannins are the first natural agonists found for TAS2R5 that display high potency

only toward this receptor. The catechol and/or galloyl groups appear to be

important structural determinants that mediate the interaction of these

polyphenolic compounds with TAS2R5. Overall, the EC50 values obtained for the

different compounds vary 100-fold with the lowest values for PGG and malvidin-

3-glucoside compounds suggesting that they could be significant polyphenols

responsible for the bitterness of fruits, vegetables and derived products even if

they are present in very low concentrations.

KEYWORDS: polyphenols, bitterness, tannins

� INTRODUCTION

It has been well-known and common sense that consumption of diets rich in

fruits, vegetables and moderate intake of red wine (i.e., polyphenol-rich foods)


are inversely correlated to cardiovascular diseases incidence and cancer risk,

probably because the polyphenols modulate several biological reactions that lead

to these disease conditions (1). In fact, during the past years polyphenol

compounds have been suggested as chemoprevention agents.

Polyphenol compounds result from plant secondary metabolism as chemical

defense against predators and are usually divided in non-flavonoids and

flavonoids, the latter being the most relevant ones. Flavonoids include very

diverse compounds such as anthocyanins and flavan-3-ols. Although they are

potentially beneficial to human health in small doses, many of these compounds

are toxic in high doses (2). Several of these compounds are responsible for major

organoleptic properties of vegetables and fruits, namely color and taste. In fact,

some of them are known to have a bitter taste and so, despite their healthy

properties, many people do not like to eat vegetables and other plant-derived

food because of the bitterness associated with polyphenols (3). Debittering of

food has been a longstanding major sensory challenge for the food industry (3),

that requires identification of those compounds making food and beverages taste

bitter. Therefore, studying the sensorial properties of polyphenol compounds is a

prominent subject relevant for people’s food choices, and the chemopreventive

potential of food.

Although bitterness in foods is usually unpleasant, there are some foodstuffs in

which it is a wanted sensory attribute, e.g. red wine and beer. In fact, most of the

sensory analysis data regarding polyphenols bitterness are related to red wine. It

is thought that the bitterness of red wine is mainly induced by polyphenol

compounds (e.g. tannins) (3-5). However, the limited available data regarding

polyphenol’s structure/bitterness relationship are rather inconsistent (5-11). In

general, these works studied the bitterness of polyphenol compounds, such as

polymeric fractions of tannic acid and tannins, as well as flavan-3-ol monomers,

dimers, and trimers, and demonstrated that larger molecules tend to be less bitter

and more astringent (8, 10). Peleg and co-workers (8) found that (-)-epicatechin

was more bitter than the stereoisomer (+)-catechin and that these both were

more bitter than the procyanidin trimers, catechin-(4-8)-catechin-(4-8)-catechin

and catechin-(4-8)-catechin-(4-8)-epicatechin. Robichaud and colleagues (10)

found that tannic acid, a commercial hydrolysable pentagalloylglucose-rich tannin

was more bitter than both, (+)-catechin and a grape seed extract which is rich in

polymeric procyanidins. However, other reports have shown that bitterness of

polyphenols increases with molecular weight (5, 9, 12). Hufnagel and Hofmann



207

(5) found that procyanidin dimers and a trimer were more bitter than (-)-

epicatechin. These authors also demonstrated by taste reconstruction and

omission experiments, that the bitterness of the red wine could be induced by

subthreshold concentrations of phenolic acid ethyl esters and flavan-3-ols.

Besides these inconsistencies, there is insufficient knowledge about the bitter

taste of polyphenol compounds that do not belong to tannin classes, such as

anthocyanins.

Bitter taste is elicited by a specific subset of taste receptor cells (TRC) localized

in the oral cavity in groups of cells called taste buds, which are embedded in the

epithelium of the gustatory papillae on the tongue and palate. These TRC are

characterized by the expression of members of the TASTE 2 Receptor (TAS2R)

gene family encoding bitter taste receptors (13-17). In humans, this gene family

codes for ~25 taste receptors (TAS2Rs) that are G protein-coupled receptors. So

far, 20 of them have been deorphaned meaning that cognate bitter compounds

have been assigned to them (14, 18-30). In general, TAS2Rs are sensitive to

several or multiple different bitter compounds, a property that has been proposed

to be the basis for recognition of the countless bitter chemicals (19, 31, 32). Due

to the presence of numerous non-synonymous single nucleotide polymorphisms

(SNPs) the TAS2R genes encode functionally distinct receptor variants that form

the basis for variations in bitterness perception in the population (23, 25, 33-35).

In order to understand better structure/bitterness relation, to overcome the

inconsistencies associated with sensorial analysis, and to identify which TAS2Rs

are activated by the various polyphenol compounds an objective, robust, and

reliable method to analyze their bitterness is required. Few recent studies used

heterologous expression experiments to successfully measure TAS2R activation

by purified bitter chemicals that are normally present in food and beverage

sources such as beer hops, cheese, and soy products (18, 26, 29, 30, 36, 37). In

the present report we use this method to examine the activation of the TAS2Rs

by several polyphenol compounds widely spread in food and beverages derived

from plants and commonly present in our daily diet (Fig.6.1).


β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose Procyanidin trimer C2

(-)-Epicatechin Malvidin-3-glucoside

Fig.6.1 Chemical structures of the four polyphenol compounds studied.

Based on their extensive prevalence in vegetable, fruits and derived products, we

chose the following six substances that belong to some of the most important

classes of polyphenols (anthocyanins, flavan-3-ols and hydrolysable tannins)

(38): (-)-epicatechin, procyanidin dimer B3 and trimer C2 (flavan-3-ols), malvidin-

3-glucoside and cyanidin-3-glucoside (anthocyanins) and pentagalloylglucose

(PGG) (hydrolysable tannins). (-)-Epicatechin (structural unit of condensed

tannins) and a number of different procyanidin dimers and trimers belong to the

condensed tannins class and are present in a variety of frequently ingested food

including red wine, cocoa, grape seeds, tea, beer and cereals (38, 39). Malvidin-

3-glucoside and cyanidin-3-glucoside belong to the anthocyanin class, being

highly present in red fruits and their derived products, red wine and olives (38,

40). Although PGG is not commonly found in a wide range of foodstuffs, it

belongs to the other class of tannins (hydrolysable tannins) and is present in

pomegranate, green tea and it can also be incorporated in red wine during the

winemaking process (by addition of commercial tannic acid) (41).

CO

O

O CO

OH

OH

OHO

CO

OH

OHOH

O

CO

OH

OH

OH

O

O

CO

OH OH

OH

OH

OH

OH

Galloyl group

O

OH

OH

OH

OH

R1H

R2

O

OH

OH

OH

OH

HH

OH

O

OH

OH

OH

OH

HH

OH

OOH

OH

OH

OH

H

H

OH

Orto-catecholgroup

O

OH

OH

OH

O

OMe

OMe

Glu

+



209

Our aim was to elucidate if the selected polyphenol compounds are activators of

human bitter taste receptors and to examine if different polyphenols activate

different TAS2Rs.


Reagents. All reagents used were of analytical grade. (+)-catechin and (-

)-epicatechin were purchased from Sigma-Aldrich, malvidin aglycon was

purchased from Extrasynthèse, sodium borohydride and tartaric acid were

purchased from Aldrich, tannic acid was purchased from Fluka Biochemica

(Switzerland), taxifolin was purchased from Extrasynthèse (Genay, France) and

Toyopearl HW-40(s) gel was purchased from Tosoh (Tokyo, Japan).

Malvidin-3-glucoside and cyanidin-3-glucoside (anth ocyanins)

isolation. Malvidin-3-glucoside and cyanidin-3-glucoside were isolated as

described in the literature (42). Briefly, these compounds were isolated from a

grape skin extract that was applied to a TSK Toyopearl HW-40(s) gel column.

The elution was made with 10% CH3OH/CH3COOH (v/v) and then the solvent

was evaporated. The resulting residue was applied to a C18 gel column and the

compounds were eluted with 10% methanol acidulated. The solution was again

evaporated and the resulting solution was mixed with distilled water, frozen and

lyophilized. The compounds purity was assessed by HPLC-MS, direct MS and

NMR analysis and was higher than 99%.

Procyanidin dimer (B3) and trimer (C2) (condensed t annins)

synthesis. The synthesis of procyanidin dimer B3 (catechin-(4-8)-catechin) and

trimer C2 (catechin-(4-8)-catechin-(4-8)-catechin) followed the procedure

described in the literature (43). Briefly, taxifolin and (+)-catechin (ratio 1:3) were

dissolved in ethanol and the mixture was treated with sodium borohydride (in

ethanol). The pH was then lowered to 4.5 by addition of CH3COOH/H2O 50%

(v/v), and the mixture stood under argon atmosphere for 30 min. The reaction

mixture was extracted with ethyl acetate. After evaporation of the solvent, water

was added, and the mixture was passed through C18 gel, washed with water,

and recovered with methanol. After evaporation of methanol, the fraction was

passed through a TSK Toyopearl HW-40(s) gel column (300 mm × 10 mm i.d.,

0.8 mL.min-1, methanol as eluent) coupled to a UV-vis detector. Several fractions

were recovered and analyzed by ESI-MS (Finnigan DECA XP PLUS) yielding


procyanidins dimers (B3 and B6) and trimer (C2). The structure was elucidated

by HPLC-MS and NMR analysis.

β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose (PGG) (h ydrolysable

tannin) synthesis. PGG was synthesized accordingly to Chen and Hagerman

(44). Briefly, 5.0 g of tannic acid was methanolyzed in 70% methanol in acetate

buffer (0.1 M, pH 5.0) at 65 °C for 15 h. The pH of the reaction mixture was

immediately adjusted to 6.0 with NaOH. Methanol was evaporated under reduced

pressure at <30 °C and water was added to maintain the volume. The solution

was extracted with 3 volumes of diethyl ether and 3 volumes of ethyl acetate. The

ethyl acetate extracts were combined and evaporated, with addition of water to

maintain the volume. The resulting suspension was centrifuged, and the

precipitate was redissolved by heating in 2% methanol solution. PGG precipitated

as the solution cooled to room temperature and was collected by centrifugation.

PGG was washed twice with an ice-cold 2% methanol solution and once with ice-

cold distilled water. The final material was lyophilized to yield a white powder with

an overall mass yield of 23%. The purity of the obtained PGG was assessed by

HPLC analysis (45) and 1H NMR spectroscopy and it was ≤ 99%.

Test compounds preparation. The compounds to be tested were either

dissolved in buffer C1 (130.0 mM NaCl, 5.0 mM KCl, 10.0 mM N-2-

hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 2.0 mM CaCl2, 10.0 mM

glucose [pH 7.4]) or in a mixture of dimethylsulfoxide (DMSO) and buffer C1 not

exceeding a final DMSO concentration of 0.1% (v/v) to avoid toxic effects on the

transfected cells.

Cell transfection and expression of TAS2Rs in heter ologous cells.

Functional expression studies were carried out as described before (18, 30).

Human embryonic kidney (HEK)-293T cells stably expressing the chimeric G

protein subunit Gα16gust44 were seeded into poly-D-lysine coated (10 µg.mL-1)

96-well plates (Greiner Bio-One, Frickenhausen, Germany) under regular cell

culture conditions [Dulbecco’s modified Eagle medium (DMEM)), 10% FCS, 1%

penicillin/streptomycin; 37ºC, 5% CO2, 95% humidity]. After 24-26 h, cells were

transfected transiently with 150 ng expression plasmids based on pEAK10 (Edge

BioSystems) or pcDNA5/FRT (Invitrogen) using 300 ng of Lipofectamine2000

(Invitrogen). In addition to the TAS2R coding sequences the plasmids contained

the first 45 amino acids of rat somatostatin receptor 3 for cell surface localization

followed by the herpes simplex virus (HSV) glycoprotein D epitope for



211

immunocytochemical detection of the receptor. The TAS2R sequences are

according to Meyerhof et al. (18).

Calcium imaging analysis. Twenty-four to 26 h after transfection, cells

were loaded with the calcium-sensitive dye Fluo-4-acetoxymethylester (2 µM,

Molecular Probes) in serum-free DMEM medium. Probenecid (Sigma-Aldrich

GmbH), an inhibitor of organic anion transport, was added at a concentration of

2.5 mM, to minimize the loss of the calcium indicator dye from cells. One hour

after loading, the wells were washed three times with C1 buffer using a Denley

Cell Washer (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were incubated

in washing buffer in the dark for 30 min between the washing steps.

Fluorescence changes were recorded at 510 nm following excitation at 488 nm

by a fluorometric imaging plate reader (FLIPR, Molecular Devices) before and

after application of the test compounds in each well. A second application of 100

nM somatostatin-14 (Bachem) activating the endogenous somatostatin receptor

type 2 was used to assess cell vitality. All experiments were performed at least in

duplicates. Mock-transfected cells (cells transfected with empty pcDNA5/FRT

vector used as negative control) were always measured in parallel on the same

microtiter plates using the same compound concentrations used to examine the

cells expressing the various TAS2Rs. All compounds were initially tested at

different concentrations for unspecific calcium responses in untransfected

HEK293T Gα16gust44 cells. Based on this pilot experiment, we used maximal

compound concentrations that were always lower than those concentrations that

generated unspecific responses in the absence of transfected receptor DNAs.

Determination of half maximal effective concentrati ons (EC 50) and

statistical analysis. Having identified responsive TAS2Rs, we examined their

concentration-dependent activation and established half maximal effective

concentrations (EC50) values for their bitter agonists. To calculate the

concentration-response curves, the fluorescence changes of mock-transfected

cells were subtracted from the corresponding values of receptor-expressing cells

by means of the FLIPR384 software (Molecular Devices, Munich, Germany). To

compensate for differences in cell density, signals were normalized to

background fluorescence for each well. Signals were recorded in at least

duplicate wells and the data averaged. Signal amplitudes were then plotted

versus log agonist concentration. EC50 values were calculated using SigmaPlot

9.01 (Systat Software Gmbh, Erkrath, Germany) by nonlinear regression using


the function (equation 1) where x is the test compound concentration and nH

the Hill coefficient. Statistical significance of the difference between the several

calculated EC50 and of the signal amplitudes was evaluated by one-way analysis

of variance, followed by the Bonferroni test. Differences were considered to be

statistically significant when P < 0.05.

� RESULTS

Identification of TAS2Rs that respond to polypheno l compounds. In

order to identify the TAS2Rs that are sensitive to the selected prototypical

polyphenols, the 25 TAS2Rs, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7,

TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R38,


TAS2R46, TAS2R30, TAS2R19, TAS2R20, TAS2R50 and TAS2R60 were

expressed individually in HEK293T cells stably expressing the chimeric G protein

Gα16gust44. This construct was designed to couple the activation of TAS2Rs in

heterologous cells to the release of Ca2+ from intracellular stores, which can be

measured using calcium-sensitive fluorescence dyes (46). Receptor-mediated

changes in intracellular calcium concentrations were detected as fluorescence

changes by means of a fluorometric imaging plate reader.

The six polyphenol compounds to be tested (-)-epicatechin, procyanidin dimer

and trimer, malvidin-3-glucoside, cyanidin-3-glucoside and PGG were

administered, at the highest possible concentrations (pre-determined in pilot

experiments, data not shown), by bath application to the 25 different receptor-

expressing cell populations. (-)-Epicatechin was employed up to 16.0 mM

whereas all the other compounds were employed only at µM range [procyanidin

trimer, 100.0 µM; malvidin-3-glucoside, 20.0 µM; PGG, 10.0 µM (Fig.6.2)]. By

tracing cytosolic calcium levels we found that cells expressing TAS2R4, TAS2R5

or TAS2R39 responded to challenge with (-)-epicatechin. Cells transfected with

DNA for TAS2R5 or TAS2R39 were also sensitive to PGG. Elevation of calcium

levels were also seen in cells expressing TAS2R5 that have been exposed to

procyanidin trimer. Notably, above chemicals are the first natural bitter

compounds found for TAS2R5. So far this specialist receptor responded only to

the synthetic compound phenantroline and may therefore be the only TAS2R that

is activated (“specific”) by natural tannins. Finally, we found that malvidin-3-

glucoside elicited signals specifically in cells transfected with DNA for TAS2R7.



213

(-)-Epicatechin Malvidin-3-glucoside

Procyanidin trimer PGG

Fig.6.2 Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are indicated by the thinner solid line. Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are indicated by the thinner solid line.

Cells expressing TAS2R1, TAS2R3, TAS2R8, TAS2R9, TAS2R10, TAS2R13,


TAS2R31, TAS2R45, TAS2R46, TAS2R30, TAS2R19, TAS2R20, TAS2R50 and

TAS2R60 did not respond to the tested compounds (data not shown). It is also

important to note that procyanidin dimer and cyanidin-3-glucoside did not activate

any receptor, at least in the highest possible concentrations tested (100 µM).

Thus, the data indicate that four of the six polyphenols compounds tested

mediate their bitterness through activation of the 4 human bitter taste receptors,

TAS2R4, TAS2R5, TAS2R7, and TAS2R39.

Functional characterization of activated TAS2Rs. In order to

investigate the activation of the bitter receptors TAS2R4, TAS2R5, TAS2R7 and

TAS2R39 by the polyphenols in greater detail, concentration-response functions

2500

MOCK

hTAS2R5

hTAS2R4

hTAS2R39

5 min

counts 1500

5 min

counts

hTAS2R7 MOCK

5 min

500 counts

hTAS2R5

MOCK

5 min

600 counts

hTAS2R5

hTAS2R39

MOCK


were recorded and the half maximal effective agonist concentrations (EC50)

established (Fig.6.3). The concentration-response curves followed sigmoid

functions. The calculated EC50 values for the compound-receptor pairs are

presented in Table 6.1 were the values with ≥ are estimates because the dose-

response curves did not saturate and so we were unable to determine an EC50

value.

Collectively, the data demonstrate that the EC50 values for the different

agonist/receptor pairs vary from the µM to the mM range. While the EC50 values

for PGG at both TAS2R5 and TAS2R39 are ≥ 8.5 and ≥ 6.6 µM, respectively,

those for (-)-epicatechin are ~1000fold higher and vary between 3.2 and 3.8 mM.

Like the EC50 values for PGG, those for malvidin-3-glucoside and procyanidin

trimer are also in the micromolar range, being 12.6 µM at TAS2R7 and 35.6 µM

at TAS2R5, respectively.

(a) (b)

(c) (d)

Fig.6.3 Concentration-response curves for HEK293T-Gα16gust44 cells transfected with DNA for the indicated TAS2R following stimulation with the indicated test compound. Error bars represent the confidential interval (P = 0.05). (a) Dose dependent activation was observed for TAS2R4, TAS2R5, and TAS2R39 by (-)-epicatechin (a), for TAS2R5 and TAS2R39 by PGG (b), for TAS2R7 by malvidin-3-glucoside (c), and for TAS2R5 by procyanidin trimer (d).

(-)-Epicatechin [mM]

0.01 0.1 1 10 100

∆F/F

-0.2

0.0

0.2

0.4

0.6

0.8hTAS2R4 hTAS2R39hTAS2R5

PGG [µM]

0.01 0.1 1 10 100

∆ F/F

-0.2

0.0

0.2

0.4

0.6

0.8

hTAS2R39hTAS2R5

Malvidin-3-glucoside [µM]

0.1 1 10 100

∆F/F

-0.2

0.0

0.2

0.4

0.6

0.8

Procyanidin trimer [µM]

0.1 1 10 100 1000

∆ F/F

-0.2

0.0

0.2

0.4

0.6

0.8hTAS2R5



215

Table 6. 1 EC50 values (µM) for the test compounds and respective receptors. The values with superscript equal letters are not significantly different (P < 0.05). Values with ≥ are estimates because the dose-response curves did not saturate.

EC50 (µM)

TAS2R4 TAS2R5 TAS2R7 TAS2R39

(-)-Epicatechin ≥ 30151.0 3210.0 +/- 42.0a - 3800.0 +/- 200.0b,c

Procyanidin trimer - 35.6 +/- 0.7d - -

PGG - ≥ 8.5 - ≥ 6.6

Malvidin-3-glucoside - - 12.6 +/- 0.7 -

Besides these pronounced differences in the EC50 values, the observed threshold

concentrations, defined as the lowest concentration that resulted in calcium

signals in receptor-transfected cells, are also largely different (Table 6.2).

Whereas PGG induced robust receptor responses already at a concentration of

3.0 µM at both TAS2R5 and TAS2R39, a 300-1000fold higher concentration of (-

)-epicatechin was required to evoke receptor responses at TAS2R4, TAS2R5,

and TAS2R39. Like PGG, threshold values for malvidin-3-glucoside and

procyanidin trimer were in the micromolar range. The lowest concentrations that

induced detectable receptor responses were 6.0 µM malvidin-3-glucoside for

TAS2R7 and 30.0 µM procyanidin trimer for the TAS2R5. Thus the data clearly

demonstrate that PGG, malvidin-3-glucoside and procyanidin trimer act as high

potency agonists on TAS2Rs, whereas (-)-epicatechin is a low potency stimulus.

Table 6.2 Threshold concentrations (µM), defined as the lowest concentration that resulted in calcium signals in receptor-transfected cells, for the test compounds.

Threshold concentrations (µM)


(-)-Epicatechin 2000.0 1000.0 - 1000.0

Procyanidin trimer - 30.0 - -

PGG - 3.0 - 3.0

Malvidin-3-glucoside - - 6.0 -

Also signal amplitudes, which are related to the efficiency of the receptor

activation, differ across receptors and the polyphenols (Table 6.3). (-)-

Epicatechin, activated TAS2R5 cognate receptor as well as TAS2R4 and

TAS2R39 with high efficacy as revealed by the change in fluorescence, i.e., the

corresponding ∆F/F values. Similarly, PGG was an efficient stimulus for

TAS2R39 and also malvidin-3-glucoside showed high efficiency at its cognate


receptor. In contrast, the observed efficacy of procyanidin trimer at its cognate

receptor is relatively low.

Table 6.3 Signal amplitudes (given as relative fluorescence changes ∆F/F) for the test compounds.

Signal amplitudes


(-)-Epicatechin 0.44 +/- 0.04a 0.59 +/- 0.11a - 0.65 +/- 0.04a

Procyanidin trimer - 0.22 +/- 0.04c - -

PGG - 0.36 +/- 0.16d - 0.61 +/- 0.04d

Malvidin-3-glucoside - - 0.54 +/- 0.09 -

� DISCUSSION

It has been well-known and common sense that consumption of diets with high

content of food rich in vitamins, minerals, fibers, and polyphenols, as well as low

levels of saturated fats are directly associated to a low incidence of

cardiovascular and cancer diseases. The well-known French Paradox that

appeared in 1992, it’s a classic example of this association (47). Since that time,

an exponential growing of epidemiological, clinical and experimental data support

the importance of a diet rich in polyphenol-rich foods (1). In fact, these

compounds have been suggested as chemoprevention agents. For instance,

pentagalloylglucose has been shown in vivo to have anti-cancer effect against

prostate, lung, and breast cancers as well as to have anti-diabetes activity (48).

Also cyanidin-3-glucoside has been shown to be able to revert human melanoma

cells from the proliferating to the differentiated state (49). Because of

polyphenols’ beneficial effects on human health, increasing the phytonutrient

content of plant foods is a potent dietary option for preventing diseases and a

challenge for the design of functional food. However, many people do not like to

eat vegetables and/or derived products especially because of their bitter taste

(3). So, the study of the bitterness properties of polyphenol compounds is a

prominent subject that could reflects in people’s food choices, and lastly in

people’s food chemoprevention.

Therefore, in this report we studied the bitterness of 6 polyphenol compounds

commonly present in human diet ((-)-epicatechin, procyanidin dimer B3 and

trimer C2, malvidin-3-glucoside, cyanidin-3-glucoside, and PGG) and showed

that the human bitter taste receptors, TAS2R4, TAS2R5, TAS2R7, and TAS2R39

are sensitive to 4 of the 6 compounds tested. The agonist-receptor pairs display



217

an interesting activation pattern. Firstly, different compounds activate the same

receptor. For example (-)-epicatechin and PGG activate TAS2R39 whereas (-)-

epicatechin, PGG and procyanidin trimer activate TAS2R5. Secondly, different

receptors are activated by the same compound. This is evidenced by (-)-

epicatechin which activates TAS2R4, TAS2R5 and TAS2R39 and PGG which

stimulates TAS2R5 and TAS2R39. These results confirm well with the

combinatorial activation patterns of TAS2Rs previously reported (18, 20).

Furthermore, by the structural diverse compounds, these data also have

implications for structure-activity relationships. They suggest that the catechol

and/or galloyl group (which has only one more hydroxyl group than catechol) are

critical features (but not essential) for the interaction of polyphenol compounds

with TAS2R5. Firstly, the compounds that activated this receptor, (-)-epicatechin,

procyanidin trimer and PGG have at least one of these groups while the other

tested compounds, malvidin-3-glucoside and cyanidin-3-glucoside, did not.

Secondly, procyanidin trimer and PGG that possess three ortho-catechol groups

and five galloyl groups, respectively, activate TAS2R5 at 100-fold lower

concentrations than does (-)-epicatechin which has only one ortho-catechol

group. However, this structural feature is not required for other compounds that

activate TAS2R5 since the synthetic ligand 1,10-phenanthroline lacks these

groups completely. In fact, it is evident that the presence of these groups is not

sufficient for the responsiveness of this receptor since procyanidin dimer (which

as two ortho-catechol groups) does not activate this receptor. However, it seems

that, when a compound activates TAS2R5, the presence of these groups could

be essential for higher responses/interaction. The importance of the hydroxyl

groups in bitter receptor activation is also referred by Roland and co-workers

(36), who have studied the activation of receptor TAS2R39 by the soy isoflavone

genistein and structurally similar isoflavonoids. These authors concluded that the

presence of three hydroxyl groups in the isoflavonoid ring system which

resembles that of the polyphenols seemed to be more favorable for TAS2R39

activation than the presence of fewer hydroxyl groups. These authors also noted

the importance of glucose residues attached to their test compounds for

TAS2R39 activation. The present report demonstrated that TAS2R39 responded

to PGG which also contains a glucoside moiety. However, malvidin-3-glucoside

and cyanidin-3-glucoside did not activate this receptor. Interestingly, the

glycosylated compounds did not activate the TAS2R16, which has been

suggested to be specific for β-glucopyranosides (19). However, these authors


(19) also found that hydrophobicity and size of the aglycons are critical for

receptor activation. The great structural differences between the compounds

employed by Bufe and co-workers and those tested here easily explain the

absence of responses of TAS2R16.

A receptor for which the presence of glucose residues seems to be important is

TAS2R7. Whereas malvidin-3-glucoside activated TAS2R7, its aglycon form

(without glucose residue) failed to do so, at least at the tested concentrations.

Our result for malvidin-3-glucoside oppose those of a sensory study that showed

that this anthocyanin does not taste bitter (50). These authors studied the

bitterness of malvidin-3-glucoside in a mixture with cyanidin-3-glucoside made in

a solution saturated with tartaric acid (pH 3.6) and containing ethanol. It is also

important to mention that anthocyanins, co-exist in solution in different forms

because of their well-known pH dependence (51, 52): at low pH (1-2) they are

essentially present in the red cationic form (AH+), but as the pH increases rapid

proton transfer reactions occur leading to the formation of blue quinonoidal bases

(A and A-) and chalcones. Therefore, the two data sets appear not to be

comparable since the use of other compounds and distinct experimental

conditions could influence the perceived bitterness and receptor responses

differently. It is important to point out that we could not test ethanol in the in vitro

assay because it is toxic for cells already at 1%.

Even though the factors that determine bitter intensity are still unknown the

concentration-response functions of the 5 polyphenols and their cognate

receptors predict that PGG appears to be a strong bitter compound in foodstuffs

if present at least in low micromolar concentrations. Firstly, it activates both

receptors, TAS2R5 and TAS2R39. Secondly, it displays high potency evident as

low EC50 values at both cognate receptors. Thirdly, it shows also high efficacy as

revealed by the high signal amplitudes of the activated receptors, in particular for

TAS2R39. Malvidin-3-glycoside and procyanidin trimer are also of high potency,

i.e., they have low EC50 values at their receptors. However, both compounds

activate one receptor with relatively low efficacy, TAS2R7 and TAS2R5,

respectively. This could mean that these polyphenol compounds elicit a weaker

bitter taste than PGG, if they are present in food in similar concentrations as

PGG. (-)-Epicatechin also appears to be a strong bitter chemical. It activates

three receptors, TAS2R4, TAS2R5, and TAS2R39 with high efficacy. The

potencies of this compound at its cognate receptors may be low relative to the

other 4 polyphenols because its EC50 values are in the millimolar range.



219

However, if (-)-epicatechin is present in food in this concentration range it would

function as a strong activator of bitterness due to its high efficacy. This is

supported by data about β-glucopyranosides which have millimolar potencies at

only one receptor, hTAS2R16, yet elicit strong bitterness and avoidance behavior

(19, 53).

Polyphenol concentrations in vegetables, fruits and derived products depend on

many factors, e.g. culture conditions and degree of ripeness. The tested

polyphenols are present in numerous plant-derived products commonly present

in our diet (e.g. red wine, beer, cocoa, red fruits), even though their

concentrations vary widely (54). For instance, (-)-epicatechin has been identified

in 125 food and beverages and it is known to be present in dark chocolate (mean

content 0.070 mg.g-1), apple (mean content 0.008 mg.g-1) and blackberry (mean

content 0.012 mg.g-1). Malvidin-3-glucoside has been identified in 21 foodstuffs

including black grapes (mean content 0.039 mg.g-1) and beans (mean content

0.0006 mg.g-1). Procyanidin trimer C2 has been identified in beer (0.3 µg.mL-1).

The wide range of concentrations is particular evident in red wine (55-57). (-)-

Epicatechin has been reported to be present in the range of 5.0 to 88.0 mg.L-1 (=

17.0 – 302.0 µM) in over 800 types of red wine (56) and malvidin-3-glucoside is

present in the range of 40.0 to 510.0 mg.L-1 (= 81.0 – 1035.0 µM) in Port wine

(55). Thus, the concentrations of (-)-epicatechin are about 10fold below its EC50

values at all cognate receptors suggesting that this polyphenol may contributes

little to the bitterness of red wine. Malvidin-3-glucoside is present at

concentrations 6-80fold higher than its EC50 value at TAS2R7 suggesting that this

receptor is robustly activated when consuming these types of wine. Regarding

procyanidin trimer and PGG used herein, there are no reported concentrations in

red wine but, as referred previously, this trimer has been reported to be present

in beer at 0.3 µg.mL-1(54). However, procyanidin trimer C1 is known to be

present in red wine at 25.6 mg.L-1 (29.6 µM) (58).

Although the existing results are controversial, some sensory evaluations

perceive smaller polyphenol molecules as bitterer than larger ones (8, 10). Peleg

and co-workers (8) studied the bitterness of several compounds including (-)-

epicatechin, procyanidin dimers and trimers at the concentration of 0.9 g.L-1 in

aqueous ethanol (1% v/v). This concentration corresponds to 3093.0 µM of (-)-

epicatechin and to 1040.0 µM of procyanidin trimer, which in the case of (-)-

epicatechin lies in the concentration range used herein, but for procyanidin trimer

is much higher than the concentration range used herein. At that concentration,


Peleg (8) found that monomers were significantly bitterer than the dimers, which

in turn were significantly bitterer than the trimers.

Hufnagel and Hofmann (5) studied the astringency and bitterness of numerous

compounds from a red wine by sensorial analysis and they demonstrated that the

bitterness of that red wine could be induced by phenolic acid ethyl esters and

flavan-3-ols (including (-)-epicatechin, procyanidin dimers and C1 trimer). But in

contrast to Peleg, they found that the larger molecules were bitterer than smaller

ones. In fact, the bitter taste threshold for (-)-epicatechin was found to be 930.0

µM, and so these data correlate with our observations for the TAS2R39.

However, the bitter taste thresholds they found for procyanidins were between

400 and 500 µM which are much higher than the values found in this work (30.0

µM). This observed correlation between the in vivo and in vitro analysis, with

sensory data showing mostly higher thresholds values in comparison to in vitro

data, is common and has been reported previously (30). On possible explanation

to this fact are the formation of complexes with proline-rich proteins in the saliva

and/or adsorption of these compounds by the oral epithelium which could alter

their effective oral concentrations differently and explain the different

concentration-response functions recorded in vivo and in cell-culture (30). We

can also not exclude the possibility that a particular fully activated TAS2Rs

evokes bitter perception to a lesser degree than another one.

Also, a sensory analysis of fractions of grape seed phenolics showed that

bitterness increases along with molecular size (9). Moreover, Lea and Arnold (12)

showed that bitterness is associated with oligomeric procyanidins reaching a

maximum with the epicatechin tetramer. This is in agreement with our results

since (-)-epicatechin monomer is less bitter that procyanidin trimer.

Apart from the molecular size of the bitter polyphenols under study, other factors

could affect the bitterness of the compounds in sensory evaluations, as referred

previously. For example, while ethanol enhances bitterness intensity, varying

wine pH has little or no effect on perceived bitterness (59). Fischer and Noble

(59) prepared eighteen wines, varying in ethanol (8%, 11%, 14% v/v), pH (2.9,

3.2, 3.8) and (+) catechin (100.0 and 1500.0 mg.L-1) using a dealcoholized white

wine concentrate. In a completely randomized design, bitterness intensity was

rated by 20 subjects and it was found that bitterness increased by all three

components. These authors have shown that an increase of 3% v/v ethanol

elevated bitterness more (around 50%) than adding 1400.0 mg.L-1 of catechin to

the same wine (which increased bitterness by 28%). Such increase in catechin is



221

not common in the winemaking industry; however differences of 3% v/v ethanol

occur frequently in table wines and will produce a significant increase in

bitterness. Unfortunately, it is impossible to examine this interesting effect of

ethanol by means of the receptor assay, because ethanol is highly toxic for the

cells above 1%.

In summary, the results show that different polyphenol compounds activate

different subsets of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors

TAS2R4, TAS2R5 and TAS2R39 whereas only two receptors, TAS2R5 and

TAS2R39, responded to PGG. In contrast, malvidin-3-glucoside and procyanidin

trimer stimulated one receptor, TAS2R7 and TAS2R5, respectively. Overall, the

EC50 values obtained for the different compounds vary 100-fold with the lowest

values for the compounds that belong to hydrolysable tannins and anthocyanins

classes, suggesting that these polyphenols could be the major compounds

responsible for the bitterness of fruits and derived products, such as red wine.



*Tel: +351 220402558. Fax: +351 220402659. E-mail: [email protected].

Acknowledgment

This work has been supported by a grant of the German Research Foudation

(DFG) to W.M. (Me 1024/2-3) and also by two grants of the Fundação para a

Ciência e Tecnologia (one PhD grant [SFRH/BD/41946/2007] and one project

grant [PTDC/AGR-ALI/67579/2006]).

� ABREVIATIONS USED DNA, deoxyribonucleic acid, DMSO, dimethylsulfoxide, H1 NMR, nuclear

magnetic resonance, HPLC, high performance liquid chromatography, PGG,

pentagalloylglucose, TRC, taste receptor cells

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CONCLUSÃO E PERSPETIVAS FUTURAS

Este trabalho centrou-se na compreensão das propriedades sensoriais de

diferentes famílias de polifenóis (amargor e adstringência) e dos mecanismos

envolvidos. Por outro lado, foi estudado de que modo é que diferentes

carboidratos vulgarmente utilizados na indústria alimentar influenciam a

adstringência no contexto do vinho tinto. Para isso, procurou-se determinar: (a),

as principais famílias de proteínas salivares que apresentam mais afinidade para

os taninos; (b), de que forma é afetada a interação taninos/proteínas salivares

quando os taninos estão numa matriz de vinho tinto; (c), como é que o

enriquecimento de um vinho tinto em taninos influência a adstringência; (d),

quais as caraterísticas (solubilidade e tamanho) dos complexos formados pelas

diferentes famílias de proteínas salivares e taninos condensados vs.

hidrolisáveis; (e), como é que os carboidratos (goma arábica, β-ciclodextrina,

pectina e ácido poligalacturónico) inibem a interação taninos/proteínas,

particularmente com as proteínas salivares; (f), quais são os recetores de sabor

amargo ativados por alguns polifenóis frequentemente encontrados em diversos

alimentos.

Para atingir estes objetivos, começou-se por identificar na saliva humana as

respetivas proteínas por uma abordagem proteómica (HPLC-DAD, ESI-MS,

SDS-PAGE e MALDI-TOF). Seguidamente, foi estudada a afinidade entre as

diferentes famílias de proteínas salivares e taninos condensados através da

interação da saliva com procianidinas isoladas de grainhas de uvas. Os

resultados obtidos num ensaio competitivo, i. e., quando todas as proteínas

estão presentes em simultâneo tal como na cavidade oral, demonstram que os

taninos condensados interagem inicialmente com as aPRPs e estaterina e só

depois com as histatinas, gPRPs e bPRPs. Este resultado foi extremamente

importante pois foi observado pela primeira vez que as aPRPs são as PRPs que

apresentam maior afinidade para os taninos, contrariamente ao que era

esperado uma vez que as bPRPs são as mais estudadas e referidas na literatura

como as mais importantes nesta interação. Na verdade, este trabalho é o

primeiro que compara estas duas classes de PRPs.

Foi estudada a interação entre as diferentes proteínas da saliva com os taninos

diretamente no vinho tinto e os resultados foram confrontados com a

adstringência determinada por um painel de provadores. Para isso, foi

suplementado um vinho tinto com diferentes concentrações de taninos


condensados extraídos de grainhas de uvas. Comparando estes resultados in

vitro com a análise sensorial in vivo verificou-se que para concentrações baixas

de taninos, a complexação/precipitação de aPRPs e estaterina está

correlacionada com a intensidade da adstringência, pois estas proteínas têm

uma taxa muito elevada de complexação/precipitação comparativamente às

outras proteínas salivares. No entanto, para concentrações maiores de taninos,

a adstringência relativa percecionada dos vinhos parece correlacionar-se com

mudanças na composição das PRPs glicosiladas.

Após o estudo inicial da afinidade relativa da interação taninos

condensados/proteínas salivares, pretendeu-se comparar a interação das

proteínas salivares com um tanino hidrolisável. Para isso, foi estudada a

interação de uma procianidina trimérica e da PGG com as proteínas salivares

por HPLC e dynamic light scattering (DLS). Os resultados mostraram que as

proteínas aPRPs e estaterina, interagem em maior extensão com ambos os

taninos, comparativamente com as gPRPs e bPRPs. As bPRPs interagem pouco

com a procianidina trimérica e as gPRPs só complexam com a PGG. Em geral, a

PGG apresentou mais tendência para a formação de complexos insolúveis

enquanto a procianidina trimérica mostrou mais tendência para a formação de

complexos solúveis (exceto com a estaterina). Os resultados indicam que os

diferentes taninos e proteínas salivares estudados apresentam mecanismos

diferentes de interação e consequentemente particularidades diferentes no

desenvolvimento da adstringência.

Após o estudo da interação taninos/proteínas salivares, estudou-se o efeito dos

carboidratos nesta interação usando dois modelos de estudo: taninos

condensados/α-amilase e taninos condensados/proteínas salivares. No primeiro

estudo foram usadas as técnicas de fluorescência, nefelometria e DLS; no

segundo monitorizou-se por HPLC as proteínas salivares que permaneciam

solúveis na presença dos taninos e após a adição dos carboidratos. Os

respetivos precipitados formados foram também analisados por SDS-PAGE.

Globalmente os resultados mostraram que todos os carboidratos reduziram a

formação de agregados insolúveis, e portanto a precipitação, observando-se que

a pectina foi sempre o carboidrato mais eficiente, seguida da goma arábica. No

primeiro estudo, a extinção da fluorescência permitiu ter evidências de qual o

mecanismo mais provável subjacente a essa ação inibidora, sugerindo que a

goma arábica e a β-ciclodextrina inibem a interação tanino/proteína pelo

mecanismo de competição, associando-se aos taninos e impedindo-os de



229

interagir com a proteína. Por outro lado, a pectina parece atuar pela formação de

um complexo ternário tanino/proteína/pectina com solubilidade acrescida em

meio aquoso. Estas observações também foram testemunhadas no estudo com

as proteínas salivares, neste caso para os carboidratos goma arábica e a

pectina.

Na parte final deste trabalho, estudou-se o sabor amargo de alguns polifenóis

pertencentes a diferentes classes [(-)-epicatequina, PGG, procianidinas dimérica

B3 e trimérica C2, malvidina-3-glucósido e cianidina-3-glucósido], identificando

quais dos 25 recetores de sabor amargo dos humanos (hTAS2Rs) são ativados

por estes compostos.

Os resultados obtidos mostraram que diferentes compostos ativam diferentes

hTAS2Rs. (-)-Epicatequina ativou três recetors hTAS2R4, hTAS2R5 e

hTAS2R39, enquanto a PGG só ativou dois recetores, hTAS2R5 e hTAS2R39.

Por outro lado, a malvidina-3-glucósido e o trímero C2 só ativaram um recetor,

hTAS2R7 e hTAS2R5, respetivamente. Um dos resultados mais notáveis é que

os taninos são os primeiros agonistas naturais encontrados para o hTAS2R5,

apresentando grande potência na ativação deste recetor. Para além disso, os

grupos catecol e/ou galhoilo parecem ser estruturalmente importantes na

mediação da interação dos polifenóis com este recetor.

Os valores obtidos de EC50 para os diferentes compostos variam cerca de 100-

vezes, sendo os valores mais baixos os da PGG e malvidina-3-glucósido. Isto

sugere que estes compostos podem ser significativamente importantes no

amargor de frutos, vegetais e produtos derivados, mesmo que estejam presentes

em concentrações muito baixas.

Os resultados deste trabalho contribuíram para uma melhor compreensão do

envolvimento das diferentes famílias de proteínas salivares no desenvolvimento

da adstringência resultante da ingestão de taninos e do papel dos carboidratos

neste fenómeno. Neste trabalho surgiram, pela primeira vez, evidências de que

as diferentes proteínas salivares estão envolvidas em diferentes fases do

desenvolvimento da adstringência. Também pela primeira vez foi demonstrada

uma grande afinidade entre os taninos presentes no vinho e as aPRPs e a

estaterina. Com base na literatura seria de esperar que as bPRPs fossem as

mais eficientes. No entanto, isto não se verificou porque na literatura existem

poucos trabalhos com as aPRPs e nenhum trabalho que englobe o estudo das

diferentes famílias de PRPs em simultâneo.


Relativamente aos carboidratos, pela primeira vez a utilização de várias técnicas

permitiu conhecer melhor os mecanismos responsáveis pela sua ação inibidora

da interação entre os polifenóis e proteínas, nomeadamente com as diferentes

proteínas salivares. O estudo experimental de sistemas ternários envolvendo

três famílias complexas de compostos como os polifenóis, as proteínas e os

carboidratos é um desafio difícil mas, neste caso em particular, a conjugação

das técnicas de fluorescência, DLS e nefelometria permitiu obter resultados

importantes.

No que diz respeito ao sabor amargo, foram identificados pela primeira vez os

hTAS2Rs ativados por alguns polifenóis. Foram obtidas pela primeira vez

evidências de que a malvidina-3-glucósido (uma antocianina) ativa um hTAS2Rs

e portanto pode ter um papel no sabor amargo dos alimentos. E, finalmente, um

dos resultados mais notáveis é que os taninos são os primeiros agonistas

naturais encontrados para o hTAS2R5, apresentando grande potência na

ativação deste recetor.

Apesar da importância dos resultados obtidos, a interação tanino/proteína ainda

é uma área pouco estudada com muitas questões por explorar. Estudos futuros

passam pela compreensão do papel fisiológico das diferentes famílias de

proteínas salivares a nível sensorial e nutricional, considerando a interação com

os polifenóis. Por outro lado, do ponto de vista nutricional, os polifenóis

apresentam propriedades biológicas importantes (prevenção cardiovascular,

anticancerígena, etc), sendo que a interação com as proteínas salivares poderá

afetar a sua biodisponibilidade. Assim, seria importante estabelecer uma relação

estrutura-atividade dos polifenóis (extraídos de frutos ou sintetizados) na

interação com diferentes famílias de proteínas salivares (PRPs básicas, acídicas,

glicosiladas, estaterina e histatinas).

A abordagem experimental passaria pela determinação das constantes de

associação e estequiometria dos agregados, na ausência e na presença de

carbohidratos existentes nos alimentos ou usados na indústria alimentar

(pectina, goma arábica, etc). Este estudo envolveria diferentes técnicas,

nomeadamente HPLC, ESI-MS e RMN. Na interpretação dos resultados seria

importante recorrer à modelação molecular de modo a determinar os principais

locais moleculares da interação tanto na proteína como no polifenol.

A nível nutricional seria importante determinar a estabilidade dos complexos

taninos/proteínas salivares no estômago e intestino e a eventual



231

biodisponibilidade dos taninos, recriando um ambiente de digestão simulado in

vitro.

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