Carotenoid and carotenoid esters : analysis and in vitro digestion in individual and co-consumed foods

Orientadores: Adriana Zerlotti Mercadante, Lilian Regina Barros MariuttiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O crescente interesse no comportamento dos carotenoides e ésteres de carotenoides durante o processo de digestão e seu impacto na saúde humana, faz dos ensaios de bioacessibilidade in vitro destes compostos um dos tópicos mais atuais de estudo na área de Alimentos e Nutrição. Aspectos desafiadores relacionados à análise de carotenoides e ésteres de carotenoides nos alimentos, requisito para determinação da sua bioacessibilidade, e aos métodos de digestão in vitro utilizados para este fim, foram abordados nesta tese. A identificação do perfil nativo de ésteres de carotenoides é frequentemente negligenciada devido à complexidade desta análise. Dentre outros fatores, interferentes lipídicos que permanecem no extrato quando a saponificação não é realizada prejudicam ou mesmo impedem a identificação dos compostos. Nesse sentido, um novo procedimento de limpeza pré-cromatográfico em duas etapas foi desenvolvido para tornar viável a análise da composição nativa de carotenoides de murici, uma fruta da Amazônia. Os interferentes (principalmente triacilglicerois) foram eficientemente removidos após separação física seguida de cromatografia em coluna aberta, possibilitando a identificação de 35 carotenoides (seis carotenoides livres, 14 monoésteres, e 15 diésteres) no extrato não saponificado de murici, por HPLC-DAD(APCI)MS/MS, enquanto apenas 6 compostos foram identificados quando a limpeza não foi realizada. Além disso, a publicação recente, pela ação INFOGEST, de um novo método de digestão in vitro de consenso internacional, chamou a atenção para a necessidade de padronização das condições de digestão simulada de alimentos em todo o mundo. O método INFOGEST, no entanto, não contempla passos chave para a determinação da bioacessibilidade de carotenoides, e tem se mostrado trabalhoso, demorado e oneroso. Diante disso, este método foi adaptado para análise de carotenoides com as etapas de separação da fração micelar e extração de carotenoides das micelas, e aplicado com sucesso na determinação da bioacessibilidade de ésteres de carotenoides de murici e de carotenoides em um amplo grupo de alimentos que são fontes destes compostos. Os resultados obtidos com o método INFOGEST adaptado foram comparados aos encontrados com métodos mais simples, tradicionalmente utilizados para estimar a bioacessibilidade de carotenoides e ésteres de carotenoides. O método INFOGEST adaptado forneceu valores de bioacessibilidade de carotenoides e ésteres de carotenoides de murici maiores do que os resultados obtidos usando o método de digestão que vinha sendo utilizado em nosso laboratório, publicado em 2014. A bioacessibilidade variou de 4 a 29%, dependendo do método utilizado e da estrutura do carotenoide, e, em geral, carotenoides livres apresentaram maior eficiência de micelarização. Quando comparado ao primeiro método de digestão in vitro adaptado para carotenoides, publicado em 1999, o método INFOGEST adaptado forneceu estimativas similares de bioacessibilidade de carotenoides tanto em alimentos individuais como combinados, e uma correlação positiva foi encontrada entre os dois conjuntos de dados. Ainda, a adição de ovo cozido à salada vegetal aumentou a bioacessibilidade in vitro de luteína e licopeno, enquanto a co-digestão com salmão promoveu maior micelarização de 'alfa'-caroteno, 'beta'-caroteno e luteína, independentemente do método de digestão empregado. Com os resultados obtidos durante este período 3 artigos foram submetidos a revisão por pares e publicados em periódicos internacionais indexadosAbstract: The interest in understanding the fate of carotenoid and carotenoid esters through the digestion process and its impact in human health is growing, making their bioaccessibility assays one of the most innovative issues of study in the field of Food and Nutrition. Challenging aspects related to the analysis of carotenoids and carotenoid esters in foods, which is a requirement for assessing their bioaccessibility, and to the in vitro digestion methods used for this purpose were addressed in this thesis. The identification of the native carotenoid profile of foods is often overlooked because of the complexity of such analysis. Among other factors, interfering lipids that remain in the extract when no saponification step is carried out impair or even preclude the compound identification. In this sense, a new pre-chromatographic two-step cleanup procedure was developed to make feasible the identification of the native carotenoid composition of murici, an Amazonian fruit. Interfering compounds (mainly triacylglycerides) were efficiently removed after physical separation followed by open column chromatography, thereby allowing the identification of 35 carotenoids (six free carotenoids, 14 monoesters and 15 diesters) in non-saponified extracts from murici by HPLC-DAD(APCI)MS/MS, whereas only 6 compounds were identified when no cleanup procedure was performed. In addition, a recent publication of a new in vitro digestion method as an international consensus by the INFOGEST action drew attention to the necessity for standardization of the in vitro digestion conditions of foods at the international level. The INFOGEST method, however, does not address crucial steps needed to assess the carotenoid bioaccessibility, and it is more laborious, time-consuming and expensive than the traditionally used ones. Therefore, the INFOGEST method was adapted for carotenoid analysis by coupling the steps of micellar fraction separation and carotenoid extraction from the micelles, which allowed the successful determination of the in vitro bioaccessibility of carotenoid esters in murici and bioaccessibility of carotenoids in a large group of carotenoid-rich foods. The results obtained with the adapted INFOGEST method were compared to those found using relatively simple and consolidated in vitro digestion models, traditionally used to estimate the bioaccessibility of carotenoids and carotenoid esters. The adapted INFOGEST method provided values of carotenoid and carotenoid ester bioaccessibility in murici higher than the results obtained using the digestion method that was being used in our laboratory, published in 2014. The carotenoid bioaccessibility ranged from 4 to 29%, depending on the digestion method and carotenoid structure, and free carotenoids overall presented higher efficiencies of micellarization than free carotenoids and monoesters. Moreover, compared with the first in vitro digestion method adapted for carotenoids, published in 1999, the adapted INFOGEST method generally provided similar estimates of carotenoid bioaccessibility during the digestion of both individual and combined foods, and a positive correlation was found between the two sets of data. Furthermore, addition of cooked egg to the vegetable salad increased the in vitro bioaccessibility of lutein and lycopene, while the co-digestion of pan-fried salmon promoted the micellarization of 'alpha'-carotene, 'beta'-carotene and lutein, regardless of the digestion method employed. Three scientific papers were published in international peer-reviewed indexed journals with the results obtained during this periodDoutoradoCiência de AlimentosDoutora em Ciência de Alimentos2013/23218-1FAPES

Potential for storage of chemically treated soybean seeds

A cultura da soja atualmente esta entre as principais commodities do agronegócio brasileiro. Diversos estudos relacionam o efeito da alta qualidade de sementes de soja na produção da cultura, entretanto a utilização de tratamento químico de sementes pode auxiliar na manutenção da qualidade durante o armazenamento e assegurar o estabelecimento de plântulas a campo. Neste contexto o presente trabalho objetivou avaliar o comportamento de diferentes formulações para tratamento de sementes durante 60 dias de armazenamento. Foram utilizados nove tratamentos de sementes, incluindo o controle (sementes não tratadas) para avaliar a influência das formulações na qualidade fisiológica de sementes de soja. As avaliações foram realizadas aos 0, 30 e 60 dias de armazenamento. Para mensurar o efeito de cada tratamento foram testadas germinação, envelhecimento acelerado, comprimento de parte aérea, comprimento de raiz, matéria seca de parte aérea e matéria seca de raiz. Observou-se que a utilização de tratamento químico não afeta negativamente o potencial de armazenamento de sementes, porem as formulações Tiametoxan, Abamectina+Tiametoxan+Fludioxonil,Fipronil+Tiofanato etílico+Piraclostrobina, Carboxamida, Metalaxyl-m+Fluioxonil, favorecem a conservação do vigor de sementes.The soybean crop is currently among the main commodities of Brazilian agribusiness. Several studies have related the effect of the high quality of soybean seeds on the production of the crop, however the use of chemical treatment of seeds can help to maintain quality during storage and ensure the establishment of field seedlings. In this context the present work aimed to evaluate the behavior of different formulations for treatment of seeds during 60 days of storage. Nine seed treatments, including control (untreated seeds) were used to evaluate the influence of the formulations on the physiological quality of soybean seeds. Evaluations were performed at 0, 30 and 60 days of storage. To measure the effect of each treatment were tested germination, accelerated aging, shoot length, root length, shoot dry matter and root dry matter. It was observed that the use of chemical treatment does not negatively affect the storage potential of seeds, but the formulations Tiametoxan, Abamectin + Tiametoxan + Fludioxonil, Fipronil + Ethyl thiophanate + Piraclostrobina, Carboxamide, Metalaxyl-m + Fluioxonil, favor conservation of vigor of seeds

Carotenoid and carotenoid esters : analysis and in vitro digestion in individual and co-consumed foods

Orientadores: Adriana Zerlotti Mercadante, Lilian Regina Barros MariuttiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O crescente interesse no comportamento dos carotenoides e ésteres de carotenoides durante o processo de digestão e seu impacto na saúde humana, faz dos ensaios de bioacessibilidade in vitro destes compostos um dos tópicos mais atuais de estudo na área de Alimentos e Nutrição. Aspectos desafiadores relacionados à análise de carotenoides e ésteres de carotenoides nos alimentos, requisito para determinação da sua bioacessibilidade, e aos métodos de digestão in vitro utilizados para este fim, foram abordados nesta tese. A identificação do perfil nativo de ésteres de carotenoides é frequentemente negligenciada devido à complexidade desta análise. Dentre outros fatores, interferentes lipídicos que permanecem no extrato quando a saponificação não é realizada prejudicam ou mesmo impedem a identificação dos compostos. Nesse sentido, um novo procedimento de limpeza pré-cromatográfico em duas etapas foi desenvolvido para tornar viável a análise da composição nativa de carotenoides de murici, uma fruta da Amazônia. Os interferentes (principalmente triacilglicerois) foram eficientemente removidos após separação física seguida de cromatografia em coluna aberta, possibilitando a identificação de 35 carotenoides (seis carotenoides livres, 14 monoésteres, e 15 diésteres) no extrato não saponificado de murici, por HPLC-DAD(APCI)MS/MS, enquanto apenas 6 compostos foram identificados quando a limpeza não foi realizada. Além disso, a publicação recente, pela ação INFOGEST, de um novo método de digestão in vitro de consenso internacional, chamou a atenção para a necessidade de padronização das condições de digestão simulada de alimentos em todo o mundo. O método INFOGEST, no entanto, não contempla passos chave para a determinação da bioacessibilidade de carotenoides, e tem se mostrado trabalhoso, demorado e oneroso. Diante disso, este método foi adaptado para análise de carotenoides com as etapas de separação da fração micelar e extração de carotenoides das micelas, e aplicado com sucesso na determinação da bioacessibilidade de ésteres de carotenoides de murici e de carotenoides em um amplo grupo de alimentos que são fontes destes compostos. Os resultados obtidos com o método INFOGEST adaptado foram comparados aos encontrados com métodos mais simples, tradicionalmente utilizados para estimar a bioacessibilidade de carotenoides e ésteres de carotenoides. O método INFOGEST adaptado forneceu valores de bioacessibilidade de carotenoides e ésteres de carotenoides de murici maiores do que os resultados obtidos usando o método de digestão que vinha sendo utilizado em nosso laboratório, publicado em 2014. A bioacessibilidade variou de 4 a 29%, dependendo do método utilizado e da estrutura do carotenoide, e, em geral, carotenoides livres apresentaram maior eficiência de micelarização. Quando comparado ao primeiro método de digestão in vitro adaptado para carotenoides, publicado em 1999, o método INFOGEST adaptado forneceu estimativas similares de bioacessibilidade de carotenoides tanto em alimentos individuais como combinados, e uma correlação positiva foi encontrada entre os dois conjuntos de dados. Ainda, a adição de ovo cozido à salada vegetal aumentou a bioacessibilidade in vitro de luteína e licopeno, enquanto a co-digestão com salmão promoveu maior micelarização de ?-caroteno, ?-caroteno e luteína, independentemente do método de digestão empregado. Com os resultados obtidos durante este período 3 artigos foram submetidos a revisão por pares e publicados em periódicos internacionais indexadosAbstract: The interest in understanding the fate of carotenoid and carotenoid esters through the digestion process and its impact in human health is growing, making their bioaccessibility assays one of the most innovative issues of study in the field of Food and Nutrition. Challenging aspects related to the analysis of carotenoids and carotenoid esters in foods, which is a requirement for assessing their bioaccessibility, and to the in vitro digestion methods used for this purpose were addressed in this thesis. The identification of the native carotenoid profile of foods is often overlooked because of the complexity of such analysis. Among other factors, interfering lipids that remain in the extract when no saponification step is carried out impair or even preclude the compound identification. In this sense, a new pre-chromatographic two-step cleanup procedure was developed to make feasible the identification of the native carotenoid composition of murici, an Amazonian fruit. Interfering compounds (mainly triacylglycerides) were efficiently removed after physical separation followed by open column chromatography, thereby allowing the identification of 35 carotenoids (six free carotenoids, 14 monoesters and 15 diesters) in non-saponified extracts from murici by HPLC-DAD(APCI)MS/MS, whereas only 6 compounds were identified when no cleanup procedure was performed. In addition, a recent publication of a new in vitro digestion method as an international consensus by the INFOGEST action drew attention to the necessity for standardization of the in vitro digestion conditions of foods at the international level. The INFOGEST method, however, does not address crucial steps needed to assess the carotenoid bioaccessibility, and it is more laborious, time-consuming and expensive than the traditionally used ones. Therefore, the INFOGEST method was adapted for carotenoid analysis by coupling the steps of micellar fraction separation and carotenoid extraction from the micelles, which allowed the successful determination of the in vitro bioaccessibility of carotenoid esters in murici and bioaccessibility of carotenoids in a large group of carotenoid-rich foods. The results obtained with the adapted INFOGEST method were compared to those found using relatively simple and consolidated in vitro digestion models, traditionally used to estimate the bioaccessibility of carotenoids and carotenoid esters. The adapted INFOGEST method provided values of carotenoid and carotenoid ester bioaccessibility in murici higher than the results obtained using the digestion method that was being used in our laboratory, published in 2014. The carotenoid bioaccessibility ranged from 4 to 29%, depending on the digestion method and carotenoid structure, and free carotenoids overall presented higher efficiencies of micellarization than free carotenoids and monoesters. Moreover, compared with the first in vitro digestion method adapted for carotenoids, published in 1999, the adapted INFOGEST method generally provided similar estimates of carotenoid bioaccessibility during the digestion of both individual and combined foods, and a positive correlation was found between the two sets of data. Furthermore, addition of cooked egg to the vegetable salad increased the in vitro bioaccessibility of lutein and lycopene, while the co-digestion of pan-fried salmon promoted the micellarization of ?-carotene, ?-carotene and lutein, regardless of the digestion method employed. Three scientific papers were published in international peer-reviewed indexed journals with the results obtained during this periodDoutoradoCiência de AlimentosDoutora em Ciência de Alimentos2013/23218-1FAPES

An In Vitro Digestion Method Adapted For Carotenoids And Carotenoid Esters: Moving Forward Towards Standardization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)In vitro digestion methods are a useful approach to predict the bioaccessibility of food components and overcome some limitations or disadvantages associated with in vivo methodologies. Recently, the INFOGEST network published a static method of in vitro digestion with a proposal for assay standardization. The INFOGEST method is not specific for any food component; therefore, we aimed to adapt this method to assess the in vitro bioaccessibility of carotenoids and carotenoid esters in a model fruit (Byrsonima crassifolia). Two additional steps were coupled to the in vitro digestion procedure, centrifugation at 20 000g for the separation of the aqueous phase containing mixed micelles and exhaustive carotenoid extraction with an organic solvent. The effect of electrolytes, enzymes and bile acids on carotenoid micellarization and stability was also tested. The results were compared with those found with a simpler method that has already been used for carotenoid bioaccessibility analysis. These values were in the expected range for free carotenoids (5-29%), monoesters (9-26%) and diesters (4-28%). In general, the in vitro bioaccessibility of carotenoids assessed by the adapted INFOGEST method was significantly higher (p < 0.05) than those assessed by the simplest protocol, with or without the addition of simulated fluids. Although no trend was observed, differences in bioaccessibility values depended on the carotenoid form (free, monoester or diester), isomerization (Z/E) and the in vitro digestion protocol. To the best of our knowledge, it was the first time that a systematic identification of carotenoid esters by HPLC-DAD-MS/MS after in vitro digestion using the INFOGEST protocol was carried out.71249925001FAPESP [2012/20988-8, 2013/23218-1, 2012/01506-2]CNPq [308484/2014-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Two-step Cleanup Procedure For The Identification Of Carotenoid Esters By Liquid Chromatography-atmospheric Pressure Chemical Ionization-tandem Mass Spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Carotenoids are naturally found in both free form and esterified with fatty acids in most fruits; however, up to now the great majority of studies only evaluated their composition after saponification. This fact is easily explained by the difficult to analyze carotenoid esters. Preliminary studies showed that cleanup procedures in the extract are necessary for further analysis by LC-MS/MS since triacylglycerols (TAGs) impair the MS detection. Considering these facts, we developed a new cleanup procedure to remove TAGs and other lipids from carotenoid fruit extracts. This procedure is based on physical removal of solid lipids at low temperature followed by open column chromatography on MgO and diatomaceous earth. Before cleanup, four carotenoid diesters and two free xanthophylls were identified in murici (Byrsonyma crassifolia), corresponding to about 65% of the total chromatogram area. After carrying out the two-step cleanup procedure, 35 carotenoids were identified, being 14 monoesters, six free carotenoids and 15 carotenoid diesters. We can conclude that this two-step procedure was successfully applied to murici, an Amazonian fruit, which contains high amounts of lipids. (C) 2016 Elsevier B.V. All rights reserved.1457116124FAPESP [2012/20988-8, 2013/23218-1, 2012/01506-2]CNPq [308484/2014-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Marigold carotenoids : much more than lutein esters

Carotenoids constitute a large group of lipophilic pigments whose health-promoting benefits have been widely recognized. Hydroxy-containing carotenoids can be found in both free form or esterified with fatty acids in several plant matrices, but the native carotenoid profile is overall poorly explored due to the difficulty of analyzing carotenoid esters. One of the main natural sources of carotenoids is the marigold flower, which has been extensively used by the industry for the production of food colorants or supplements, both often manufactured with no saponification process. Although lutein esters are well established as the major compounds naturally found in marigold petals and their products, carotenoid esters other than the lutein ones have not been extensively examined. We carried out a comprehensive identification of carotenoids and carotenoid esters from marigold petals by LC-DAD-(APCI + )MS/MS. Whereas 18 carotenoids were identified in the saponified extract, 56 were identified when no saponification procedure was carried out: 6 free carotenoids, 20 monoesters and 30 diesters. This is the first time that esters of zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and beta-cryptoxanthin are identified in marigold. The structural information obtained through characteristic fragmentation patterns and diagnostic fragments in MS and MS/MS spectra (APCI+) sustained the differentiation between carotenoid esters with similar characteristics. Therefore, the separation of carotenoids by reversed-phase liquid chromatography using C-30 columns in combination with DAD and APCI-MS/MS detection allowed high sensitivity and selectivity for carotenoid ester analysis119653664CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP455748/2014-42013/23218-1; 2015/15238-8The authors acknowledge the financial support of FAPESP (grants #2013/23218-1 and #2015/15238-8), CNPq (455748/2014-4) and FAEPEX-Unicamp (2084/17

Caracterização físico-química de variedades de amora-preta da região sul do Brasil.

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