universidade estadual de campinas faculdade de engenharia de...
TRANSCRIPT
UNIVERSIDADE ESTADUAL DE CAMPINAS
Faculdade de Engenharia de Alimentos
FLÁVIA REGINA DE FARIA
Degradação de polifenóis e formação de compostos de sabor no processamento
de chocolate a partir de amêndoas de cacau fermentadas e não fermentadas
CAMPINAS
2019
FLÁVIA REGINA DE FARIA
Degradação de polifenóis e formação de compostos de sabor no processamento
de chocolate a partir de amêndoas de cacau fermentadas e não fermentadas
Dissertação apresentada à
Faculdade de Engenharia de
Alimentos da Universidade Estadual
de Campinas como parte dos
requisitos exigidos para a obtenção
do título de Mestra em Tecnologia de
Alimentos
Orientadora: PROFA. DRA. PRISCILLA EFRAIM
ESTE TRABALHO CORRESPONDE À
VERSÃO FINAL DA DISSERTAÇÃO
DEFENDIDA PELA ALUNA FLÁVIA
REGINA DE FARIA E ORIENTADA PELA
PROFA. DRA. PRISCILLA EFRAIM
CAMPINAS
2019
FICHA CATALOGRÁFICA
COMISSÃO EXAMINADORA
Profa. Dra. Priscilla Efraim (Orientadora)
Universidade Estadual de Campinas
Dra. Adriana Barreto Alves (Membro Titular)
Laboratório Federal de Defesa Agropecuária (LFDA – SP)
Dra. Juliana Campos Hashimoto (Membro Titular)
Universidade Estadual de Campinas
A ata da defesa com as respectivas assinaturas dos membros encontra-se no SIGA/Sistema de
Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.
DEDICATÓRIA
Dedico este trabalho aos meus pais, por todo amor, apoio e incentivo que sempre
demonstraram.
AGRADECIMENTOS
Agradeço à Deus, pela vida.
À minha família, em especial aos meus pais, pelo apoio e incentivo e ao Santiago, pelo
companheirismo e apoio durante a realização do projeto.
À Profa. Dra. Priscilla Efraim, pela orientação, dedicação e compreensão.
À Universidade Estadual de Campinas, pelas oportunidades de formação desde o Curso
Técnico, a Graduação e o Mestrado.
À Faculdade de Tecnologia SENAI Horácio Augusto da Silveira pela disponibilização de
instalações e recursos para a realização do projeto e aos meus colegas da Instituição pelo
apoio.
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior - Brasil (CAPES) – Código de Financiamento 001
A todos que contribuíram para a realização deste trabalho, obrigada!
RESUMO
O chocolate é um alimento mundialmente consumido e apreciado por seu sabor inigualável. Além
da questão sensorial, o chocolate é visto, em muitos países, como um alimento que pode
propiciar benefícios à saúde, se consumido de forma balanceada na dieta. Tais propriedades são
observadas em função da presença de compostos fenólicos nas sementes de cacau, porém seus
teores são diminuídos durante o processamento do fruto. A degradação dos flavanóis nas etapas
de processo tem sido objeto de estudo para sustentar o desenvolvimento de chocolates com
maiores teores de polifenóis, porém, a formação de sabor é complexa e envolve diversas
transformações químicas e bioquímicas que ocorrem durante o processamento. Enquanto a
etapa de fermentação é apontada como uma das principais responsáveis pela perda de
polifenóis, é também considerada como fundamental para a formação de precursores de sabor,
assim, o objetivo deste estudo foi analisar a degradação de flavanóis e a formação de compostos
voláteis durante o processamento de amêndoas fermentadas e não fermentadas de cacau,
obtidas da mesma fonte, até a obtenção de chocolates, variando as condições de conchagem.
Os teores de epicatequina, catequina e procianidina B2 foram determinados empregando
cromatografia líquida de alta eficiência (CLAE). A extração dos compostos voláteis foi realizada
pela técnica de micro extração em fase sólida e o perfil foi analisado por cromatografia gasosa
com detecção por espectrometria de massas. Os chocolates foram submetidos à análise
sensorial para avaliação da aceitação dos produtos. As amêndoas não fermentadas
apresentaram teores de flavanóis cinco vezes maiores que as amêndoas fermentadas e a etapa
de torração ocasionou diminuição expressiva no teor de epicatequina e de procianidina B2. Os
perfis inicias de compostos voláteis obtidos nas amêndoas apresentaram diferenças
significativas e os principais compostos reconhecidos como típicos na composição de sabor de
chocolates foram formados durante a torração das amêndoas fermentadas. O emprego de maior
tempo de conchagem não resultou em alteração significativa nos teores dos compostos fenólicos
analisados, porém impactou na redução de compostos voláteis, que foi percebida na análise
sensorial dos chocolates. Os chocolates produzidos a partir de amêndoas não fermentadas não
foram bem aceitos, principalmente em decorrência do amargor e adstringência pronunciados
devido ao alto teor de polifenóis e da falta de sabor de típico nos produtos.
Palavras chave: cacau, chocolate, polifenóis, sabor e aroma.
ABSTRACT
Worldwide, chocolate is appreciated for its unique and complex flavor. Beyond the sensory
appeal, chocolate consumption, in many countries, has been associated to health benefits when
composing a balanced diet. Phenolic compounds found on cocoa beans are responsible for
antioxidant properties, but its contents decrease during processing stages. At the same time that
degradation of flavanols has been studied to optimize process conditions and support the
development of chocolates with higher contents of polyphenols, flavor formation is complex and
involves chemical and biochemical transformations that occur during process. Since extensive
degradation of polyphenols is reported on fermentation whilst the formation of flavor precursors
is relevant on this step, the aim of this study was to analyze flavanols degradation and volatile
compounds formation during the processing of fermented and non-fermented cocoa beans from
the same source up to chocolate varying conching times. Epicatechin, catechin and procyanidin
B2 were quantified by HPLC and volatile compounds were extracted by SPME and analyzed by
GC-MS. Sensory evaluation of chocolates was employed to assess the acceptance of the
products. The quantity of flavanols was initially five-fold higher on non-fermented beans compared
to fermented ones and results showed an important loss of epicatechin and procyanidin B2 during
the roasting process. Volatile composition profile from fermented and non-fermented samples
were significantly different and the main flavor-active compounds were formed during the roasting
of fermented beans. Longer conching period at the same temperature did not cause a significant
variation on the flavanols contents but reduction on volatiles were observed and noticed on the
sensory evaluation. Chocolates produced from unfermented beans were not well accepted mainly
because of the astringency and bitterness caused, probably by the high content of flavanols and
the lack of chocolate flavor.
Keywords: cocoa, chocolate, polyphenols, flavor
SUMÁRIO
1. INTRODUÇÃO .................................................................................................................................. 10
2. OBJETIVO ......................................................................................................................................... 12
2.1. Objetivo geral ............................................................................................................................ 12
2.2. Objetivos específicos ............................................................................................................... 12
3. REVISÃO BIBLIOGRÁFICA ........................................................................................................... 13
3.1. Processamento de cacau........................................................................................................ 13
3.2. Compostos fenólicos no cacau .............................................................................................. 14
3.3. Formação de sabor no processamento de cacau ............................................................... 15
4. ARTIGO - Flavanols degradation and volatile flavor compounds formation during the
processing of fermented and non-fermented cocoa beans ............................................................... 18
Abstract .................................................................................................................................................. 18
Highlights ............................................................................................................................................... 18
4.1. Introduction ................................................................................................................................ 19
4.2. Material and methods .............................................................................................................. 20
4.2.1. Cocoa samples and post harvesting process .............................................................. 20
4.2.2. Cut test ............................................................................................................................... 20
4.2.3. Processing ......................................................................................................................... 21
4.2.4. Catechin, Epicatechin and Procyanidin B2 quantification - HPLC............................ 21
4.2.5. Volatile aroma profiles – HS-SPME-GC-MS ................................................................ 22
4.2.6. Sensory analysis .............................................................................................................. 22
4.3. Results and discussion ............................................................................................................ 23
4.4. Conclusions ............................................................................................................................... 35
Acknowledgments ................................................................................................................................ 36
References ............................................................................................................................................ 37
5. CONCLUSÃO GERAL ..................................................................................................................... 43
REFERÊNCIAS GERAIS ........................................................................................................................ 44
ANEXO I – Parecer substanciado do comitê de ética em pesquisa da UNICAMP ....................... 50
ANEXO II – Declaração de cadastro no Sistema Nacional de Gestão do Patrimônio Genético . 56
10
1. INTRODUÇÃO
O cacau é reconhecido como um dos alimentos com maior teor de compostos
fenólicos e o consumo de seus derivados tem despertado interesse em relação aos
benefícios à saúde associados (COOPER et al., 2008; MCSHEA et al., 2008).
A demanda global por cacau vem aumentando impulsionada por diversos fatores
como o crescimento do mercado asiático e a grande presença de derivados de cacau em
diversos alimentos. Além disso, observa-se uma maior procura por chocolates amargos
e até o emprego de derivados em cosméticos e pela indústria farmacêutica, que tem
interesse nas suas propriedades antioxidantes (BEG et al., 2017).
O chocolate, produtos com chocolate ou com pó de cacau são as principais formas
de consumo de derivados de cacau. Dados indicam maior crescimento na demanda de
cacau em relação ao aumento no consumo de chocolate, o que possivelmente é reflexo
do aumento de procura por produtos com maiores teores de cacau (ICCO, 2012).
O chocolate apresenta sabor único e complexo, derivado da combinação de
compostos resultantes de transformações químicas e bioquímicas durante o
processamento, que são influenciadas pelas características do fruto, do cultivo e das
condições de processo empregadas (APROTOSOAIE; LUCA; MIRON, 2016).
Durante o pré-processamento das sementes de cacau, que inclui as etapas de
fermentação e secagem para obtenção das amêndoas, ocorrem perdas significativas nos
compostos fenólicos, sendo apontadas reduções de 75% no teor de epicatequinas
(ALBERTINI et al., 2015). Porém, nessas etapas também são formados precursores de
sabor, como aminoácidos livres e açúcares redutores, que darão origem aos produtos da
reação de Maillard e degradação de Strecker, principais responsáveis pelo perfil de sabor,
através de aquecimento na etapa de torração durante o processamento das amêndoas
para a obtenção de liquor de cacau (AFOAKWA et al., 2008).
Na fabricação do chocolate, que emprega o liquor de cacau como principal
ingrediente, a conchagem é uma etapa com reconhecida importância na melhoria de
sabor e textura do produto, na qual ocorrem eliminação de voláteis como ácido acético,
redução da umidade e interação de componentes. Porém, também ocorre perda e
alteração no perfil de compostos fenólicos, com estudo menos explorado (DI MATTIA et
al., 2014; BORDIGA et al., 2015).
Reconhecendo que o processamento de chocolate a partir de amêndoas não
fermentadas pode impactar negativamente no perfil de sabor do chocolate, porém
11
positivamente em uma maior retenção de compostos fenólicos de interesse em relação
aos benefícios à saúde e ainda que o número de trabalhos que combinam a avaliação da
degradação de compostos fenólicos com a formação de sabor no produto é escasso, o
presente projeto propõe estudar conjuntamente os dois efeitos.
12
2. OBJETIVO
2.1. Objetivo geral
Avaliar a degradação de compostos fenólicos e a formação de compostos voláteis
relacionados com o sabor de chocolates produzidos a partir de amêndoas de cacau
fermentadas e não fermentadas, variando o tempo de conchagem no processo.
2.2. Objetivos específicos
• Obter liquor de cacau a partir de amêndoas de cacau fermentadas e não
fermentadas e utilizá-los para a produção de chocolates.
• Empregar duas condições de tempo de conchagem durante o processamento de
chocolates produzidos a partir de amêndoas de cacau fermentadas e não
fermentadas.
• Quantificar a perda dos principais compostos fenólicos presentes no cacau
durante o processamento das amêndoas até os chocolates.
• Avaliar os perfis de compostos voláteis nas amêndoas de cacau fermentadas e
não fermentadas, nos derivados intermediários e nos chocolates obtidos.
• Avaliar a aceitação sensorial dos chocolates obtidos.
13
3. REVISÃO BIBLIOGRÁFICA
3.1. Processamento de cacau
O método tradicional de processamento de cacau (Figura 1) compreende as
etapas de pré-processamento das sementes (fermentação e secagem); processamento
das amêndoas de cacau, que envolve torração para obtenção de nibs de cacau, moagem,
que originará o liquor de cacau, prensagem do liquor para a obtenção de manteiga de
cacau e pó de cacau, sendo que os primeiros são os ingredientes normalmente
empregados na fabricação de chocolate. Já a fabricação de chocolate compreende as
etapas de formulação, com os seguintes ingredientes: liquor e manteiga de cacau, açúcar
e, opcionalmente, leite em pó ou outros derivados de leite, mistura dos ingredientes,
refino, conchagem, temperagem, moldagem, resfriamento, desmoldagem e embalagem,
de forma que cada etapa tem papel importante no desenvolvimento do sabor e da textura
que caracterizam o produto (BECKETT, 2009).
Figura 1 – Diagrama esquemático do processamento de cacau até a obtenção de chocolate
Fonte: Adaptado de BECKETT (2009)
14
3.2. Compostos fenólicos no cacau
A presença de polifenóis em alimentos de origem vegetal está associada com a
cor e o sabor desses produtos, com destaque para as notas de amargor e adstringência
conferidas por componentes do grupo; porém atualmente os estudos têm focado nos
potenciais efeitos benéficos à saúde atribuídos ao consumo desses alimentos (SOTO-
VACA et al., 2012)
As amêndoas de cacau fermentadas e secas possuem cerca de 6% (base seca)
de compostos fenólicos em sua composição, sendo que os principais componentes do
grupo encontrados nas amêndoas são os flavanóis, que representam cerca de 37% do
total de compostos fenólicos principalmente representados pelas epicatequinas (35%),
procianidinas (58%) e antocianinas (4%) (WOLLGAST; ANKLAM, 2000).
Variações no teor de polifenóis em cacau são atribuídas a diferentes condições de
cultivo (NIEMENAK et al., 2006), região geográfica (CARRILLO; LONDOÑO-LONDOÑO;
GIL, 2014), variações genéticas e diferentes metodologias de determinação
(WOLLGAST; ANKLAM, 2000).
São elencados na literatura diversos efeitos benéficos à saúde humana
associados ao consumo dos compostos fenólicos presentes no cacau e em seus
derivados. Entre eles, destacam-se principalmente aqueles relacionados à saúde
cardiovascular e danos inflamatórios, devido à capacidade antioxidante desses
compostos, com efeito potencial no aumento da resistência do organismo ao estresse
oxidativo (ANDÚJAR et al., 2012). Além disso, estudos indicam que os produtos de cacau
ricos em flavanóis melhoram a função endotelial e a sensibilidade à insulina e estariam
relacionados com a prevenção de doenças (SOTO-VACA et al., 2012).
O processamento do cacau afeta de forma qualitativa e quantitativa o perfil de
polifenóis, e diversos estudos apresentam o efeito das condições de processo na
degradação desses componentes.
As etapas de pré-processamento do cacau para obtenção de amêndoas
fermentadas e secas ocasionam perdas significativas nos compostos fenólicos originais
do fruto. Durante a fermentação do cacau, são relatadas perdas de mais de 70% no teor
de epicatequinas (CAMU et al., 2008), cuja degradação segue a tendência de degradação
dos compostos fenólicos totais (ALBERTINI et al., 2015).
Estudos indicam diferentes alterações no teor de polifenóis quando são
empregados métodos de secagem natural ou artificial nas amêndoas de cacau, sendo
que em alguns casos a secagem natural mostrou maior retenção de compostos fenólicos
15
(EFRAIM et al., 2010; DI MATTIA et al., 2013) enquanto a secagem artificial mostrou
maior percentual de retenção em outras condições estudadas, de forma que a cinética de
degradação durante a etapa foi descrita como de primeira ordem (TEH et al., 2015).
No processamento das amêndoas de cacau para a produção de liquor, são
observadas perdas de até 30% dos polifenóis, principalmente atribuídas à exposição ao
calor e oxigênio na etapa de torração (BORDIGA et al., 2015). A etapa de torração é
apontada como de grande influência no perfil de polifenóis presentes, sendo que o tipo
de fermentação preliminar (AFOAKWA et al., 2015) e as condições de processo
influenciam na extensão das alterações (ŻYŻELEWICZ et al., 2016). Diferentes
temperaturas de torração resultam em diferentes perfis de flavanóis nos produtos obtidos
devido à diferentes extensões de epimerização (KOTHE; ZIMMERMANN; GALENSA,
2013).
Poucos estudos relatam o efeito da conchagem nos compostos fenólicos durante
o processamento de chocolate. A variação de temperatura na faixa de 60 a 80ºC foi
relatada como não significativa na alteração do perfil de compostos fenólicos (GÜLTEKIN-
ÖZGÜVEN; BERKTAŞ; ÖZÇELIK, 2016). Em estudo comparativo entre duas diferentes
condições de conchagem – longa (60ºC por 12 h) e curta (6 h a 90ºC mais 1 h a 60ºC) –
os autores não encontraram diferença significativa no teor total de polifenóis, porém a
avalição dos perfis finais desses compostos nas duas amostras obtidas sugeriu que
ocorreu polimerização durante a etapa (DI MATTIA et al., 2014).
Em estudo para otimização do processamento das amêndoas de cacau, incluindo
a etapa de alcalinização para a obtenção de chocolate, foi observado que o emprego da
temperatura mínima testada (115ºC) durante a torração do material em pH mínimo (7,0 –
correspondente ao menor pH alvo na alcalinização testado) resultaram na maior retenção
de compostos fenólicos e maior capacidade antioxidante no produto dentre as condições
testadas (GÜLTEKIN-ÖZGÜVEN; BERKTAŞ; ÖZÇELIK, 2016), confirmando que as
etapas de torração e alcalinização (quando realizada) são as mais impactantes no perfil
de polifenóis durante o processamento das amêndoas de cacau fermentadas e secas
para a produção de liquor de cacau (MAZOR JOLIĆ et al., 2011)
3.3. Formação de sabor no processamento de cacau
O chocolate apresenta sabor único e complexo e o perfil sensorial é resultado da
combinação de componentes voláteis e não voláteis (APROTOSOAIE; LUCA; MIRON,
2016).
16
O perfil de compostos de sabor presentes nos derivados de cacau é influenciado
pela origem e variedade do cacau e pelas condições de processamento (TRAN et al.,
2015), sendo que as etapas de fermentação, torração e conchagem têm impacto
relevante no sabor do chocolate (OWUSU; PETERSEN; HEIMDAL, 2012)
Dentre os componentes não voláteis que influenciam no sabor do cacau e seus
derivados, os de maior destaque são as metilxantinas (teobromina e cafeína) e os
polifenóis, que conferem notas de amargor e adstringência, além das proteínas e
açúcares, diretamente envolvidos na formação de compostos voláteis que caracterizam
o sabor do produto (APROTOSOAIE; LUCA; MIRON, 2016).
Já foram identificados cerca de 600 compostos voláteis componentes do aroma
do cacau, sendo que a maior parte dos compostos voláteis responsáveis pelas
características de sabor do chocolate são derivados dos precursores de aroma gerados
durante o pré-processamento do fruto (fermentação e secagem) e os principais
compostos de sabor são resultantes da reação de Maillard e degradação de Strecker, que
ocorrem principalmente durante a torração das amêndoas (AFOAKWA et al., 2008).
Dentre as classes de compostos voláteis desejáveis e indesejáveis encontradas,
destacam-se as pirazinas, ésteres, aldeídos, cetonas, álcoois e fenóis (APROTOSOAIE;
LUCA; MIRON, 2016).
Durante a fermentação, a microbiota mista de leveduras, bactérias lácticas e
acéticas tem papel fundamental na formação de precursores e dos compostos voláteis
em si. Pelo menos 39 diferentes compostos identificados e relacionados com notas de
sabor específicas estão relacionados com alterações ocasionadas pela fermentação,
como por exemplo alterações de pH que ocorrem principalmente devido à degradação de
ácido cítrico e geração de ácidos láctico e acético (RODRIGUEZ-CAMPOS et al., 2011).
Importantes transformações bioquímicas ocorrem durante as etapas de
fermentação e secagem, como a formação de açúcares redutores e a hidrólise de
proteínas. Ainda durante o pré-processamento, ocorre a oxidação enzimática de
compostos fenólicos a quinonas, que leva à diminuição do amargor e da adstringência.
Os valores de pH e temperatura alcançados devido à atividade microbiana são fatores
determinantes para obtenção de condições ótimas para a atuação das enzimas
responsáveis pelas reações descritas (APROTOSOAIE; LUCA; MIRON, 2016).
Na torração, a exposição de aminoácidos livres e açúcares redutores formados
durante a fermentação a altas temperaturas, induz à reação de Maillard e à degradação
de Strecker, que dão origem aos principais componentes da fração volátil responsáveis
17
pelo sabor do produto, como as pirazinas – responsáveis por mais de 27% dos
componentes identificados e com reconhecida importância sensorial (OWUSU;
PETERSEN; HEIMDAL, 2012).
Durante a conchagem são observados importantes aumentos nos teores de
alguns componentes como pirazinas e diminuição de outros voláteis, como alguns
aldeídos resultantes da degradação de Strecker (COUNET et al., 2002). Além disso, a
conchagem é reconhecida pela contribuição na melhoria do sabor do chocolate em função
da eliminação de compostos responsáveis por sabores residuais não apreciados, como
ácidos livres (AFOAKWA et al., 2008).
18
4. ARTIGO - Flavanols degradation and volatile flavor compounds formation during
the processing of fermented and non-fermented cocoa beans
(O artigo será submetido à revista “Food Research International”)
Authors: FARIA, Flávia Regina de; EFRAIM, Priscilla
Abstract
At the same time that degradation of flavanols has been studied to optimize process conditions
and support the development of chocolates with higher contents of polyphenols, flavor formation
is complex and involves chemical and biochemical transformations that occur during process.
Since extensive degradation of polyphenols is reported on fermentation whilst the formation of
flavor precursors is relevant on this step, the aim of this study was to analyze flavanols
degradation and volatile compounds formation during the processing of fermented and non-
fermented cocoa beans from the same source up to chocolate varying conching times.
Epicatechin, catechin and procyanidin B2 were quantified by HPLC and volatile compounds were
extracted by SPME and analyzed by GC-MS. Sensory evaluation of chocolates was employed to
assess the acceptance of the products. The quantity of flavanols was initially five-fold higher on
non-fermented beans compared to fermented ones and results showed an important loss of
epicatechin and procyanidin B2 during the roasting process. Volatile composition profile from
fermented and non-fermented samples were significantly different and the main flavor-active
compounds were formed during the roasting of fermented beans. Longer conching period at the
same temperature did not cause a significant variation on the flavanols contents but reduction on
volatiles were observed and noticed on the sensory evaluation. Chocolates produced from
unfermented beans were not well accepted mainly because of the astringency and bitterness
caused, probably by the high content of flavanols and the lack of chocolate flavor.
Highlights
• Chocolates from non-fermented and fermented cocoa beans were processed in parallel
and analyzed
• Evolution of epicatechin, procyanidin B2 and catechin contents and volatile compounds
profile during process stages from beans to chocolates were tracked
• Longer conching did not impact flavanols contents but products were different on aroma
profile and sensory perception
Keywords: Unfermented cocoa, epicatechin, conching, chocolate flavor, pyrazines
19
4.1. Introduction
Cocoa is recognized as a relevant source of phenolic compounds and the consumption
of cocoa products has called attention to positive health benefits associated to dietary
flavonoid intake (Cooper, Donovan, Waterhouse, & Williamson, 2008; McShea et al.,
2008), especially regarding to cardiovascular and inflammatory diseases, metabolic
disorders and cancer prevention due to their antioxidant properties (Andújar, Recio, Giner,
& Ríos, 2012). Nevertheless, during cocoa processing and chocolate manufacturing,
important losses and changes are reported on the polyphenols profile (Di Mattia et al.,
2013; Bordiga et al., 2015).
Worldwide, chocolate is appreciated for its unique and complex flavor derived from
various compounds formed from biochemical and chemical reactions during its
processing, which are influenced by cocoa genotype, farming practices, post-harvesting
conditions and manufacturing stages (Aprotosoaie, Luca, & Miron, 2016).
Fermentation is crucial for flavor formation as it provides some volatile compounds
and some precursors for further Maillard reaction (free amino acids and reducing sugars)
in roasting (Afoakwa, Paterson, Fowler, Ryan, & Afoakwa, 2008). However, there is a
relevant reduction of polyphenol content, up to 80 – 90% in the first 48 h of fermentation
(Albertini et al., 2015). During cocoa drying, degradation of phenolic compounds and the
influence of process conditions have been studied (Efraim et al., 2010; Di Mattia et al.,
2013; Teh et al., 2015; Alean, Chejne, & Rojano, 2016).
Heat exposure of cocoa beans during roasting is important for flavor formation as
pyrazines and aldehydes formed by Maillard reaction are reported as the main flavor-
active components on chocolate (Afoakwa et al., 2008). On the other hand, roasting is
responsible for significant loss of total polyphenols, as oxidative processes are
accelerated (Bordiga et al., 2015).
During chocolate manufacturing, conching process also influences the final flavor as
volatile compounds responsible for some off flavors, such as acetic acid, are reduced
(Afoakwa et al., 2008; Owusu, Petersen, & Heimdal, 2012) but some losses on key
components like pyrazines were also previously reported (Albak & Tekin, 2016).
Regarding to the effect on phenolic compounds, previous results show no or little
impact of conching process on polyphenols contents (Di Mattia et al., 2014; Gültekin-
Özgüven, Berktaş, & Özçelik, 2016).
20
Recognizing that chocolate processing from non-fermented cocoa beans could have
a negative impact on the final flavor as the same time that a higher level of phenolic
compounds on the product could be achieved, and that there are few studies that measure
both impacts in parallel, the aim of this study was to analyze flavanols degradation and
volatile flavor compounds formation during processing stages from fermented and non-
fermented cocoa beans to chocolate, varying conching times.
4.2. Material and methods
4.2.1. Cocoa samples and post harvesting process
Samples of fermented and non-fermented cocoa beans were acquired from
Agricola Cantagalo (Bahia, Brazil). A spontaneous fermentation of a 240 kg batch was
carried on the traditional procedure for the region – inside wooden boxes – for a period of
six days. The cocoa mass (seeds with pulp) under fermentation was revolved according
to the variation of its temperature after 46, 70 and 94 hours for oxygenation and mixing.
After the fermentation period, cocoa beans were sun-dried under movable roofs during six
days in a temperature range from 25 to 40°C to a final moisture content of 6%. Apart from
that, non-fermented pulped cocoa seeds from the same harvesting batch were directly
sun-dried under the same conditions until they reached the same moisture content of the
fermented batch (11 days). Temperature evolution of batches on fermentation and drying
stages were recorded.
4.2.2. Cut test
Cocoa beans were visually assessed using the cut test (Wood & Lass, 1985). A
total of 300 beans were cut lengthwise through the middle to expose the maximum cut
surface of the cotyledons. Both halves were examined under artificial light and placed in
one of the following categories: fully brown (fermented); partly brown, partly purple (partly
fermented); purple (under-fermented) or slaty (not fermented). The compartmentation
degree was also evaluated. Results were expressed as a percentage and all analyses
were done triplicate.
21
4.2.3. Processing
Fermented and non-fermented cocoa beans were roasted in a pilot scale rotatory
roaster (JAF Inox, Tambaú, Brazil) for 70min at 120°C and broken into nibs on a knife mill
(ICMA, Campinas, Brazil) with sieves with holes of 6 mm in diameter. Nibs were separated
from shells and germs by a winnower machine (Capco, Ipswich, UK) and ground in a ball
mill (CAO B5, Caotech, Wormerveer, The Netherlands) to produce cocoa liquor.
Two different chocolates were produced from each liquor one made from the
fermented and the other from the non-fermented beans, with the same recipe varying
conching duration (4 h or 16 h). Ingredients (65% liquor, 34.6% sugar and 0.4% soy
lecithin) were mixed and refined on a ball mill (CAO B5, Caotech) up to particle size bellow
25 µm. The conching step was carried out at 70°C on a laboratory conche (CWC 5,
Caotech, Wormerveer, The Netherlands) for 4 or 16 h. Afterwards, chocolate masses
were tempered on a laboratory temperer (Tabletop Temperer, ACMC Chocolate
Tempering Machine) by cooling the mass from 45°C to 27°C under agitation and slightly
heating them until 31°C. Temper index was verified using a temperimeter (ChocoMeter,
Aasted) and values from 4.0 to 6.0 were accepted. The pre-crystalized chocolate mass
was molded into bars, refrigerated for crystallization, removed from molds and stored at
20°C for sensory analysis, and at -18°C for flavanols and volatile compounds
determinations.
4.2.4. Catechin, Epicatechin and Procyanidin B2 quantification - HPLC
Catechin, epicatechin and procyanidin B2 were determined using a High Pressure
Liquid Chromatograph (Shimadzu LC-10, Shimadzu Scientific Instruments, Columbia,
USA) with degasser, quaternary pump (LC-10AT VP), column oven (CTO-10AS VP),
manual injector (Rheodyne model 7725i, with a 20 μL loop), diode array detector SPD-
M20A VP and an interface SCL-10A, operated with the software Class VP Workstation
version 6.14. Samples were defatted using hexane and flavanols extraction was
performed with aqueous methanol as proposed by Machonis, Jones, Schaneberg, Kwik-
Uribe and Dowell (2014). The extract supernatant was filtered through PTFE 0.45 µm
syringe filter (Millipore Corporation, Bedford, USA), diluted on mobile phase (1:1) and
immediately injected into the chromatograph. The separation was performed on Nova-Pak
C18 column (3.9 mm x 150 mm, 4 µm) (Waters, Milford, USA) at a temperature of 35°C,
with isocratic elution of the mobile phase, composed by a 20 mmol L-1 ammonium acetate
22
buffer (pH 4.00 adjusted with glacial acetic acid) and methanol (85:15, v/v) with a flow rate
of 0.8mL min-1. The analytes were quantified by external standard calibration with the peak
areas calculated at 279 nm. All determinations were carried out in triplicate, average
values and standard deviations were calculated and analyses of variation (one-way
ANOVA) followed by Tukey´s test were applied to verify which samples differed from
others (p<0.05).
4.2.5. Volatile aroma profiles – HS-SPME-GC-MS
Volatile compounds of cocoa beans and derivates were extracted by headspace
solid phase microextraction (HS-SPME) as proposed by Ducki, Miralles-Garcia, Zumbé,
Tornero & Storey (2008): sample (2 g) was placed in a 20 mL hermetically sealed vial and
incubated for 10 min at extraction temperature of 60 °C for conditioning, after that, a
divinylbenzene/ carboxen on polydimethylsiloxane on a StableFlex fibre (DVB/CAR/
PDMS SPME) (Supelco, Sigma-Aldrich) was exposed to the headspace for 15 min at 60
°C and desorbed for 5 min at 250 °C in the gas chromatographer liner.
The volatiles extracted were analyzed on a Shimadzu GCMS-QP2010S gas
chromatographer using splitless injection, helium as a carrier gas (2 mL min-1), and a 100m
capillary column with a 0.25 mm (i.d.) and 0.25 µm film thickness (Model CP7420, Agilent
Technologies). The following temperature program was used: start at 40 °C for 5 min,
followed by an increase at 10 °C min-1 to 250 °C and held at 250 °C for 15 min. Injector
and transfer lines were maintained at 250 °C, electron ionization energy was −70 eV and
with a 1200V in the detector. One mass spectra scan every 0.5 s was acquired.
Identification of volatile organic compounds in the headspace was done using US National
Institute of Standards and Technology Mass Spectral Library (NIST08). Three identical
samples were prepared for each analysis and the average results followed by standard
deviation of peak areas for each compound were reported.
4.2.6. Sensory analysis
Four different chocolates produced were submitted to sensory evaluation by an
untrained panel of 70 chocolate consumers, aged between 18 to 52, who were asked
about aroma, chocolate flavor, bitterness, acidity and overall acceptability by giving scores
in a nine-point hedonic scale corresponding to their liking of each attribute evaluated. They
were also asked about buying intention in a five-point scale. ANOVA and Tukey´s test
23
were employed to analyze tabulated results and determine if there was significant
difference (p<0.05) between samples for each attribute. The procedure of sensory
evaluation was previously approved by a Human Research Ethics Committee for human
surveys (CAAE: 76190517.2.0000.5404).
4.3. Results and discussion
The mass (seeds with pulp) temperature data during the fermentation step (batch that
was fermented) is presented in Figure 1.
Fig. 1. Evolution of temperature (●) during fermentation period. Vertical lines indicate when cocoa
mass was revolved
Records of temperature showed a typical behavior during spontaneous cocoa mass
fermentation (Figure 1), according to traditional practices employed in Bahia (Brazil)
(Passos, Lopez, & Silvia, 1984; Papalexandratou, Vrancken, de Bruyne, Vandamme, &
de Vuyst, 2011), thus indicating the expected course of fermentation process. There was
a substantial increase of temperature during the first 40 h of fermentative process, some
decreases after mixing operations were observed and a maximum value of 46.8°C was
found at the end of process.
Cut test indicated that the cotyledons of the cocoa beans presented fermentation
degree in accordance with the expected for this study proposition. Fermented cocoa
sample presented more than 60% of fully brown beans, approximately 30% of partly
24
fermented and less than 5% of purple or slaty (not fermented). These percentages were
similar to those reported by Afoakwa, Quao, Budu, Takrama, & Salia (2012) when
analyzing cocoa beans fermented for 6 days, depending on preconditioning time. The
authors have reported that reductions in the purple beans were noted between the 4th
and 6th days of fermentation at the same time that brown beans were noted to increase.
On the other hand, non-fermented sample was mainly composed by fully purple
beans, 20% showed some degree of fermentation (partly purple, partly brown) and only
6% of beans were fully brown (well-fermented), thus, some incomplete fermentative
process may have occurred during sun-drying period, mainly in the beginning of the step
while high moisture content was present.
Evolution of epicatechin, catechin and procyanidin B2 during processing stages
from cocoa beans to chocolate are presented in Figure 2, and detailed data is presented
in Tables 1 and 2.
(a) (b)
Fig. 2. Contents of catechin (■), epicatechin (■) and procyanidin B2 (■) from different process stages: (a)
non-fermented cocoa beans (NFB), non-fermented roasted cocoa nibs (NFN), cocoa liquor from non-
fermented nibs (NFL), chocolate with 4h of conching from non-fermented liquor (ChNF 4h) and chocolate
with 16h of conching from non-fermented liquor (ChNF 16h); (b) fermented cocoa beans (FB), fermented
roasted cocoa nibs (FN), cocoa liquor from fermented nibs (FL), chocolate with 4h of conching from
fermented liquor (ChF 4h) and chocolate with 16h of conching from fermented liquor (ChF 16h).
Quantifications of selected phenolic compounds showed a degradation tendency
during process stages, particularly for epicatechin content that has been used as an index
0
5
10
15
20
25
30
35
40
45
50
NFB NFN NFL ChNF 4h ChNF 16h
mg.
g-1
0
1
2
3
4
5
6
7
8
9
FB FN FL ChF 4h ChF 16h
mg.
g-1
25
of the processing extent (Camu et al., 2008; Payne, Hurst, Miller, Rank, & Stuart, 2010;
Di Mattia et al., 2013).
Epicatechin and procyanidin B2, which have been previously reported as the main
flavan-3-ol on cocoa beans (Oracz, Nebesny, & Żyżelewicz, 2015; Quiroz-Reyes &
Fogliano, 2018), presented similar decreasing behavior from fermented and non-
fermented samples although contents were initially five-fold higher on non-fermented
sample and seven-fold higher on final chocolate in comparison to those produced from
fermented beans (Figure 2).
In a recent study, Dwijatmoko, Nurtama, Yuliana, & Misnawi (2018) analyzed
polyphenols from various cocoa clones during fermentation and found that unfermented
beans had higher total polyphenols, total flavonoids, epicatechin, and catechin content
than fermented ones; for the cocoa clone with the highest content of phenolic compounds,
they also observed a great decrease of epicatechin (52.5 mg/g for unfermented to 10.5
mg/g for fermented beans) and catechin (2.0 mg/g to 0.68 mg/g) during fermentation;
these epicatechin and catechin contents are similar to the values reached on this study
(Table 1). Although different initial contents of flavanols were found, probably due to
different clones (Dwijatmoko et al., 2018), epicatechin concentration was also reported to
reduce, with more than 70% loss after 144 h of fermentation (Camu et al., 2008).
Substantial decreases (>80%) in catechin and epicatechin levels were similarly observed
in fermented versus unfermented beans and the losses extents were dependent on the
length of fermentation (Payne et al., 2010).
From non-fermented cocoa beans to chocolate (Fig.2a and Table 1) there was a
substantial loss of epicatechin and procyanidin B2 with a major impact during roasting
process as epicatechin and procyanidin B2 levels decreased by 53% and 47%
respectively. On the other hand, catechin has increased by 395% following the results
related by Payne, Hurst, Miller, Rank, & Stuart (2010), who had also found that roasting
(120°C) caused the epicatechin content of unfermented cocoa beans to drop (82% loss)
whereas catechin raised (by 640-696%). This increasing trend of catechin level during
roasting has been associated to a probable epimerization of (-)-epicatechin (Kothe,
Zimmermann, & Galensa, 2013; Ioannone et al., 2015; Oracz et al., 2015; Żyżelewicz et
al., 2016; Quiroz-Reyes & Fogliano, 2018).
Results (Fig. 2b and Table 2) also showed a significant loss of epicatechin (74%)
and procyanidin B2 (69%) during roasting of fermented cocoa beans. Some available data
suggested that within flavanols, the greatest degradations during roasting occur on (-)-
26
epicatechin and procyanidin B2 (Żyżelewicz et al., 2016) and even if the levels of (-)-
epicatechin are reported to decrease in a time and temperature-dependent manner
(Stanley et al., 2018), the extent of the epimerization reaction and the impact on flavanols
contents can vary strongly between different cocoa beans when the same roasting
conditions are employed (Kothe et al., 2013).
However, catechin content did not present the same behavior during fermented
beans processing as a decrease occurred during roasting followed by increase from nibs
to liquor. Żyżelewicz et al. (2016) monitored the contents of (-)-epicatechin, (+)-catechin
and procyanidin B2 before and after a combined grinding-conching process and found
that in most of the previous roasting condition applied, these compounds did not present
significant variation, but in some cases, monomers compounds presented increase or
decrease during grinding-conching stage that could be associated with procyanidins
degradation. In the present study, increase in catechin content during grinding also could
be due to epimerization, or procyanidins degradation as some temperature increase
occurred during grinding process as a result of intense friction in ball mill, but others
phenolic compounds, including epimers should be quantified to confirm that.
Table 1. Contents of flavanols (mg/g) during process stages from non-fermented cocoa beans
Catechin Epicatechin Procyanidin B2
Non-fermented cocoa beans 1.89 + 0.15d 43.45 + 0.94a 15.49 + 0.38a
Non-fermented roasted cocoa nibs 9.37 + 0.17a 20.35 + 0.55b 8.26 + 0.38b
Cocoa liquor from non-fermented nibs 8.17 + 0.18b 18.48 + 0.68c 8.62 + 0.86b
Chocolate with 4h of conching 4.47 + 0.59c 10.37 + 1.68d 4.54 + 0.41c
Chocolate with 16h of conching 4.17 + 0.20c 9.74 + 0.17d 4.19 + 0.26c
Values are expressed as mean ± standard deviation (n=3). Different letters within the same column indicate statistical differences (one-way ANOVA and Tukey’s test, p < 0.05)
Table 2. Contents of flavanols (mg/g) during process stages from fermented cocoa beans
Catechin Epicatechin Procyanidin B2
Fermented cocoa beans 0.63 + 0.08b 8.24 + 0.31ª 2.87 + 0.11ª
Fermented roasted cocoa nibs 0.32 + 0.03d 2.16 + 0.21b 0.90 + 0.08b
Cocoa liquor from fermented nibs 0.75 + 0.04a 2.29 + 0.03b 1.03 + 0.08b
Chocolate with 4h of conching 0.46 + 0.04c 1.50 + 0.12c 0.62 + 0.05c
Chocolate with 16h of conching 0.41 + 0.00c 1.40 + 0.03c 0.53 + 0.01c
Values are expressed as mean ± standard deviation (n=3). Different letters within the same column indicate statistical differences (one-way ANOVA and Tukey’s test, p < 0.05)
27
From cocoa liquor to chocolate there was a decrease on the contents of the three
analyzed flavanols for both fermented and non-fermented resulting products (Tables 1
and 2), but the dilution effect should be taken into account with the addition of sugar
(Bordiga et al., 2015). Considering that chocolate formulation employed 65% of liquor,
there was an average loss of 15% of analyzed flavanols on chocolate manufacturing
stages for non-fermented liquor derivates and less than 10% loss for fermented beans
chocolates. Few studies have focused attention on the effect of conching on polyphenols;
Albak & Tekin (2016) reported 3% of total polyphenols loss during a three-phase conching
for dark chocolate (dry phase: 2h at 50°C, pasty phase: 4h at 80°C and final phase: 1h
with linear decrease in temperature from 80°C to 45°C), and a previous investigation on
procyanidin contents during conching has shown that depending on time-temperature
conditions, there was a little tendency to condensation reaction of procyanidins during
process (Di Mattia et al., 2014).
There was no significant difference between catechin, epicatechin and procyanidin
B2 contents for different conching times tested (4 h and 16 h) at the same temperature
either for chocolates produced from fermented and non-fermented cocoa beans (Tables
1 and 2). A former study reported no effect of conching on phenolic compounds and a
variation on conching temperature (60 °C to 80 °C) did not presented significative
difference on results (Gültekin-Özgüven et al., 2016). Since there is great variation on
conching process conditions and equipment, more studies should be carried out to fully
understand the role of sole conching on polyphenols profile of final chocolate.
Volatile compounds evolution during process from cocoa beans (fermented and
non-fermented) to chocolates were analyzed and results were expressed on peak areas
(Tables 3 and 4), which allows the comparison between samples. As expected, non-
fermented cocoa beans presented different volatile composition compared to fermented
ones. Different fermentation methods and further roasting and conching process
conditions impact volatile flavor compounds formation and levels (Owusu et al., 2012).
The volatile composition of raw cocoa beans is very simple and mainly comprises
alcohols, aldehydes and ketones, during fermentation the total concentration of volatiles
increases considerably (Gill, Macleod, & Moreau, 1984). Esters were previously reported
as one of the main groups of volatile compounds formed during fermentation (Koné et al.,
2016) and the comparison between cocoa beans volatile profiles showed a higher
presence of esters and acids on the fermented sample compared to the unfermented one.
28
Acetic acid presented the highest peak area among volatile compounds from the
fermented cocoa beans and, although the amount of acetic acid decreased during the
processing stages, it also had the highest peak between volatiles found on chocolates
produced from fermented beans. Batista, Ramos, Dias, Pinheiro, & Schwan (2016) also
reported acetic acid as the main volatile acid detected in fermented beans from Bahia
(Brazil) and was also present in chocolate in a lower relative concentration.
The amount of acetic acid on unfermented beans account for only 15% of the
relative quantity presented by the fermented cocoa beans (Tables 3 and 4). Acetic acid is
produced during cocoa fermentation and is an important compound to flavor formation as
its diffusion into the cotyledons stimulates enzymatic reactions that generate flavor
precursors (Afoakwa et al., 2008). Rodriguez-Campos, Escalona-Buendía, Orozco-Avila,
Lugo-Cervantes, & Jaramillo-Flores (2011) reported that the amount of acetic acid
increased in the first two days of fermentation when the greatest amount was found and
then remained high during fermentation and even increased during drying process. As
acetic acid was not reported in unfermented nibs (Ho, Zhao, & Fleet, 2014) the fraction
detected on non-fermented beans could had been formed during drying stage, as long
time sun-drying process was applied. Acetic acid is considered an off-flavor in chocolate
and its reduction during processing is desirable (Ascrizzi, Flamini, Tessieri, & Pistelli,
2017). The roasting and conching steps resulted in important decrease of acetic acid
content on fermented beans process and longer conching period effectively lowered its
levels.
The compound 2,3-butanediol was found in both samples, but the fermented
beans presented a higher relative quantity in comparison to non-fermented ones. Its
derivate, 2,3-butanedione, was only detected on fermented cocoa beans. Volatile alcohols
produced during fermentation were reported as precursors to other compounds, i.e. 2,3-
butanediol to produce 2,3-butanedione (Rodriguez-Campos et al., 2011). The same
authors proposed that the oxidation of 3-methyl-1-butanol to 3-methyl-1-butanol acetate
could be used to evaluated the degree of fermentation. In accordance to that, on this study
it was observed that 3-methyl-butanol was only present on non-fermented cocoa beans
and 3-methyl-1-butanol acetate was present on both fermented and non-fermented
samples, but on higher relative content in the fermented beans and its derived products.
29 Table 3. Volatile compounds found on different process stages from non-fermented cocoa beans to chocolate (peak area x 103)
Compounda NFB NFN NFL ChNF4h ChNF16h Flavor descriptionb
Alcohols
Ethanol 2741 + 161 1152 + 56 967 + 24 553 + 28 491 + 24
3-Buten-2-ol, 2-methyl- 1059 + 57 ND ND ND ND
1-Heptanol, 2-propyl- ND 351 + 4 319 + 10 ND ND 2-Pentanol 29523 + 1824 23209 + 1516 21421 + 1730 540 + 22 176 + 16 Green, mild green
2,6-Octadien-1-ol, 2,7-dimethyl- ND 175 + 10 184 + 17 ND ND
1-Butanol, 3-methyl- 1821 + 90 1409 + 42 1242 + 67 ND ND Malty
2-Pentanol, 4-methyl- 517 + 32 514 + 2 491 + 30 ND ND
1-Pentanol 145 + 12 168 + 11 151 + 13 57 + 2 56 + 2 Wizened
2-Heptanol 18317 + 1180 19681 + 394 18674 + 964 3797 + 11 202 + 8 Fruity
3-Ethyl-2-pentanol 1067 + 9 843 + 30 889 + 75 ND ND
2-Nonanol 1095 + 37 1461 + 97 1187 + 97 742 + 20 171 + 12
2,3-Butanediol 3000 + 158 1062 + 76 2383 + 57 495 + 40 ND Sweet chocolate
4-Nonanol ND 156 + 8 52 + 5 ND ND
alpha-Methylbenzyl alcohol 55 + 4 58 + 3 52 + 4 34 + 1 ND
Phenylethyl Alcohol 876 + 34 966 + 74 757 + 25 495 + 5 197 + 6 Honey, floral
Aldehydes
Butanal, 2-methyl- 227 + 8 1830 + 111 1339 + 133 ND ND Sweet chocolate
Butanal, 3-methyl- ND ND ND 115 + 8 112 + 2 Sweet chocolate
Pentanal ND ND ND 81 + 3 95 + 1
Hexanal ND ND ND 81 + 4 52 + 5
Ketones
Acetone 552 + 12 ND ND ND ND
2-Butanone 162 + 7 ND ND ND ND
2-Pentanone 17017 + 745 15046 + 1072 11347 + 1045 184 + 1 103 + 8 Fruity
2,3-Pentanedione ND 383 + 13 206 + 15 ND ND Bitter 2-Hexanone, 4-hydroxy-5-methyl-3-propyl- 241 + 6 218 + 12 219 + 4 ND ND
3-Penten-2-one ND 290 + 10 185 + 8 ND ND
2-Hexanone, 4-methyl- 139 + 13 154 + 6 152 + 6 41 + 2 ND
2,3-Octanedione ND 1197 + 102 608 + 50 ND ND
2-Nonanone 2611 + 150 3997 + 190 2977 + 238 1553 + 33 189 + 8
Acetophenone 217 + 6 265 + 20 222 + 14 113 + 3 ND Floral
3,6-Heptanedione 68 + 7 ND ND ND ND
Acids
30
Acetic acid 15715 + 1091 18105 + 317 19931 + 769 4344 + 189 3373 + 249 Sour, vinegar-like
Esters
Acetic acid, methyl ester ND 1417 + 72 1430 + 86 ND ND
1-Propen-2-ol, acetate ND 684 + 24 745 + 22 ND ND
Ethyl Acetate 999 + 67 1025 + 74 715 + 57 ND ND Fruity, pineapple
1-Butanol, 3-methyl-, acetate 514 + 27 623 + 12 636 + 26 ND ND
Vinyl butyrate ND 779 + 39 810 + 50 ND ND
Butanedioic acid, 2,3-bis(acetyloxy)- ND 574 + 20 605 + 34 241 + 18 127 + 10
1-Butanol, 3-methyl-, benzoate 55 + 2 71 + 5 62 + 2 49 + 1 42 + 0 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate ND 509 + 21 440 + 14 244 + 14 203 + 14 1,2-Benzenedicarboxylic acid, diheptyl ester 550 + 16 344 + 13 339 + 12 200 + 9 149 + 6
Pyrazines
Pyrazine, 2,5-dimethyl- ND 523 + 26 343 + 28 ND ND Roasted, nutty
Furans, furanones, pyrans, pyrones
Furan, tetrahydro-2-methyl- ND 64 + 4 54 + 3 ND ND
3(2H)-Furanone, dihydro-2-methyl- ND 367 + 12 292 + 19 ND ND
Furfural ND 1078 + 74 609 + 19 ND ND Almond, nutty
2-Furanmethanol ND 133 + 3 140 + 5 37 + 1 32 v 1
2H-Pyran-2-one, tetrahydro- ND 35 + 3 23 + 2 ND ND 2(3H)-Furanone, dihydro-3-hydroxy-4,4-dimethyl-, ND 117 + 4 115 + 2 48 + 3 ND
Butyrolactone 1221 + 80 1538 + 116 1738 + 69 686 + 34 257 + 9
Pyrroles
Ethanone, 1-(1H-pyrrol-2-yl)- ND 50 + 4 37 + 1 23 + 0 ND
Amides
Methacrylamide ND 37 + 1 52 + 5 ND ND
3-Butenamide ND ND ND 35 + 2 37 + 2
Others
Dimethyl sulfide 47 + 2 341 + 17 181 + 5 ND ND
Ethanol, 2-(2-ethoxyethoxy)- 90 + 7 1125 + 100 1134 + 61 817 + 56 499 + 27 a Compounds tentatively identified by comparison of mass spectra to NIST08 library b Flavor description from literature matches (Rodriguez-Campos et al., 2011; Tran, et al., 2015; Aprotosoaie et al., 2016)
Values are expressed as mean ± standard deviation (n=3). Non-fermented cocoa beans (NFB), non-fermented roasted cocoa nibs (NFN), cocoa liquor from non-
fermented nibs (NFL), chocolate with 4h of conching from non-fermented liquor (ChNF 4h) and chocolate with 16h of conching from non-fermented liquor (ChNF
16h)
31 Table 4. Volatile compounds found on different process stages from fermented cocoa beans to chocolate (peak area x 103)
Compounda FB FN FL ChF4h ChF16h Flavor descriptionb
Alcohols
Ethanol 399 + 39 372 + 33 372 + 40 191 + 13 171 + 14
2-Pentanol 817 + 36 ND ND ND ND Green, mild green
2-Heptanol 3023 + 103 5558 + 379 7562 + 530 2138 + 119 ND Fruity
2-Heptanol, 3-methyl- 446 + 19 ND ND ND ND
2-Nonanol ND 818 + 51 932 + 49 782 + 50 163 + 10
2,3-Butanediol 9181 + 354 5477 + 182 13866 + 338 4437 + 109 901 + 48 Sweet chocolate
alpha-Methylbenzyl alcohol ND 68 + 1 72 + 3 55 + 1 ND
Phenylethyl Alcohol 477 + 23 472 + 33 480 + 14 343 + 5 335 + 6 Honey, floral
Aldehydes
Propanal, 2-methyl- ND 487 + 23 490 + 25 ND ND Sweet chocolate
Butanal, 3-methyl- 273 + 7 4741 + 263 5183 + 159 286 + 10 287 + 12 Sweet chocolate
Ketones
Acetone 122 + 7 ND ND ND ND
2-Heptanone, 3-methyl- ND 200 + 17 201 + 17 ND ND
2,3-Butanedione 1095 + 75 586 + 56 549 + 38 171 + 8 148 + 10 Buttery
2-Pentanone ND 388 + 7 258 + 7 ND ND Fruity
2,3-Pentanedione ND 142 + 9 130 + 7 ND ND Bitter
2-Pentanone, 4-hydroxy- ND 1542 + 10 2215 + 120 ND ND
2-Butanone, 3-hydroxy- 15750 + 1060 6404 + 77 6420 + 95 962 + 56 583 + 32
2-Nonanone 965 + 58 2329 + 123 2416 + 145 1686 + 69 239 + 20
Acetophenone ND 174 + 1 182 + 10 115 + 5 ND Floral
Acids
Acetic acid 100546 + 4273 69214 + 3133 68512 + 3042 30659 + 1803 15520 + 981 Sour, vinegar-like
Propanoic acid, 2-methyl- 1026 + 33 ND ND ND ND Floral 2-Acetylamino-3-hydroxy-propionic acid 226 + 11 ND ND ND ND
Butanoic acid, 3-methyl- 770 + 23 435 + 29 462 + 23 ND ND
Esters
Acetic acid, methyl ester 694 + 27 914 + 89 932 + 50 ND ND
Ethyl Acetate 1711 + 22 1607 + 133 1589 + 41 ND ND Fruity, pineapple
Acetic acid, butyl ester 130 + 8 707 + 53 702 + 57 ND ND Fruity
2-Pentanol, acetate ND 2786 + 114 2581 + 94 181 + 17 ND
1-Butanol, 3-methyl-, acetate 871 + 42 4393 + 137 4253 + 198 379 + 21 ND
Butanedioic acid, 2,3-bis(acetyloxy)- ND 1137 + 22 1162 + 47 311 + 10 180 + 11
32 Pentanoic acid, 2-hydroxy-4-methyl-, methyl ester ND 245 + 13 265 + 9 ND ND
3-Hydroxy-2-butanone, acetate 2392 + 96 2908 + 98 4181 + 111 ND ND
2-Furanmethanediol, dipropionate ND 161 + 14 160 + 13 ND ND
1-Methoxy-2-propyl acetate 8466 + 460 8960 + 142 12302 + 388 5445 + 237 281 + 18
Acetic acid, 2-phenylethyl ester 127 + 12 273 + 3 292 + 27 241 + 7 133 + 10 Honey, floral 1,2-Benzenedicarboxylic acid, diheptyl ester 478 + 20 554 + 19 435 + 24 359 + 22 248 + 9
Pyrazines
Pyrazine, 2,5-dimethyl- ND 826 + 34 780 + 48 168 + 4 ND Roasted, nutty
Pyrazine, 2,3-dimethyl- ND 396 + 1 406 + 3 117 + 2 ND Caramel, sweet chocolate
Pyrazine, trimethyl- ND 3466 + 49 3314 + 205 1541 + 109 74 + 3 Roasted, nutty
Pyrazine, tetramethyl- ND 1773 + 91 2106 + 74 1473 + 58 178 + 9 Sweet chocolate
2,3,5-Trimethyl-6-ethylpyrazine ND 250 + 4 243 + 23 194 + 4 45 + 3 Sweet chocolate
Furans, furanones, pyrans, pyrones
2-Furanmethanol ND 198 + 8 274 + 10 81 + 2 74 + 2 2,5-Dimethyl-4-hydroxy-3(2H)-furanone ND 182 + 1 186 + 12 104 + 2 ND Fruity, nutty
Butyrolactone 428 + 8 704 + 32 746 + 4 319 + 6 253 + 3 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- ND 528 + 23 501 + 43 292 + 15 ND 2(3H)-Furanone, dihydro-3-hydroxy-4,4-dimethyl-, ND 155 + 5 156 + 4 149 + 11 76 + 4 Coconut, nutty
Pyrroles
Ethanone, 1-(1H-pyrrol-2-yl)- ND 344 + 26 332 + 18 222 + 8 91 + 6
Amides
Methacrylamide ND 42 + 3 43 + 3 37 + 2 35 + 2
Others
Dimethyl sulfide 45 + 3 154 + 14 99 + 8 ND ND
Propanenitrile, 3-(1-methylethoxy)- ND 983 + 12 1063 + 51 556 + 17 ND
Ethanol, 2-(2-ethoxyethoxy) ND 644 + 58 683 + 43 550 + 44 467 + 44 1,3,4-Oxadiazole, 2-(acetyloxy)-2,5-dihydro-2,5,5-trimethyl- ND 465 + 9 557 + 42 434 + 29 74 + 3
a Compounds tentatively identified by comparison of mass spectra to NIST08 library b Flavor description from literature matches (Rodriguez-Campos et al., 2011; Tran, et al., 2015; Aprotosoaie et al., 2016)
Values are expressed as mean ± standard deviation (n=3). Fermented cocoa beans (FB), fermented roasted cocoa nibs (FN), cocoa liquor from fermented nibs
(FL), chocolate with 4h of conching from fermented liquor (ChF 4h) and chocolate with 16h of conching from fermented liquor (ChF 16h)
33
A negative correlation between procyanidins content and volatile compounds
(some pyrazines) formation during roasting has been previously reported (Counet,
Ouwerx, Rosoux, & Collin, 2004) and the obtained results were in agreement to that, as
non-fermented beans with high contents of procyanidins showed a deficient formation of
pyrazines during roasting.
Roasting plays an important role as substantial changes on individual contents are
reported, some components are lost and others are formed, notably pyrazines (Gill et al.,
1984; Ho et al., 2014). Although some previous studies found formation of pyrazines
during fermentation (Puziah, Jinap, Sharifah, & Asbi, 1998), on the collected results they
were only detected after roasting, but in the unfermented and roasted beans it was just
observed 2,5 dimethyl pyrazine while there were four other pyrazines on the fermented
and roasted nibs (Figure 3).
Three Strecker aldehydes with strong chocolate notes previously reported, 2-
methylpropanal, 3-methylbutanal and 2-methylbutanal, respectively derived from valine,
leucine and isoleucine (Counet, Callemien, Ouwerx, & Collin, 2002) were detected on the
fractions of different step process analyzed; 2-methylbutanal was only present in
unfermented cocoa beans, increased during roasting and was lost during conching; 2-
methylpropanal emerged on roasting of fermented beans and was also lost during
conching, on the other hand 3-methylbutanal was present in all fractions during fermented
beans process to chocolates and was also found in a smaller relative quantity on
chocolates produced from unfermented cocoa. Although conched chocolate from
fermented cocoa had significant smaller peak area of 3-methylbutanal, longer conching
did not vary its content, therefore for this aldehyde, the first four hours of conching had
more impact on its amount than the continuing period.
Cocoa liquors presented almost the same volatile composition as the respective
roasted nibs, but the volatile composition of derived chocolates indicated that the
manufacturing stages from liquor to chocolate caused a decrease in number and relative
content of volatile compounds. When a longer conching was employed, a decreasing
tendency on volatiles was observed even for some flavor-active chocolate components
(Figure 3). Albak & Tekin (2016) also reported differences in number and levels of aroma
compounds during conching step and a decrease in pyrazines during the role process, but
some components increased and some were formed in the course of conching, which was
not observed in this study. Since there is an important variation on conching methods and
conditions, more studies regarding conching effect on volatile composition should be done
34
to understand the role of conching conditions on volatile components on chocolate
manufacturing.
Fig. 3. Peak areas of pyrazines from different process stages: fermented cocoa beans (FB), fermented
roasted cocoa nibs (FN), cocoa liquor from fermented nibs (FL), chocolate with 4h of conching from
fermented liquor (ChF 4h) and chocolate with 16h of conching from fermented liquor (ChF 16h).
Sensory evaluation of chocolate produced from non-fermented cocoa showed low
acceptability and no statistical difference on liking between two conching extents analyzed
for all five attributes (Table 5), following that, buying intention was very low as around 90%
of respondents stated they would not buy those products.
Flavanols are recognized as bitter and astringent (Serra Bonvehí & Ventura Coll,
1997) and unfermented and partly fermented cocoa beans were previously associated
with excessively astringent and bitter taste due to the high polyphenol content (Misnawi,
Selamat, Bakar, & Saari, 2002). Misnawi, Jinap, Jamilah, & Nazamid (2004) found that a
high content of polyphenols prior to roasting significantly decreased the intensity of
perceived cocoa flavor of the resultant liquor, and they proposed a possible binding effect
of polyphenol on aroma precursors and aroma compounds formed during roasting, as a
result of lower contents of free amino acids and reducing sugars, with the increase in
polyphenol concentration and/ or sensory interference from its strong astringent and bitter
sensations.
0
500
1000
1500
2000
2500
3000
3500
4000
FB FN FL ChF 4h ChF 16h
Pea
k ar
ea x
10
3
Pyrazine, 2,5-dimethyl- Pyrazine, 2,3-dimethyl- Pyrazine, trimethyl-
Pyrazine, tetramethyl- 2,3,5-Trimethyl-6-ethylpyrazine
35
Table 5. Results of sensory evaluation of chocolates by an untrained panel of 70 consumers
Attribute ChNF 4h ChNF 16h ChF 4h ChF 16h
Aroma 5.4 c 5.8 c 7.5 a 6.8 b
Chocolate flavor 3.0 b 3.1 b 6.2 a 6.1 a
Bitterness 2.7 b 3.0 b 6.0 a 6.4 a
Acidity 3.3 c 3.2 c 4.9 b 5.6 a
Overall acceptability 2.7 b 2.9 b 6.1 a 6.2 a Results are expressed in terms of mean values on nine-point liking score. Different letters within
the same line indicate statistical differences (p < 0.05). Chocolate from non-fermented cocoa with
4h of conching (ChNF 4h) and with 16h of conching (ChNF 16h); chocolate from fermented cocoa
with 4h of conching (ChF 4h) and with 16h of conching (ChF 16h).
In contrast, chocolates produced from fermented beans were better graded for the
same panel and differences on aroma and acidity were perceived when distinct conching
periods were employed. Acidity was better rated when longer conching was applied in
accordance to results previously discussed of volatile composition, as chocolate conched
for 16h showed a smaller relative content for acetic acid. For aroma evaluation, when a
shorter conching process was performed, acceptability increased, also in line with volatile
profile results that showed a more complex composition for this product.
4.4. Conclusions
Fermented and non-fermented sun-dried cocoa beans presented different
flavanols contents and volatile profile compositions, reaffirming the role of fermentation on
the degradation of polyphenols and the formation of volatile compounds.
Processing of fermented and non-fermented cocoa beans for chocolate resulted
on a similar negative percentual impact on the content of epicatechin and procyanidin B2;
during roasting the main decrease on those flavanols levels and the major changes on
volatile compounds profiles were detected. There was no difference on flavanols contents
when chocolates where conched for 4 or 16 h; nevertheless, volatile compounds showed
smaller relative quantities when a longer conching process was employed, which caused
a distinctive sensory perception of aroma and acidity on chocolates produced from
fermented cocoa.
Cocoa flavanols have called attention for their possible health benefits associated
to antioxidant properties, and even though chocolates produced from unfermented cocoa
beans had seven-fold higher content of epicatechin and procyanidin B2 when compared
36
to their fermented cocoa beans counterpart, they were not well accepted on a sensory
evaluation due to their intense bitterness and astringency and lack of chocolate flavor
perception, which is consistent with high polyphenols contents and volatile composition
results.
Acknowledgments
The authors thank to Faculdade de Tecnologia SENAI “Horácio Augusto da Silveira”, where
part of experimental work was done and CAPES for the financial support. This study was
financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –
Brasil (CAPES) – Finance Code 001.
37
References
Afoakwa, E. O., Paterson, A., Fowler, M., & Ryan, A. (2008). Flavor formation and character
in cocoa and chocolate: A critical review. Critical Reviews in Food Science and Nutrition,
48(9), 840–857. https://doi.org/10.1080/10408390701719272
Afoakwa, E. O., Quao, J., Budu, A. S., Takrama, J. F., & Salia, F. K. (2012). Influence of
pulp-preconditioning and fermentation on fermentative quality and appearance of Ghanaian
cocoa (Theobroma cacao) beans. International Food Research Journal, 19(1), 127–133.
Albak, F., & Tekin, A. R. (2016). Variation of total aroma and polyphenol content of dark
chocolate during three phase of conching. Journal of Food Science and Technology, 53(1),
848–855. https://doi.org/10.1007/s13197-015-2036-4
Albertini, B., Schoubben, A., Guarnaccia, D., Pinelli, F., Della Vecchia, M., Ricci, M., …
Blasi, P. (2015). Effect of Fermentation and Drying on Cocoa Polyphenols. Journal of
Agricultural and Food Chemistry, 63(45), 9948–9953.
https://doi.org/10.1021/acs.jafc.5b01062
Alean, J., Chejne, F., & Rojano, B. (2016). Degradation of polyphenols during the cocoa
drying process. Journal of Food Engineering, 189, 99–105.
https://doi.org/10.1016/j.jfoodeng.2016.05.026
Andújar, I., Recio, M. C., Giner, R. M., & Ríos, J. L. (2012). Cocoa polyphenols and their
potential benefits for human health. Oxidative Medicine and Cellular Longevity, 2012.
https://doi.org/10.1155/2012/906252
Aprotosoaie, A. C., Luca, S. V., & Miron, A. (2016). Flavor Chemistry of Cocoa and Cocoa
Products-An Overview. Comprehensive Reviews in Food Science and Food Safety, 15(1),
73–91. https://doi.org/10.1111/1541-4337.12180
Ascrizzi, R., Flamini, G., Tessieri, C., & Pistelli, L. (2017). From the raw seed to chocolate:
Volatile profile of Blanco de Criollo in different phases of the processing chain.
Microchemical Journal, 133, 474–479. https://doi.org/10.1016/j.microc.2017.04.024
Batista, N. N., Ramos, C. L., Dias, D. R., Pinheiro, A. C. M., & Schwan, R. F. (2016). The
impact of yeast starter cultures on the microbial communities and volatile compounds in
cocoa fermentation and the resulting sensory attributes of chocolate. Journal of Food
Science and Technology, 53(2), 1101–1110. https://doi.org/10.1007/s13197-015-2132-5
38
Bordiga, M., Locatelli, M., Travaglia, F., Coïsson, J. D., Mazza, G., & Arlorio, M. (2015).
Evaluation of the effect of processing on cocoa polyphenols: Antiradical activity,
anthocyanins and procyanidins profiling from raw beans to chocolate. International Journal
of Food Science and Technology, 50(3), 840–848. https://doi.org/10.1111/ijfs.12760
Camu, N., De Winter, T., Addo, S. K., Takrama, J. S., Bernaert, H., & De Vuyst, L. (2008).
Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations
on the flavour of chocolate. Journal of the Science of Food and Agriculture, 88(13), 2288–
2297. https://doi.org/10.1002/jsfa.3349
Cooper, K. a, Donovan, J. L., Waterhouse, A. L., & Williamson, G. (2008). Cocoa and
health: a decade of research. The British Journal of Nutrition, 99(1), 1–11.
https://doi.org/10.1017/S0007114507795296
Counet, C., Callemien, D., Ouwerx, C., & Collin, S. (2002). Use of Gas
Chromatography−Olfactometry To Identify Key Odorant Compounds in Dark Chocolate.
Comparison of Samples before and after Conching. Journal of Agricultural and Food
Chemistry, 50(8), 2385–2391. https://doi.org/10.1021/jf0114177
Counet, C., Ouwerx, C., Rosoux, D., & Collin, S. (2004). Relationship between procyanidin
and flavor contents of cocoa liquors from different origins. Journal of Agricultural and Food
Chemistry, 52(20), 6243–6249. https://doi.org/10.1021/jf040105b
Di Mattia, C., Martuscelli, M., Sacchetti, G., Beheydt, B., Mastrocola, D., & Pittia, P. (2014).
Effect of different conching processes on procyanidin content and antioxidant properties of
chocolate. Food Research International, 63, 367–372.
https://doi.org/10.1016/j.foodres.2014.04.009
Di Mattia, C., Martuscelli, M., Sacchetti, G., Scheirlinck, I., Beheydt, B., Mastrocola, D., &
Pittia, P. (2013). Effect of Fermentation and Drying on Procyanidins, Antiradical Activity and
Reducing Properties of Cocoa Beans. Food and Bioprocess Technology, 6(12), 3420–3432.
https://doi.org/10.1007/s11947-012-1028-x
Ducki, S., Miralles-Garcia, J., Zumbé, A., Tornero, A., & Storey, D. M. (2008). Evaluation of
solid-phase micro-extraction coupled to gas chromatography-mass spectrometry for the
headspace analysis of volatile compounds in cocoa products. Talanta, 74(5), 1166–1174.
https://doi.org/10.1016/j.talanta.2007.08.034
39
Dwijatmoko, M. I., Nurtama, B., Yuliana, N. D., & Misnawi. (2018). Characterization of
Polyphenols from Various Cocoa ( Theobroma cacao L .) Clones During Fermentation.
Pelita Perkebunan, 34(2), 104–112.
Efraim, P., Pezoa-García, N. H., Jardim, D. C. P., Nishikawa, A., Haddad, R., & Eberlin, M.
N. (2010). Influência da fermentação e secagem de amêndoas de cacau no teor de
compostos fenólicos e na aceitação sensorial. Ciência e Tecnologia de Alimentos, 30, 142–
150. https://doi.org/10.1590/S0101-20612010000500022
Gill, M. S., Macleod, A. J., & Moreau, M. (1984). Volatile components of cocoa with
particular reference to glucosinolate products. Phytochemistry, 23(9), 1937–1942.
https://doi.org/10.1016/S0031-9422(00)84945-6
Gültekin-Özgüven, M., Berktaş, İ., & Özçelik, B. (2016). Influence of processing conditions
on procyanidin profiles and antioxidant capacity of chocolates: Optimization of dark
chocolate manufacturing by response surface methodology. LWT - Food Science and
Technology, 66, 252–259. https://doi.org/10.1016/j.lwt.2015.10.047
Ho, V. T. T., Zhao, J., & Fleet, G. (2014). Yeasts are essential for cocoa bean fermentation.
International Journal of Food Microbiology, 174, 72–87.
https://doi.org/10.1016/j.ijfoodmicro.2013.12.014
Ioannone, F., Di Mattia, C. D., De Gregorio, M., Sergi, M., Serafini, M., & Sacchetti, G.
(2015). Flavanols, proanthocyanidins and antioxidant activity changes during cocoa
(Theobroma cacao L.) roasting as affected by temperature and time of processing. Food
Chemistry, 174, 256–262. https://doi.org/10.1016/j.foodchem.2014.11.019
Koné, M. K., Guéhi, S. T., Durand, N., Ban-Koffi, L., Berthiot, L., Tachon, A. F., … Montet,
D. (2016). Contribution of predominant yeasts to the occurrence of aroma compounds
during cocoa bean fermentation. Food Research International, 89, 910–917.
https://doi.org/10.1016/j.foodres.2016.04.010
Kothe, L., Zimmermann, B. F., & Galensa, R. (2013). Temperature influences epimerization
and composition of flavanol monomers, dimers and trimers during cocoa bean roasting.
Food Chemistry, 141(4), 3656–3663. https://doi.org/10.1016/j.foodchem.2013.06.049
40
Machonis, P., Jones, M., Schaneberg, B., Kwik-Uribe, C., & Dowell, D. (2014). Method for
the Determination of Catechin and Epicatechin Enantiomers in Cocoa-Based Ingredients
and Products by High-Performance Liquid Chromatography: First Action 2013.04. Journal of
AOAC International, 97(2), 506–509. https://doi.org/https://doi.org/10.5740/jaoacint.13-351
McShea, A., Ramiro-Puig, E., Munro, S. B., Casadesus, G., Castell, M., & Smith, M. A.
(2008). Clinical benefit and preservation of flavonols in dark chocolate manufacturing.
Nutrition Reviews, 66(11), 630–641. https://doi.org/10.1111/j.1753-4887.2008.00114.x
Misnawi, A., Jinap, S., Jamilah, B., & Nazamid, S. (2004). Sensory properties of cocoa liquor
as affected by polyphenol concentration and duration of roasting. Food Quality and
Preference, 15(5), 403–409. https://doi.org/10.1016/S0950-3293(03)00097-1
Misnawi, Selamat, J., Bakar, J., & Saari, N. (2002). Oxidation of polyphenols in unfermented
and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase. Journal of
the Science of Food and Agriculture, 82(5), 559–566. https://doi.org/10.1002/jsfa.1075
Oracz, J., Nebesny, E., & Żyżelewicz, D. (2015). Changes in the flavan-3-ols, anthocyanins,
and flavanols composition of cocoa beans of different Theobroma cacao L. groups affected
by roasting conditions. European Food Research and Technology, 241(5), 663–681.
https://doi.org/10.1007/s00217-015-2494-y
Owusu, M., Petersen, M. A., & Heimdal, H. (2012). Effect of fermentation method, roasting
and conching conditions on the aroma volatiles of dark chocolate. Journal of Food
Processing and Preservation, 36(5), 446–456. https://doi.org/10.1111/j.1745-
4549.2011.00602.x
Papalexandratou, Z., Vrancken, G., de Bruyne, K., Vandamme, P., & de Vuyst, L. (2011).
Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a
restricted species diversity of lactic acid bacteria and acetic acid bacteria. Food
Microbiology, 28(7), 1326–1338. https://doi.org/10.1016/j.fm.2011.06.003
Passos, F. M. L., Lopez, A. S., & Silvia, D. O. (1984). Aeration and its influence on the
microbial sequence in cacao fermentations in Bahia. Journal of Food Science, 49, 1470–
1474.
41
Payne, M. J., Hurst, W. J., Miller, K. B., Rank, C., & Stuart, D. A. (2010). Impact of
Fermentation , Drying , Roasting , and Dutch Processing on Epicatechin and Catechin
Content of Cacao Beans and Cocoa Ingredients. Journal of Agricultural and Food
Chemistry, 58, 10518–10527. https://doi.org/10.1021/jf102391q
Puziah, H., Jinap, S., Sharifah, K., & Asbi, A. (1998). Changes in Free Amino Acid, Peptide-
N, Sugar and Pyrazine Concentration during Cocoa Fermentation. J Sci Food Agric, 78,
535–542.
Quiroz-Reyes, C. N., & Fogliano, V. (2018). Design cocoa processing towards healthy cocoa
products : The role of phenolics and melanoidins. Journal of Functional Foods, 45(January),
480–490. https://doi.org/10.1016/j.jff.2018.04.031
Rodriguez-Campos, J., Escalona-Buendía, H. B., Orozco-Avila, I., Lugo-Cervantes, E., &
Jaramillo-Flores, M. E. (2011). Dynamics of volatile and non-volatile compounds in cocoa
(Theobroma cacao L.) during fermentation and drying processes using principal
components analysis. Food Research International, 44(1), 250–258.
https://doi.org/10.1016/j.foodres.2010.10.028
Serra Bonvehí, J., & Ventura Coll, F. (1997). Evaluation of bitterness and astringency of
polyphenolic compounds in cocoa powder. Food Chemistry, 60(3), 365–370.
https://doi.org/10.1016/S0308-8146(96)00353-6
Stanley, T. H., Buiten, C. B. Van, Baker, S. A., Elias, R. J., Anantheswaran, R. C., &
Lambert, J. D. (2018). Impact of roasting on the fl avan-3-ol composition , sensory-related
chemistry , and in vitro pancreatic lipase inhibitory activity of cocoa beans. Food Chemistry,
255, 414–420. https://doi.org/10.1016/j.foodchem.2018.02.036
Teh, Q. T. M., Tan, G. L. Y., Loo, S. M., Azhar, F. Z., Menon, A. S., & Hii, C. (2015). The
Drying Kinetics and Polyphenol Degradation of Cocoa Beans. Journal of Food Process
Engineering, 1–8. https://doi.org/10.1111/jfpe.12239
Tran, P. D., Van de Walle, D., De Clercq, N., De Winne, A., Kadow, D., Lieberei, R., … Van
Durme, J. (2015). Assessing cocoa aroma quality by multiple analytical approaches. Food
Research International, 77, 657–669. https://doi.org/10.1016/j.foodres.2015.09.019
Wood, G. A. R., & Lass, R. A. (1985). Cocoa. (G. A. R. Wood & R. A. Lass, Eds.) (Fourth).
Oxford, UK: Blackwell Science Ltd. https://doi.org/10.1002/9780470698983
42
Żyżelewicz, D., Krysiak, W., Oracz, J., Sosnowska, D., Budryn, G., & Nebesny, E. (2016).
The influence of the roasting process conditions on the polyphenol content in cocoa beans ,
nibs and chocolates. Food Research International, 89(2), 918–929.
https://doi.org/https://doi.org/10.1016/j.foodres.2016.03.026
43
5. CONCLUSÃO GERAL
As amêndoas de cacau não fermentadas e secas ao sol apresentaram uma
composição de voláteis e níveis de flavanóis distintos das amêndoas fermentadas obtidas
do mesmo lote de frutos e secas sob as mesmas condições, reafirmando a importância
da fermentação na formação de sabor e na degradação de polifenóis.
O processamento das amêndoas, fermentadas e não fermentadas, para a
produção de chocolate resultou em diminuição percentual similar nos teores de
epicatequina e procianidina B2, e, dentre as etapas, a torra foi responsável pelo maior
impacto tanto na degradação de flavanóis como na formação de compostos voláteis.
Não houve diferença significativa nos teores dos polifenóis analisados entre os
chocolates que foram submetidos à 4 ou 16 h de conchagem sob as mesmas condições;
porém foi observada menor quantidade relativa dos compostos voláteis quando a
conchagem prolongada foi empregada e essa diferença foi percebida na avaliação
sensorial dos chocolates produzidos a partir de amêndoas fermentadas.
A presença de flavanóis no cacau e seus derivados tem aumentado o interesse
em relação aos possíveis benefícios à saúde associados à sua capacidade antioxidante
e, apesar dos chocolates produzidos a partir de amêndoas não fermentadas terem
apresentado teores de epicatequina e de procianidina B2 sete vezes superiores àqueles
encontrados em chocolates produzidos em paralelo com amêndoas fermentadas, os
produtos não foram bem aceitos em avaliação sensorial, devido à alta percepção de
amargor e adstringência conferida pelos polifenóis e à falta de reconhecimento do sabor
de chocolate. Os resultados da análise sensorial foram condizentes com os perfis de
compostos voláteis obtidos.
Assim, sugere-se que condições intermediárias de processo sejam estudadas e
que os derivados do processamento de amêndoas não fermentadas de cacau, como o
liquor, poderiam ser empregados parcialmente em formulações de alimentos, com o
intuito de aumentar os teores de flavanóis sem comprometer totalmente a percepção
sensorial dos produtos.
44
REFERÊNCIAS GERAIS
AFOAKWA, E. O. et al. Flavor formation and character in cocoa and chocolate: A critical review.
Critical Reviews in Food Science and Nutrition, v. 48, n. 9, p. 840–857, 2008.
AFOAKWA, E. O. et al. Influence of pulp-preconditioning and fermentation on fermentative
quality and appearance of Ghanaian cocoa (Theobroma cacao) beans. International Food
Research Journal, v. 19, n. 1, p. 127–133, 2012.
AFOAKWA, E. O. et al. Roasting effects on phenolic content and free-radical scavenging
activities of pulp preconditioned and fermented cocoa (Theobroma cacao) beans. African
Journal of Food, Agriculture, Nutrition and Development, v. 15, n. 1, p. 9635–9650, 2015.
ALBAK, F.; TEKIN, A. R. Variation of total aroma and polyphenol content of dark chocolate
during three phase of conching. Journal of Food Science and Technology, v. 53, n. 1, p.
848–855, 2016.
ALBERTINI, B. et al. Effect of Fermentation and Drying on Cocoa Polyphenols. Journal of
Agricultural and Food Chemistry, v. 63, n. 45, p. 9948–9953, 2015.
ALEAN, J.; CHEJNE, F.; ROJANO, B. Degradation of polyphenols during the cocoa drying
process. Journal of Food Engineering, v. 189, p. 99–105, 2016.
ANDÚJAR, I. et al. Cocoa polyphenols and their potential benefits for human health. Oxidative
Medicine and Cellular Longevity, v. 2012, 2012.
APROTOSOAIE, A. C.; LUCA, S. V.; MIRON, A. Flavor Chemistry of Cocoa and Cocoa
Products-An Overview. Comprehensive Reviews in Food Science and Food Safety, v. 15, n.
1, p. 73–91, 2016.
ASCRIZZI, R. et al. From the raw seed to chocolate: Volatile profile of Blanco de Criollo in
different phases of the processing chain. Microchemical Journal, v. 133, p. 474–479, 2017.
ASUERO, A. G.; SAYAGO, A.; GONZÁLEZ, A. G. The Correlation Coefficient: An Overview.
Critical Reviews in Analytical Chemistry, v. 36, n. July, p. 41–59, 2006.
BATISTA, N. N. et al. The impact of yeast starter cultures on the microbial communities and
volatile compounds in cocoa fermentation and the resulting sensory attributes of chocolate.
Journal of Food Science and Technology, v. 53, n. 2, p. 1101–1110, 2016.
45
BECKETT, S. T. Traditional Chocolate Making. In: BECKETT, S. T. (Ed.). . Industrial
Chocolate Manufacture and Use. 4. ed. Oxford, UK: Wiley-Blackwell, 2009. p. 1–9.
BEG, M. S. et al. Status, supply chain and processing of cocoa - A review. Trends in Food
Science & Technology, v. 66, p. 108–116, Aug. 2017.
BORDIGA, M. et al. Evaluation of the effect of processing on cocoa polyphenols: Antiradical
activity, anthocyanins and procyanidins profiling from raw beans to chocolate. International
Journal of Food Science and Technology, v. 50, n. 3, p. 840–848, 2015.
BRAGA, S. C. G. N. Avaliação dos perfis de sementes de cacau e derivados obtidos por
HS-SPME e cromatografia gasosa bidimensional abrangente utilizando ferramentas
quimiométricas. [s.l.] Universidade Estadual de Campinas, 2016.
CAMU, N. et al. Fermentation of cocoa beans: influence of microbial activities and polyphenol
concentrations on the flavour of chocolate. Journal of the Science of Food and Agriculture,
v. 88, n. 13, p. 2288–2297, Oct. 2008.
CARRILLO, L. C.; LONDOÑO-LONDOÑO, J.; GIL, A. Comparison of polyphenol,
methylxanthines and antioxidant activity in Theobroma cacao beans from different cocoa-
growing areas in Colombia. Food Research International, v. 60, p. 273–280, 2014.
COOPER, K. A et al. Cocoa and health: a decade of research. The British journal of nutrition,
v. 99, n. 1, p. 1–11, 2008.
COUNET, C. et al. Use of Gas Chromatography−Olfactometry To Identify Key Odorant
Compounds in Dark Chocolate. Comparison of Samples before and after Conching. Journal of
Agricultural and Food Chemistry, v. 50, n. 8, p. 2385–2391, Apr. 2002.
COUNET, C. et al. Relationship between procyanidin and flavor contents of cocoa liquors from
different origins. Journal of Agricultural and Food Chemistry, v. 52, n. 20, p. 6243–6249,
2004.
DI MATTIA, C. et al. Effect of Fermentation and Drying on Procyanidins, Antiradical Activity and
Reducing Properties of Cocoa Beans. Food and Bioprocess Technology, v. 6, n. 12, p. 3420–
3432, 2013.
DI MATTIA, C. et al. Effect of different conching processes on procyanidin content and
antioxidant properties of chocolate. Food Research International, v. 63, p. 367–372, 2014.
46
DUCKI, S. et al. Evaluation of solid-phase micro-extraction coupled to gas chromatography-
mass spectrometry for the headspace analysis of volatile compounds in cocoa products.
Talanta, v. 74, n. 5, p. 1166–1174, 2008.
DWIJATMOKO, M. I. et al. Characterization of Polyphenols from Various Cocoa ( Theobroma
cacao L .) Clones During Fermentation. Pelita Perkebunan, v. 34, n. 2, p. 104–112, 2018.
EFRAIM, P. et al. Influência da fermentação e secagem de amêndoas de cacau no teor de
compostos fenólicos e na aceitação sensorial. Ciência e Tecnologia de Alimentos, v. 30, p.
142–150, 2010.
GILL, M. S.; MACLEOD, A. J.; MOREAU, M. Volatile components of cocoa with particular
reference to glucosinolate products. Phytochemistry, v. 23, n. 9, p. 1937–1942, 1984.
GÜLTEKIN-ÖZGÜVEN, M.; BERKTAŞ, İ.; ÖZÇELIK, B. Influence of processing conditions on
procyanidin profiles and antioxidant capacity of chocolates: Optimization of dark chocolate
manufacturing by response surface methodology. LWT - Food Science and Technology, v.
66, p. 252–259, 2016.
HO, V. T. T.; ZHAO, J.; FLEET, G. Yeasts are essential for cocoa bean fermentation.
International Journal of Food Microbiology, v. 174, p. 72–87, 2014.
HU, S. J.; KIM, B. Y.; BAIK, M. Y. Physicochemical properties and antioxidant capacity of raw,
roasted and puffed cacao beans. Food Chemistry, v. 194, p. 1089–1094, 2016.
ICCO. International Cocoa Organization - The world cocoa economy: past and present.
Disponível em: <https://www.icco.org/about-us/international-cocoa-agreements/cat_view/30-
related-documents/45-statistics-other-statistics.html>. Acesso em: 20 oct. 2016.
IOANNONE, F. et al. Flavanols, proanthocyanidins and antioxidant activity changes during
cocoa (Theobroma cacao L.) roasting as affected by temperature and time of processing. Food
Chemistry, v. 174, p. 256–262, 2015.
KONÉ, M. K. et al. Contribution of predominant yeasts to the occurrence of aroma compounds
during cocoa bean fermentation. Food Research International, 2016.
KOTHE, L.; ZIMMERMANN, B. F.; GALENSA, R. Temperature influences epimerization and
composition of flavanol monomers, dimers and trimers during cocoa bean roasting. Food
Chemistry, v. 141, n. 4, p. 3656–3663, 2013.
47
MACHONIS, P. et al. Method for the Determination of Catechin and Epicatechin Enantiomers in
Cocoa-Based Ingredients and Products by High-Performance Liquid Chromatography: First
Action 2013.04. Journal of AOAC International, v. 97, n. 2, p. 506–509, 2014.
MAZOR JOLIĆ, S. et al. Changes of phenolic compounds and antioxidant capacity in cocoa
beans processing. International Journal of Food Science & Technology, v. 46, n. 9, p.
1793–1800, Sep. 2011.
MCSHEA, A. et al. Clinical benefit and preservation of flavonols in dark chocolate
manufacturing. Nutrition Reviews, v. 66, n. 11, p. 630–641, 2008.
MISNAWI et al. Oxidation of polyphenols in unfermented and partly fermented cocoa beans by
cocoa polyphenol oxidase and tyrosinase. Journal of the Science of Food and Agriculture, v.
82, n. 5, p. 559–566, 2002.
MISNAWI, A. et al. Sensory properties of cocoa liquor as affected by polyphenol concentration
and duration of roasting. Food Quality and Preference, v. 15, n. 5, p. 403–409, 2004.
NIEMENAK, N. et al. Comparative study of different cocoa (Theobroma cacao L.) clones in
terms of their phenolics and anthocyanins contents. Journal of Food Composition and
Analysis, v. 19, n. 6–7, p. 612–619, 2006.
ORACZ, J.; NEBESNY, E.; ŻYŻELEWICZ, D. Changes in the flavan-3-ols, anthocyanins, and
flavanols composition of cocoa beans of different Theobroma cacao L. groups affected by
roasting conditions. European Food Research and Technology, v. 241, n. 5, p. 663–681,
2015.
OWUSU, M.; PETERSEN, M. A.; HEIMDAL, H. Effect of fermentation method, roasting and
conching conditions on the aroma volatiles of dark chocolate. Journal of Food Processing
and Preservation, v. 36, n. 5, p. 446–456, 2012.
PAPALEXANDRATOU, Z. et al. Spontaneous organic cocoa bean box fermentations in Brazil
are characterized by a restricted species diversity of lactic acid bacteria and acetic acid
bacteria. Food Microbiology, v. 28, n. 7, p. 1326–1338, 2011.
PASSOS, F. M. L.; LOPEZ, A. S.; SILVIA, D. O. Aeration and its influence on the microbial
sequence in cacao fermentations in Bahia. Journal of Food Science, v. 49, p. 1470–1474,
1984.
48
PAYNE, M. J. et al. Impact of Fermentation , Drying , Roasting , and Dutch Processing on
Epicatechin and Catechin Content of Cacao Beans and Cocoa Ingredients. Journal of
Agricultural and Food Chemistry, v. 58, p. 10518–10527, 2010.
PUZIAH, H. et al. Changes in Free Amino Acid, Peptide-N, Sugar and Pyrazine Concentration
during Cocoa Fermentation. J Sci Food Agric, v. 78, p. 535–542, 1998.
QUIROZ-REYES, C. N.; FOGLIANO, V. Design cocoa processing towards healthy cocoa
products : The role of phenolics and melanoidins. Journal of Functional Foods, v. 45, n.
January, p. 480–490, 2018.
RODRIGUEZ-CAMPOS, J. et al. Dynamics of volatile and non-volatile compounds in cocoa
(Theobroma cacao L.) during fermentation and drying processes using principal components
analysis. Food Research International, v. 44, n. 1, p. 250–258, 2011.
SERRA BONVEHÍ, J.; VENTURA COLL, F. Evaluation of bitterness and astringency of
polyphenolic compounds in cocoa powder. Food Chemistry, v. 60, n. 3, p. 365–370, 1997.
SOTO-VACA, A. et al. Evolution of good polyphenolics from color and flavor problems to health
benefits Evolution of Phenolic compounds from Color and Flavor Problems to Health Benefits.
Journal of Agricultural and Food Chemistry, v. 60, n. 2, p. 6658−6677, 2012.
STANLEY, T. H. et al. Impact of roasting on the fl avan-3-ol composition , sensory-related
chemistry , and in vitro pancreatic lipase inhibitory activity of cocoa beans. Food Chemistry, v.
255, p. 414–420, 2018.
STONE, H.; SIDEL, J. L. Sensory evaluation practices. [s.l.] Elsevier Academic Press, 2004.
TEH, Q. T. M. et al. The Drying Kinetics and Polyphenol Degradation of Cocoa Beans. Journal
of Food Process Engineering, p. 1–8, 2015.
TRAN, P. D. et al. Assessing cocoa aroma quality by multiple analytical approaches. Food
Research International, v. 77, p. 657–669, 2015.
WOLLGAST, J.; ANKLAM, E. Review on polyphenols in Theobroma cacao: Changes in
composition during the manufacture of chocolate and methodology for identification and
quantification. Food Research International, v. 33, n. 6, p. 423–447, 2000.
49
WOOD, G. A. R.; LASS, R. A. Cocoa. Fourth ed. Oxford, UK: Blackwell Science Ltd, 1985.
ŻYŻELEWICZ, D. et al. The influence of the roasting process conditions on the polyphenol
content in cocoa beans , nibs and chocolates. Food Research International, v. 89, n. 2, p.
918–929, 2016.
50
ANEXO I – Parecer substanciado do comitê de ética em pesquisa da UNICAMP
51
52
53
54
55
56
ANEXO II – Declaração de cadastro no Sistema Nacional de Gestão do Patrimônio Genético