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
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
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.
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.
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.
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
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
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) fermented cocoa beans (NFB), fermented roasted cocoa nibs (NFN), cocoa liquor from non-fermented nibs (NFL), chocolate with 4h of conching from non-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 m g.g -1 0 1 2 3 4 5 6 7 8 9 FB FN FL ChF 4h ChF 16h m g.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-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 non-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), fermented roasted cocoa nibs (NFN), cocoa liquor from non-fermented nibs (NFL), chocolate with 4h of conching from non-non-fermented liquor (ChNF 4h) and chocolate with 16h of conching from non-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
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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 Peak ar ea x 10 3
Pyrazine, 2,5-dimethyl- Pyrazine, 2,3-dimethyl- Pyrazine, trimethyl-Pyrazine, tetramethyl- 2,3,5-Trimethyl-6-ethylpyrazine
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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
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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.
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