RESSALVA Atendendo solicitação da autora, o texto completo desta dissertação será disponibilizado somente a partir de 27/02/2027. UNIVERSIDADE ESTADUAL PAULISTA – UNESP Instituto de Biociências, Letras e Ciências Exatas - Campus de São José do Rio Preto Maria Eduarda Carvalho Vargas Extrato fenólico de cálices de vinagreira (Hibiscus sabdariffa L.): avaliação da estabilidade da cor e das antocianinas durante o armazenamento, e sua aplicabilidade em iogurte São José do Rio Preto 2025 Maria Eduarda Carvalho Vargas Extrato fenólico de cálices de vinagreira (Hibiscus sabdariffa L.): avaliação da estabilidade da cor e das antocianinas durante o armazenamento, e sua aplicabilidade em iogurte Dissertação apresentada à Universidade Estadual Paulista (UNESP), Instituto de Biociências, Letras e Ciências Exatas, São José do Rio Preto, para obtenção do Título de Mestre em Alimentos, Nutrição e Engenharia de Alimentos. Área de Concentração: Alimentos e Nutrição Orientadora: Profª. Drª. Ellen Silva Lago-Vanzela Coorientador: Prof. Dr. Roberto da Silva Coorientadora: Dra. Yara Paula Nishiyama-Hortense São José do Rio Preto 2025 Sistema de geração aut.omática de fichas catalográficas da Unesp. Dados fornecidos pelo autor(a). V297e Vargas, Maria Eduarda Carvalho Extrato fenólico de cálices de vinagreira (Hibiscus sabdariffa L.): avaliação da estabilidade da cor e das antocianinas durante o armazenamento, e sua aplicabilidade em iogurte / Maria Eduarda Carvalho Vargas. -- , 2025 128 p. : il., tabs. Dissertação (mestrado) - Universidade Estadual Paulista (UNESP), Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto, Orientadora: Ellen Silva Lago-Vanzela Coorientadora: Yara Paula Nishiyama-Hortense 1. Hibiscus sabdariffa L. 2. Extratos fenólicos. 3. Bioingredientes. I. Título Maria Eduarda Carvalho Vargas Extrato fenólico de cálices de vinagreira (Hibiscus sabdariffa L.): avaliação da estabilidade da cor e das antocianinas durante o armazenamento, e sua aplicabilidade em iogurte Dissertação apresentada à Universidade Estadual Paulista (UNESP), Instituto de Biociências, Letras e Ciências Exatas, São José do Rio Preto, para obtenção do título de Mestre em Alimentos, Nutrição e Engenharia de Alimentos. Área de Concentração: Alimentos e Nutrição Data da defesa: 27/02/2025 Banca Examinadora: Profª.Drª. Ellen Silva Lago Vanzela Universidade Estadual Paulista “Júlio de Mesquita Filho” – Campus São José do Rio Preto Orientador Profª.Drª. Iasnaia Maria De Carvalho Tavares Universidade Estadual do Sudoeste da Bahia Profª.Drª. Natália Soares Janzantti Universidade Estadual Paulista “Júlio de Mesquita Filho” – Campus São José do Rio Preto São José do Rio Preto 27 de fevereiro de 2025 AGRADECIMENTOS Primeiramente, agradeço a Deus, por conduzir meus passos e me proporcionar mais esta conquista. Aos meus pais, Márcia e Danilo, por serem a base de tudo. Sem eles, nada disso seria possível. Obrigada pelo apoio, amizade, ajuda e compreensão. Aos meus irmãos, Valmir, Rafaela e Vivian, que compartilham da mesma luta, dedicação e persistência. Às minhas avós, Raimunda e Urbana, pelo incentivo e apoio constante. Saibam que as senhoras são, para mim, o real exemplo de força e luta. Aos demais membros da minha família, que acompanharam de perto toda a minha jornada. À minha orientadora, Profª. Drª. Ellen Silva Lago Vanzela, pela paciência, incentivo e disposição em me orientar, além dos valiosos conhecimentos transmitidos ao longo de todo o processo. Aos meus coorientadores Prof. Dr. Roberto da Silva e Drª Yara Paula Nishiyama-Hortense, pela orientação e paciência durante toda a trajetória. Aos professores e técnicos do Departamento de Engenharia de Alimentos, pela orientação e apoio durante o desenvolvimento deste trabalho. Aos amigos do Laboratório de Frutas, Victória, Francielli, Natália, Juma, Mariana, Anelisa, Ingrid, Aline, Mery e Carla, agradeço a amizade, os momentos de descontração, pela ajuda e pelos ensinamentos práticos e teóricos. Aos amigos de Laboratórios de Carnes (Marcello, Carlos Alberto e Viviane) e a Natália de Leites, obrigada pelos cafés, conversas e risadas que ajudaram a manter minha sanidade durante esse período. Aos meus amigos de jornada, Fabiana, Lariane, Lorena e Paulo Henrique, por sempre me incentivarem a crescer. À Simone, Devair e Livia, por me acolherem e me receberem. Obrigada pela amizade e pelos momentos compartilhados. Agradeço sinceramente à Drª Izabel Cristina dos Santos, pesquisadora da EPAMIG, por sua valiosa ajuda e pelo envio das fotos presentes neste trabalho. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES), processo nº. 8887.828314/2023-00. A todos que, de alguma forma, contribuíram para a realização deste trabalho e me incentivaram ao longo do caminho, o meu sincero agradecimento. RESUMO Os cálices de vinagreira (CV, Hibiscus sabdariffa L.) são fontes importantes de compostos fenólicos (CF), cujos extratos apresentam potencial para uso como bioingredientes na produção de alimentos. Este estudo teve como objetivo caracterizar físico-quimicamente os cálices in natura e desidratados (CVD) e desenvolver estratégias para melhorar a aplicabilidade dos extratos fenólicos obtidos a partir dos CVD. As abordagens empregadas incluíram a adição de açúcar e a desidratação por secagem em leito de espuma, com posterior aplicação em iogurtes. A caracterização físico-química dos CVD revelou uma umidade de 11,61% e uma atividade de água de 0,56, ambos em conformidade com a legislação brasileira para vegetais desidratados. Para a extração dos CF dos CVD, três soluções extratoras foram avaliadas: metanol acidificado, etanol 60% e água. A extração metanólica resultou na maior concentração de compostos fenólicos totais (CFT, mg EAG·g⁻¹ de CVD, em base seca) (17,48), seguida pela extração aquosa (16,15) e pela solução hidroalcoólica (14,51), evidenciando a viabilidade da extração aquosa para obtenção de extratos fenólicos alimentícios. A análise por cromatografia líquida de alta eficiência com detectores de arranjo de diodos e espectrometria de massas multidimensional (HPLC-DAD-ESI-MSn) indicou que a concentração total de antocianinas (mg cy-3-glc·kg⁻¹) variou entre 2726,09 (extrato hidroalcoólico) e 4598,70 (extrato aquoso), sendo a delfinidina-3-sambubiosídeo e a cianidina-3-sambubiosídeo as principais antocianinas identificadas. Com base nesses resultados, os extratos aquosos foram empregados na formulação de xaropes e bioingredientes em pó (BIP). Os xaropes, preparados com diferentes concentrações de sacarose (5%, 15%, 25% e 35%), foram envasados em frascos herméticos, armazenados a 4 ºC por 30 dias e avaliados quanto à estabilidade das antocianinas totais (ANT), dos CFT e dos parâmetros cromáticos (L*, C* e h°) nos tempos 0, 15 e 30 dias. O extrato controle (sem sacarose) apresentou retenção de 91,06% das ANT após o armazenamento, enquanto nos xaropes os valores variaram entre 78,41% e 91,20%, com maiores perdas em amostras com menor teor de sacarose. Em relação aos CFT, o extrato controle reteve 93,61%, enquanto nos xaropes a retenção mínima foi de 94,98%. A coloração foi mais estável nos xaropes com maior teor de sacarose, apresentando um ∆E de 5,48 após o armazenamento, indicando manutenção da tonalidade. Para a produção do BIP, a espuma formulada a partir dos extratos fenólicos e aditivos (emulsificante, estabilizante e maltodextrina) demonstrou propriedades adequadas para desidratação, incluindo densidade, estabilidade e percentual de expansão. O produto final apresentou concentração de antocianinas e CFT de 48,50 mg de mv- 3,5-diglc·g-1 e 295 mg EAG·g-1, respectivamente. Após análise dos parâmetros de cor foi possível constatar que o BIP produzido resultou em tonalidade de cor avermelhada, característica que foi preservada após sua incorporação ao iogurte. Visualmente, os iogurtes contendo BIP apresentaram coloração mais atrativa em comparação aos enriquecidos com xarope ou extrato controle. Os resultados demonstram que os extratos fenólicos de CVD, com ou sem adição de sacarose, podem ser armazenados sob refrigeração por até 30 dias, mantendo ainda importante concentração de antocianinas e CFT, assim como coloração vermelha atrativa. Além disso, a obtenção do BIP amplia o potencial de aplicação da vinagreira em diferentes matrizes alimentícias, representando uma alternativa promissora para a indústria de alimentos. Palavras-chave: Hibiscus sabdariffa L; extratos fenólicos; bioingredientes. ABSTRACT The calyces of roselle (CV, Hibiscus sabdariffa L.) are important sources of phenolic compounds (PC), whose extracts have potential for use as bioingredients in food production. This study aimed to physicochemically characterize the fresh and dehydrated calyces (CVD) and develop strategies to improve the stability and applicability of the phenolic extracts obtained from CVD. The approaches employed included the addition of sugar and dehydration by foam-mat drying, followed by application in yogurts. The physicochemical characterization of CVD revealed a moisture content of 11.61% and a water activity of 0.56, both in compliance with Brazilian regulations for dehydrated plant materials. For the extraction of PC from CVD, three extracting solutions were evaluated: acidified methanol, 60% ethanol, and water. Methanolic extraction resulted in the highest concentration of total phenolic compounds (TPC, mg GAE·g⁻¹ of CVD, dry basis) (17.48), followed by aqueous extraction (16.15) and the hydroalcoholic solution (60% ethanol) (14.51), demonstrating the feasibility of aqueous extraction for obtaining food-grade phenolic extracts. High-performance liquid chromatography with diode-array detection and multidimensional mass spectrometry (HPLC- DAD-ESI-MSn) analysis indicated that the total anthocyanin concentration (mg cy-3-glc·kg⁻¹) ranged from 2726.09 (60% ethanol extract) to 4598.70 (aqueous extract), with delphinidin-3- sambubioside and cyanidin-3-sambubioside identified as the main anthocyanins. Based on these results, aqueous extracts were used in the formulation of syrups and powdered bioingredients (PBI). The syrups, prepared with different sucrose concentrations (5%, 15%, 25%, and 35%), were packaged in hermetically sealed bottles, stored at 4°C for 30 days, and evaluated for the stability of total anthocyanins (ANT), TPC, and color parameters (L*, C*, and h°) at 0, 15, and 30 days. The control extract (without sucrose) retained 91.06% of ANT after storage, while retention in the syrups ranged from 78.41% to 91.20%, with higher losses observed in samples with lower sucrose content. Regarding TPC, the control extract retained 93.61%, while in the syrups, the minimum retention was 94.98%. Color stability was greater in syrups with higher sucrose content, showing a ΔE of 5.48 after storage, indicating color maintenance. For PBI production, the foam formulated from phenolic extracts and additives (emulsifier, stabilizer, and maltodextrin) exhibited suitable properties for dehydration, including density, stability, and expansion percentage. Although drying at 80°C resulted in 13% retention of ANT and 24% retention of TPC, the PBI maintained an intense red color (15.61°), which was preserved after incorporation into yogurt (25.44°). Visually, yogurts containing PBI presented a more attractive color compared to those enriched with syrup or control extract. The results demonstrate that phenolic extracts from CVD, with or without sucrose addition, can be refrigerated for up to 30 days while maintaining their stability and technological functionality. Additionally, the production of PBI expands the potential application of roselle in various food matrices, representing a promising alternative for the food industry. Keywords: Hibiscus sabdariffa L; phenolic extracts; bioingredients. LISTA DE FIGURAS Pág. Figura 1.1 - Flores de H. acetossela (A), H. sabdariffa L. (B) e de H. rosa- sinensis (C). 25 Figura 1.2 - Folhas e frutos de vinagreira (H. sabdariffa L.). 26 Figura 1.3 - (A) Flores, folhas e caules de vinagreira (H. sabdariffa L.) (B) Folha de vinagreira; (C) Caule e frutos imaturos de vinagreira; (D) Frutos imaturos; (E) Morfologia dos frutos imaturos; (F) Cápsula seca e sementes. 27 Figura 1.4 - Principais compostos fenólicos presentes nos cálices de vinagreira. 31 Figura 1.5 - Antocianinas já relatadas em cálices de vinagreira. 36 Figura 2.1 - (A) Colheita dos frutos imaturos; (B) Remoção das cápsulas dos frutos imaturos; (C) Obtenção dos cálices in natura (D) Secagem ao sol de cálices; (E) Secagem à sombra de cálices; (F) Cálices desidratados. 55 Figura 2.2 - Cromatograma HPLC-DAD (detecção 520 nm) de extratos de cálices de vinagreira desidratados. A atribuição dos picos está apresentada na Tabela 2.2. 67 Figura 2.3 - Retenção das ANT (A) e CFT (B) no extrato controle (E0) e nos xaropes com 5% (E5), 15% (E15), 25% (E25) e 35% (E35) de sacarose armazenados a 4°C por 30 dias. 73 Figura 2.4 - Análise de Componentes Principais (ACP) do extrato controle (E0) e dos xaropes com 5% (E5), 15% (E15), 25% (E25) e 35% (E35) de sacarose armazenados a 4°C por 30 dias. (A) Gráfico de cargas mostrando a contribuição das variáveis para os componentes principais e (B) Gráfico de escores exibindo a distribuição das amostras ao longo dos componentes principais. 79 Figura 3.1 - A) Localização do corte basal no fruto imaturo; (B) Demonstração do corte lateral no cálice; (C) Remoção da cápsula com as sementes; (D) Cálice in natura 84 Figura 3.2 - Produção da espuma (A), espuma disposta na bandeja (B) para a subsequente etapa de secagem (C) e produto final (D). 88 Figura 3.3 - Extratos de cálices de vinagreira desidratados em diferentes faixa de pH (1,5 a 9,5). 95 Figura 3.4 - Amostras de bioingredientes e iogurtes incorporados: ES e iogurte (A), EA e iogurte (B), XC e iogurte (C) e, BIP e iogurte (D). 99 LISTA DE TABELAS Pág. Tabela 1.1 - Exemplo das antocianidinas (forma de cátion flavílio) mais comuns. 32 Tabela 1.2 - Flavonóis já relatados em vinagreira. 38 Tabela 1.3 - Flavan-3-óis presentes nos cálices de vinagreira: (+)-catequina (C); (-)-epicatequina (E); (-)galato-3-epicatequina (ECG); (+)- galocatequina (GC); (-)-epigalocatequina (EGC); (+)-galato-3- galocatequina (GCG); (-)-galato-3-epigalocatequina (EGCG). *G: ácido gálico. 41 Tabela 1.4 - Padrão de substituição dos ácidos fenólicos. 43 Tabela 1.5 - Vários tipos de substituição do ácido quínico com os ácidos hidroxicinâmicos (caféico, p-cumárico e ferúlico). *C (ácido caféico); F (ácido ferúlico) e p-Co (p-cumárico). 44 Tabela 2.1 - Características físico-química dos extratos de CVD. 61 Tabela 2.2 - Características cromatográficas e espectrais de massa das antocianinas identificadas em extratos de cálices de vinagreira desidratados por HPLC-DAD-ESI-MSn (modo de ionização positivo), e quantificação (média ± desvio padrão, n = 2). Picos numerados como na Figura 2.2. 68 Tabela 2.3 - Características físico-químicas (média ± desvio-padrão) do extrato controle e xaropes armazenados a 4 ºC no tempo inicial (dia 0) e tempo final (dia 30). 70 Tabela 2.4 - CFT e ANT (média ± desvio-padrão) do extrato controle e xaropes de cálices de vinagreira desidratados armazenados a 4 ºC por 30 dias. 128 Tabela 2.5 - Parâmetros cromáticos (média ± desvio-padrão) e ∆E do extrato e dos xaropes armazenados a 4 ºC por 30 dias. 76 Tabela 3.1 - Características físico-química dos cálices de vinagreira in natura e desidratados (médias ± desvio padrão). 90 Tabela 3.2 - Parâmetros de cor ( L* , C*, hº) e △E de extratos de cálices de vinagreira desidratados em diferentes soluções de pH. 94 Tabela 3.3 - Características físico-química do bioingrediente a partir de extratos de cálices de vinagreira desidratados 97 Tabela 3.4 - Parâmetros cromáticos (L*, C* e hº) dos ES, EA, BIP e XC antes e após a incorporação ao iogurte. 99 LISTA DE ABREVIAÇÕES E SIGLAS 3-ACQ ácido 3-cafeoilquínico 3-AFQ ácido 3-feruloilquínico 3-p-ACoCQ ácido 3-p-cumaroilquínico 3,4-diACQ ácido 3,4-di-cafeoilquínico 3,5-diACQ ácido 3,5-di-cafeoilquínico 4-ACQ ácido 4-cafeoilquínico 4-AFQ ácido 4-feruloilquínico 4-p-ACoCQ ácido 4-p-cumaroilquínico 4,5-diACQ ácido 4,5-di-cafeoilquínico 5-ACQ ácido 5-cafeoilquínico 5-AFQ ácido 5-feruloilquínico 5-p-ACoCQ ácido 5-p-cumaroilquínico A Base quinoidal ACP Análise de Componentes Principais ANP Antocianina Poliméricas ANT Antocianinas Totais AT Acidez Titulável Aw Atividade de água B Hemicetal B.S. Base seca BIP Bioingrediente em pó C Catequina C* Croma CF Compostos Fenólicos CFT Compostos Fenólicos Totais CH Chalcona CIE Commission Internationale de l‘Eclairage Cy Cianidina Cy-3-(2”-xyl)glic Cianidina-3-(2’’-xiloil)-glicosídeo Cy-3-glc Cianidina-3-glicosídeo Cy-3-rutin Cianidina-3-rutinsideo Cy-3-samb Cianidina-3-sambubiosídeo Cy-3,5-diglic Cianidina-3,5-diglicosideo CV Cálices de vinagreira CVD Cálices de vinagreira desidratados DAD Detectores de arranjo de diodos DCNTs Doenças Crônicas Não Transmissíveis Dp Delfinidina Dp-3-(2”-xyl)glic Delfinidina-3-(2’’-xiloil)-glicosídeo Dp-3-glc Delfinidina-3-glicosídeo Dp-3-samb Delfinidina-3-sambubiosídeo E Epicatequina E0 Extrato sem sacarose E5 Extrato com 5% de sacarose E15 Extrato com 15% de sacarose E25 Extrato com 25% de sacarose E35 Extrato com 35% de sacarose EA Extrato adoçado com 35% de sacarose EAG Ácido Gálico ECA Enzima Conversora de Angiotensina ECG (-) galato-3-epigalocatequina EGC (-) – epigalocatequina EGCG (-)galato-3-epigalocatequina EM Emustab ES Extrato não adoçado ESI-MSn Espectrometria de massas multidimensional FDA Food and Drug Administration G Gossipetina GC (+) -galocatequina GCG (+) galato-3-galocatequina h(º) Hue HA+ Cátion Flavílio HPLC Cromatografia Líquida de Alta Eficiência I-BIP Iogurte adicionado com bioingrediente em pó I-EA Iogurte adicionado com extrato adoçado com 35% sacarose I-ES Iogurte adicionado com extrato sem sacarose I-XC Iogurte adicionado com xarope comercial de hibicus IL-6 Interleucina-6 K Kaempferol K-3-cumglic Kaempferol-3-cumarilglicosídeo K-3-rutin Kaempferol-3-rutinosídeo K-3-samb Kaempferol-3-sambubiosídeo L* Claridade M Miricetina M-3-Ag Miricetina-3- arabinogalactose M-3-pent-7-hex Miricetina-3-pentossil-7-hexosídeo M-3-samb Miricetina-3-sambubiosídeo MA Maltodextrina 10 DE MCP-1 Proteína Quimiotática de Monócitos-1 Mv Malvidina Mv-3,5-diglic Malvidina-3,5-diglicosídeo NO Óxido Nítrico p-Co ácido p-cumárico PANC Plantas Alimentícias Não Convencionais PC1 Componente Principal 1 PC2 Componente Principal 2 Pg Pelargonidina Pn Peonidina Pt Petunidina Q Quercetina Q-3-(6-acetil-glic) Quercetina-3- (6-acetil-glicosídeo) Q-3-gal Quercetina-3-galactosíde Q-3-glic Quercetina-3-glicosídeo Q-3-pent-7-hex Quercetina-3-pentossil-7-hexosídeo Q-3-rutin Quercetina-3-rutinosídeo Q-3-samb Quercetina-3-sambubiosídeo Q-galoil-hex Quercetina-galoil-hexosídeo SPN Super Liga Neutra SS Sólidos Solúveis TNF-α Fator de Necrose Tumoral Alfa UV Ultravioleta UV-Vis Ultravioleta-visível XC Xarope comercial de Hibiscus SUMÁRIO 1 INTRODUÇÃO GERAL 21 2 OBJETIVOS 22 2.1 OBJETIVOS GERAIS 22 2.2 OBJETIVOS ESPECÍFICOS 22 3 ESTRUTURA DO TRABALHO 24 CÁLICES DE VINAGREIRA (HIBISCUS SABDARIFFA L.): COMPOSTOS FENÓLICOS E ESTRATÉGIAS PARA O USO COMO CORANTE NATURAL E BIOINGREDIENTES – UMA REVISÃO. 25 1 PANORAMA GERAL DE VINAGREIRA (HIBISCUS SABDARIFFA L.) 25 2 PRINCIPAIS CF PRESENTES NOS CÁLICES DE VINAGREIRA 30 2.1 ANTOCIANINAS 32 2.2 FLAVONÓIS 37 2.3 FLAVAN-3-ÓIS E PROANTOCIANIDINAS 40 2.4 ÁCIDOS FENÓLICOS 42 3 ESTRATÉGIAS PARA USO DE CÁLICES DE VINAGREIRA DESIDRATADOS COMO CORANTES NATURAIS E BIOINGREDIENTE 46 4 CONSIDERAÇÕES FINAIS 50 EXTRAÇÃO, CARACTERIZAÇÃO E ESTABILIDADE DE COMPOSTOS FENÓLICOS DE CÁLICES DE VINAGREIRA DESIDRATADOS (HIBISCUS SABDARIFFA L.) PARA DESENVOLVIMENTO DE XAROPES ALIMENTÍCIOS. 51 1 INTRODUÇÃO 53 2 MATERIAIS E MÉTODOS 54 2.1 REAGENTES QUÍMICOS E PRODUTOS 54 2.2 OBTENÇÃO DE CÁLICES DE VINAGREIRA DESIDRATADOS 55 2.3 POTENCIAL DOS CÁLICES DE VINAGREIRA DESIDRATADOS PARA OBTENÇÃO DE EXTRATOS FENÓLICOS 56 2.4 IDENTIFICAÇÃO E QUANTIFICAÇÃO DAS ANTOCIANINAS EM EXTRATOS FENÓLICOS DE CÁLICES DE VINAGREIRA DESIDRATADOS POR HPLC-DAD-ESI-MSN 57 2.5 OBTENÇÃO DE XAROPES A PARTIR DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS E AVALIAÇÃO DOS COMPOSTOS FENÓLICOS E DA COR, BEM COMO MONITORAMENTO DAS CARACTERÍSTICAS FÍSICO- QUÍMICAS, DURANTE ARMAZENAMENTO REFRIGERADO 58 2.6 ANÁLISE ESTATÍSTICA 59 3 RESULTADOS E DISCUSSÕES 61 3.1 POTENCIAL DOS CÁLICES DE VINAGREIRA DESIDRATADOS PARA OBTENÇÃO DE EXTRATOS FENÓLICOS 61 3.2 IDENTIFICAÇÃO E QUANTIFICAÇÃO DAS ANTOCIANINAS EM EXTRATOS FENÓLICOS DE CÁLICES DE VINAGREIRA DESIDRATADOS POR HPLC-DAD-ESI-MSN 64 3.3 AVALIAÇÃO DOS COMPOSTOS FENÓLICOS E DA COR, BEM COMO MONITORAMENTO DAS CARACTERÍSTICAS FÍSICO- QUÍMICAS, DURANTE ARMAZENAMENTO REFRIGERADO DO EXTRATO CONTROLE E DOS XAROPES 69 3.3.1 Análise de Componentes Principais (ACP) 78 4 CONCLUSÃO 80 CÁLICES DE VINAGREIRA (HIBISCUS SABDARIFFA L.): CARACTERIZAÇÃO FÍSICO-QUÍMICA, OBTENÇÃO DE BIOINGREDIENTES E APLICAÇÃO EM IOGURTE. 81 1 INTRODUÇÃO 82 2 MATERIAIS E MÉTODOS 83 2.1 MATÉRIAS-PRIMAS E PRODUTOS 83 2.2 CARACTERIZAÇÃO FÍSICO-QUÍMICA DOS CÁLICES DE VINAGREIRA (IN NATURA E DESIDRATADOS) 84 2.3 OBTENÇÃO DE EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS 85 2.4 DETERMINAÇÃO DOS PARÂMETROS DE CROMATICIDADES DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS EM DIFERENTES VALORES DE PH 86 2.5 OBTENÇÃO DA ESPUMA E DO BIOINGREDIENTE EM PÓ A PARTIR DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS 86 2.5.1 CARACTERIZAÇÃO FÍSICO-QUÍMICA DO BIOINGREDIENTE EM PÓ A PARTIR DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS 88 2.6 APLICAÇÃO DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS E DO BIOINGREDIENTE EM PÓ EM IOGURTE 89 2.7 ANÁLISE ESTATÍSTICA 89 3 RESULTADOS E DISCUSSÕES 90 3.1 CARACTERIZAÇÃO FÍSICO-QUÍMICA DOS CÁLICES DE VINAGREIRA (IN NATURA E DESIDRATADOS) 90 3.2 PARÂMETROS CROMÁTICOS DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS EM DIFERENTES VALORES DE PH 94 3.3 CARACTERIZAÇÃO DAS ESPUMAS E DOS BIOINGREDIENTES EM PÓ 96 3.4 APLICAÇÃO DOS EXTRATOS DE CÁLICES DE VINAGREIRA DESIDRATADOS E DO BIOINGREDIENTE EM PÓ EM IOGURTE 98 4 CONCLUSÃO 101 CONCLUSÃO GERAL 103 REFERÊNCIAS 104 ANPÊNDICE A- CFT E ANT EM EXTRATO CONTROLE E XAROPES DE CÁLICES DE VINAGREIRA DESIDRATADOS ARMAZENADOS A 4 ºC POR 30 DIAS. 128 21 1 INTRODUÇÃO GERAL A busca por sistemas alimentares mais sustentáveis e seguros tem impulsionado o interesse por espécies vegetais subutilizadas, especialmente em países com rica biodiversidade, como o Brasil. Nesse contexto, as Plantas Alimentícias Não Convencionais (PANC), incluindo a vinagreira (Hibiscus sabdariffa L.), emergem como alternativas promissoras para diversificação alimentar e desenvolvimento de produtos inovadores. A valorização dessas espécies negligenciadas não apenas amplia as opções nutricionais disponíveis para a população, mas também fortalece práticas agrícolas sustentáveis e estimula o aproveitamento de recursos naturais de forma mais eficiente (Kinupp; Lorenzi, 2014; Mariutti et al., 2021). Paralelamente, mudanças nos padrões de consumo têm levado à crescente demanda por alimentos mais saudáveis, livres de aditivos sintéticos e visualmente atrativos, impulsionando a substituição de corantes artificiais por alternativas naturais (Albuquerque et al., 2020; Gordillo et al., 2018). Nesse cenário, os pigmentos naturais pertencentes ao grupo dos flavonoides, como as antocianinas, destacam-se por sua versatilidade, conferindo coloração intensa e propriedades bioativas aos alimentos. Entretanto, desafios associados à estabilidade desses compostos durante a extração, o processamento e o armazenamento ainda limitam sua ampla utilização na indústria de alimentos (Cheng et al., 2023; Gordillo et al., 2018; Sampaio et al., 2021; Teixeira et al., 2023; Wijesekara; Xu, 2024). A vinagreira se sobressai como uma cultura anual de fácil cultivo e amplamente disseminada no Brasil, inclusive no estado de São Paulo. Seus cálices vermelhos são ricos em compostos fenólicos, principalmente antocianinas, conferindo-lhes potencial para aplicação como corante natural em produtos alimentícios (Mariod; Tahir; Mahunu, 2021). Além da função corante, esses compostos apresentam atividade antioxidante, estando associados à redução do risco de doenças não transmissíveis, como enfermidades cardiovasculares e câncer (Coelho; Amorim, 2019; Da-Costa-Rocha et al., 2014; Nowicka; Wojdylo, 2019; Teixeira et al., 2023). Apesar do seu potencial, a incorporação da vinagreira em formulações alimentícias apresenta desafios significativos. Nos cálices, os compostos fenólicos (CF) estão naturalmente protegidos pela complexa estrutura celular da planta. Durante o processamento, a ruptura dessas estruturas leva à descompartimentalização das organelas, desencadeando reações químicas e bioquímicas que afetam a estabilidade desses compostos. A estrutura química das antocianinas, que lhes confere propriedades funcionais, também as torna vulneráveis a processos oxidativos, térmicos e enzimáticos, comprometendo sua retenção e bioatividade ao longo da cadeia produtiva (Da-Silva; Lago-Vanzela; Baffi, 2015). Como consequência, ocorre uma redução progressiva dos CF originalmente presentes na matriz vegetal, acompanhada da formação de 22 derivados poliméricos que impactam tanto as características sensoriais quanto a funcionalidade dos produtos elaborados (Debelo; Ferruzi, 2020). Além disso, em formulações contendo múltiplos ingredientes, interações entre macronutrientes e micronutrientes podem influenciar a estabilidade desses compostos, afetando diretamente suas propriedades tecno-funcionais e sua eficácia como bioativos (Da-Silva; Lago-Vanzela; Baffi, 2015). Dessa forma, fatores como a estrutura química, a concentração e as interações com a matriz alimentar são determinantes para a viabilidade de aplicação da vinagreira em produtos diferenciados (Nagar et al., 2021; Ribas- Agustí et al., 2018). Para contornar esses desafios e viabilizar a aplicação dos extratos fenólicos obtidos da vinagreira em alimentos, este estudo adotou duas abordagens principais: o desenvolvimento de xaropes adoçados e a obtenção de extratos desidratados por secagem em leito de espuma. A formulação de xaropes adoçados busca facilitar a incorporação dos compostos bioativos em diferentes produtos alimentícios, tornando-os mais acessíveis à indústria. No entanto, a estabilidade desses xaropes ao longo do armazenamento deve ser avaliada para garantir a preservação da cor e das concentrações dos compostos bioativos presentes (Aaby; Amundsen, 2023). Paralelamente, a secagem do extrato tem como objetivo a obtenção de pós solúveis e estáveis, e com elevada importante concentração de CF. Dentre as técnicas de secagem, o leito de espuma se destaca como uma alternativa viável, de fácil execução e baixo custo, permitindo a ampliação do uso desses extratos em diversas formulações alimentares sem comprometer sua funcionalidade (Tavares et al., 2015; 2017; 2019). 103 CONCLUSÃO GERAL Os resultados obtidos neste trabalho evidenciam a viabilidade e o potencial dos uso dos CV para a produção e aplicação de bioingredientes. A caracterização físico-química dos CV in natura e desidratados revelou a influência do processo de secagem, o qual proporcionou parâmetros seguros de umidade (11,61%) e Aw (0,56). Além disso, foram observadas significativas concentrações de AT (18,50 g ácido málico·100 g-1 ), SS (1,93 °Brix), CFT (21,90 mg EAG·g-1), ANT (6,10 mg de mv-3,5-diglc·g-1 ) e ANP (3,30 mg de mv-3,5-diglc·g-1), destacando a potencialidade dos CVD como matéria-prima rica em CF. Para validar o potencial dos CVD na obtenção de extratos fenólicos, a extração com metanol acidificado (controle) apresentou maior retenção de CFT (17,48 mg EAG·g⁻¹, B.S.). Por outro lado, a extração com água demonstrou um bom potencial, alcançando valores médios de 16,15 mg EAG·g⁻¹ (B.S.). A análise por HPLC-DAD-ESI-MSn revelou que a concentração de total antocianinas (mg cy-3-glc·kg-1) variou entre 2726,09 (extrato 60% etanol) e 4598,70 (extrato aquoso), sendo identificadas como majoritárias a delfinidina-3-sambubiosídeo e a cianindina-3-sambubiosídeo. Esses resultados reforçam a extração aquosa como uma alternativa viável, segura e sustentável para a obtenção de extratos fenólicos e seus bioingredientes. Os xaropes apresentaram retenções de CFT entre 94,94% (E15) e 113,86% (E35) e de ANT entre 78,41% (E15) e 91,20% (E35), mantendo os parâmetros cromáticos, especialmente nas formulações com maiores concentrações de sacarose (E25 e E35). Os extratos sem sacarose (E0) também demonstraram boa estabilidade desses compostos, com retenções finais de 93,61% para CFT e 91,06% para ANT. Ambos os tipos de extrato mantiveram sua estabilidade durante 30 dias de armazenamento refrigerado (4 ºC). A secagem em leito de espuma, utilizada para a obtenção do BIP, resultou em uma coloração vermelha preservada (15,61º), mesmo após a incorporação ao iogurte (25,44º). Dessa forma, as estratégias adotadas neste estudo – extração com água, adição de sacarose e secagem em leito de espuma – mostraram-se eficazes na preservação dos compostos bioativos e das propriedades cromáticas dos extratos de CVD. Essas abordagens ampliam as possibilidades de aplicação dos CV como bioingrediente em diversos produtos alimentícios, consolidando-se como uma alternativa sustentável para a indústria alimentícia. 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