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Avaliação Do Potencial De Sementes Amazônicas Para Obtenção De Polissacarídeos

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Avaliação Do Potencial De Sementes Amazônicas Para Obtenção De Polissacarídeos AVALIAÇÃO DO POTENCIAL DE SEMENTES AMAZÔNICAS PARA OBTENÇÃO DE POLISSACARÍDEOS Rutelene C. Pinheiro(1), Lina F. Ballesteros (2), Miguel A. Cerqueira(2,3), Antonio M. C. Rodrigues(1), Luiza H. M. Silva(1) e José A. Teixeira(2) 1-Instituto Tecnológico, Programa de Pós- Graduação em Ciência e Tecnologia de Alimentos, Universidade Federal do Pará, Campos Guamá, 66075-900, Belém, Pará, Brasil. Telefone: +55 (91) 3201-8988 – e-mail: [email protected] 2- Centro de Engenharia Biológica, Universidade do Minho, Campos Gualtar, 4710-057, Braga, Portugal. 3– Laboratório Ibérico Internacional de Nanotecnologia, Av. Mestre José Veiga, S/n, 4715-330, Braga, Portugal. RESUMO – Cupuaçu (Theobroma grandiflorum), tucumã (Astrocaryum aculeatum), pupunha (Bactris gasipaes) e abricó (Mammea americana L.) são frutos originários da região amazônica e suas sementes normalmente são descartadas ou utilizadas para alimentação animal, adubo e plantio de novas mudas. O interesse no aproveitamento de sementes e em pesquisas utilizando plantas como fonte de componentes ativos teve um notável aumento nos últimos anos, e uma atenção particular foi dada aos polissacarídeos, os quais possuem diversas aplicações industriais. Visando explorar novas fontes de origem vegetal para atender a crescente demanda industrial quanto ao uso de polissacarídeos foram avaliados nesta pesquisa, dois métodos de extração (sólido-líquido) utilizando etanol como solvente orgânico em sementes integrais e desengorduradas. A caracterização química estrutural das sementes foi avaliada por FTIR e as frações polissacarídicas extraídas foram analisadas por cromatografia de alta eficiência (HPLC) as quais apresentaram destaque para açúcares de manose e glicose. ABSTRACT – Cupuassu (Theobroma grandiflorum), tucumã (Astrocaryum aculeatum), peach palm (Bactris gasipaes), and mammee apple (Mammea americana L.) are fruits from the Amazon region and their seeds are normally discarded or used as animal feed, fertilizer, or for planting new trees. The interest in using seeds and plants as sources of active compounds has greatly increased in recent years and particular attention has been given to polysaccharides, which have several industrial applications. Aiming to explore new plant sources to meet the growing industrial demand for polysaccharides, this research assessed two extraction methods (solid-liquid) using ethanol as organic solvent on whole and degreased seeds. The chemical structural characterization of the seeds was assessed by Fourier transform infrared spectroscopy and the polysaccharide fractions extracted were analyzed by high- efficiency liquid chromatography, which yielded expressive results for mannose and glucose. PALAVRAS-CHAVE: Theobroma grandiflorum, Astrocaryum aculeatum, Bactris gasipaes, Mammea americana L., aproveitamento de resíduos. KEYWORDS: Theobroma grandiflorum, Astrocaryum aculeatum, Bactris gasipaes, American Mammea L., waste recovery. 1. INTRODUÇÃO Cupuaçu (Theobroma grandiflorum), tucumã (Astrocaryum aculeatum), pupunha (Bactris gasipaes) e abricó (Mammea americana L.) são frutos originários da região amazônica (Vriesmann e Petkowicz, 2009; Sagrillo et al., 2015), sendo também encontrados em outras localidades (Jobim et al., 2014; Ordónez-Santos et al., 2015). Suas sementes normalmente são descartadas ou utilizadas para alimentação animal, adubo e plantio de novas mudas (Bolanho et al.,, 2015) com exceção da semente de cupuaçu que serve para produção de um alimento similar ao chocolate, chamado “cupulate” que tem um bom potencial de mercado como exótico substituto para o chocolate (Pugliese et al., 2013) podendo também ser utilizada na formulação de produtos para uso cosmético (Oliveira e Genovese, 2013). O interesse em polissacarídeos obtidos por meio de plantas tem aumentado nos últimos anos, devido as suas propriedades físico-químicas, atividades biológicas, propriedades antimicrobianas, fácil disponibilidade e baixo custo de extração (Tian et al., 2011, Nayaka et al., 2015) e podem ser isolados a partir de várias partes do vegetal (Jana et al., 2011; Malviya e Kulkarni, 2012; Oliveira et al., 2012). A composição em polissacarídeos das sementes estudadas não é reportada na literatura, há apenas alguns estudos referentes à polpa de cupuaçu (Vriesmann e Petkowicz, 2009) e farinha de pupunha (Bolanho et al., 2015), porém são limitados. Com intuito de explorar a composição polissacarídica de novas fontes vegetais, foi realizada neste estudo a caracterização química estrutural das sementes integrais e desengorduradas, bem como a extração de polissacarídeos das mesmas por diferentes tratamentos. 2. MATERIAL E MÉTODOS 2.1 Matérias-primas e produtos químicos Os frutos foram adquiridos na cidade de Bélem estado do Pará, e após o despolpamento as sementes foram limpas e a parte de interesse (endosperma) foi seca a 60 °C/24 h em estufa com circulação de ar, o material seco foi triturado, acondicionado em recipiente plástico e armazenado a temperatura ambiente (25 °C). Os açúcares glicose, arabinose, galactose, xilose e manose foram adquiridos da Sigma-Aldrich Traning Co Ltda. e o solvente utilizado de grau analítico. 2.2 Caracterização estrutural das sementes Para melhor entendimento dos grupos de ligações químicas e arranjo dos constituintes presentes em cada semente, foram separadas amostras integrais e desengorduradas as quais foram avaliadas por espectroscopia de infravermelho de Fourier (FTIR) utilizando o espectrômetro Perkin- Elmer 16 PC (Boston, USA), equipado com célula de composição diamante atenuado reflectância total (ATR). As medições foram registradas com um intervalo de 4000 números de onda a 600 cm-1e 16 leituras por amostra. 2.3 Procedimentos de extração dos polissacarídeos Foram realizados dois procedimentos de extração conforme mostra a Figura 1 os quais avaliam a eficácia do etanol como agente precipitante para as amostras integrais e desengorduradas. A remoção da fração lipídica das amostras foi realizada em equipamento Soxhlet durante 4 h de extração utilizando éter de petróleo como solvente (1:5(m/v)). A inativação enzimática e os procedimentos de extração foram realisados de acordo com Cerqueira et al. (2009) com algumas modificações. Inativação enzimática As sementes integrais e desengorduradas foram suspensas em etanol na proporção de 1:3 (w/v) e aquecidas a 70 °C/15 min, o etanol foi removido e o precipitado utilizado nas etapas seguintes. Figura 1- Fluxograma de extração de polissacarídeos aplicado para diferentes sementes. Sementes (secas e trituradas) Inativação enzimática Remoção do etanol Procedimento 1 Procedimento 2 Adição H2O ((1:5) /24 h) Adição H2O ((1:5) /24 h) Agitação (1h) Fração Sólida Etanol (1:2) Etanol (1:3/12 h) Precipitado Precipitado H2O (1:2) H2O (1:5/20h) Liofilização Agitação (1h) FI Etanol (1:2/12h) GI Precipitado H2O (1:2) Liofilização FII GII Procedimento 1 Após remoção do etanol (inativação enzimática) foi adicionado ao precipitado água destilada (1:5 (w/v)) o qual permaneceu em contato por 24 horas em temperatura de refrigeração (4°C). Terminado o período de repouso a mistura foi colocada sob agitação com rotação de 200 rpm em temperatura ambiente (25 °C) com duração de 1 hora. Posteriormente foi realizada outra lavagem em etanol (1:2 (m /v)) e ao precipitado foi adicionado água (1:2 (m /v)). As amostras foram congeladas e liofilizadas. Para melhor entendimento as amostras contendo lipídeos foram representadas pela letra F e as desengorduradas por G. Procedimento 2 De igual modo ao primeiro procedimento, após o período de 24h de repouso foram realizadas sucessivas lavagens com etanol e água. Na primeira lavagem as amostras foram deixadas por 12h em etanol (1:3 (m /v)) a 4°C, após a centrifugação foram adicionados ao precipitado água ultra pura (1:5 (m /v)) com repouso de 20 h, a seguir a mistura foi submetida à agitação (200 rpm/1h) em temperatura ambiente. A segunda lavagem consistiu de adição de etanol (1:2 (m /v)) por 12 h, seguida de adição de água destilada (1:2 (m/v)), as amostras foram congeladas e liofilizadas. Para melhor entendimento as amostras foram codificadas por FII e GII. 3. RESULTADOS E DISCUSSÃO 3.1 Caracterização estrutural das sementes Conforme mostrado na Figura 2 às amostras apresentaram três regiões de absorbância principais, a primeira corresponde a comprimentos de onda na faixa 3000- 2800 cm-1, a segunda banda em 1800-1600 cm-1 e a terceira em 1200-1000 cm-1. Absorções em picos específicos podem ser identificadas para cada componente em particular. Os picos em 2900 cm-1 são característicos às ligações C-H, em especial do grupo metileno das moléculas de lipídeos (Barbosa, 2008), conforme esperado este pico foi menor para as amostras desengorduradas (Fig. 2-B). A banda em 1700 cm-1 identificados nas amostras é atribuída a grupos carbonila (C=O) de acordo com Ren et al. (2014) este grupo funcional apresenta absorção no espectro infravermelho na faixa de 1800-1500 cm-1 e podem estar relacionados à presença de proteínas (Barbosa, 2008), aminoácidos livres e ácido clorogênico (Ribeiro et al., 2010). Os espectros de FTIR também identificaram picos a 1000 e 1200 cm-1 que são característicos da região de hidratos de carbono (750-1300 cm-1) segundo os autores esta região é específica para polissacarídeos (Sen e Erboz, 2010). Figura 2- Espectros FTIR obtidas das sementes integrais (A) e desengorduradas (B). Abrico Abrico Peachpalm Peachpalm Tucuma Tucuma Cupuassu Cupuassu Transmittance(%) Transmittance (%) 4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 -1
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