Desenvolvimento de metodologias para análise simultânea de ácido sórbico, natamicina, nisina e tilosina em produtos lácteos por cromatografia líquida acoplada à espectrometria de massas

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2015.A deterioração dos alimentos de origem animal está relacionada com uma vasta gama de reações, destacando-se as de origem microbiológica. Dentre as substâncias antimicrobianas utilizadas para inibir micro-organismos em produtos lácteos industrializados destacam-se: as bacteriocinas, os antibióticos macrolídeos polienos e os ácidos orgânicos. Com o intuito de garantir a segurança alimentar e a conformidade com a legislação, reprimindo fraudes, métodos de detecção rápida e simultânea para a determinação desses compostos estão em alta demanda. Neste trabalho, foram desenvolvidos métodos com simplificada extração, curto tempo de análise cromatográfica (cerca de dez minutos) e aplicados a diversas matrizes lácteas para determinação simultânea de natamicina, ácido sórbico, nisina e tilosina por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). Todos os parâmetros de validação avaliados ficaram de acordo com os critérios estabelecidos pela Decisão 657/2002/EC. Os métodos foram utilizados para avaliar a conformidade de 135 amostras comerciais, o que demonstra versalidade e aplicabilidade. A ocorrência de não conformidades em 30% das amostras demonstra a importância de controlar o uso de conservantes em produtos lácteos. Muitas das amostras violadas não apresentavam o conservante declarado no rótulo. Métodos rápidos e com confiabilidade (validados) como os desenvolvidos neste trabalho, constituem de ferramentas importantes para a fiscalização de alimentos no Brasil, contribuindo para a segurança alimentar.Abstract : The deterioration of foods of animal origin is due to several reactions, especially of microbiological origin. Among the antimicrobial substances used to inhibit microorganisms in processed dairy products are bacteriocins, macrolide polyene antibiotics and organic acids. In order to ensure food safety and compliance with the laws, fast and simultaneous detection methods for determining these compounds are in high demand. In this study, we developed methods with simplified extraction, short chromatographic analysis time (about ten minutes), which were applied to various dairy products for simultaneous determination of natamycin, sorbic acid, nisin and tylosin by liquid chromatography-tandem mass spectrometry (lc-ms/ms). All validation parameters evaluated were in accordance to the criteria established by commisssion decision 657/2002/ec. The methods were used to assess the compliance of 135 commercial samples, demonstrating its applicability and versatility. The occurrence of non-compliance in 30% of the samples demonstrates the importance of controlling the use of preservatives in dairy products. Many of the violated samples did not have the preservative informed on the label. Fast and reliable methods as the developed in this work proved to be a significant tool for surveillance of dairy products in brazil, contributing to improve food safety

Interações de conservantes no processamento de carnes: formação de compostos carcinogênicos, métodos de controle e agentes inibitórios

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2019.O câncer é a segunda maior causa de morte humana na atualidade. É considerada uma doença multifatorial, podendo estar associado a fatores ambientais como a dieta. Esta tese, composta por cinco capítulos, teve como principal objetivo elucidar a formação dos mutagênicos 2-metil-1,4-dinitro-pirrol (DNMP) e do ácido etilnitrólico (ENA) na reação entre conservantes utilizados no processamento de carnes. Para isso, diversas ferramentas analíticas e modelos estatísticos foram desenvolvidos com êxito proporcionando inovação química a esse cenário. Os resultados obtidos corroboram a decisão da Agência Internacional de Pesquisa sobre Câncer (IARC) de classificar as carnes processadas como cancerígenas para humanos. Mais de 100 amostras comerciais foram analisadas e 38% continham adição irregular de nitrito e sorbato. Além da utilização excessiva de conservantes, ENA (> 5,00 µg kg-1) e DNMP (> 12,0 µg kg-1) foram detectados em amostras de mortadela, bacon, linguiça calabresa e patê. Além disso, mortadelas produzidas em escala piloto com 0,3% de nitrito e 0,034% de sorbato, sem eritorbato de sódio, apresentaram altas concentrações de ENA e DNMP. Esses resultados alertam sobre um grande problema de saúde pública, demandando incremento da fiscalização e Ciência dos Alimentos para sua resolução. Dessa forma, o micro-organismo Lactobacillus bulgaricus (LB-UFSC 001) foi empregado em um tratamento experimental para que, através da fermentação, pudesse biodegradar ENA e DNMP na mortadela fabricada em escala piloto. Houve diferença significativa (p 5.00 µg kg-1) and DNMP (> 12.0 µg kg-1) were detected in samples of mortadella, bacon, pepperoni, and pâté. Moreover, high concentrations of ENA and DNMP were quantitated in mortadella produced on a pilot scale with 0.3% nitrite and 0.034% sorbate without sodium erythorbate. These results warn of a major public health problem, demanding the improvement of official inspection and food science for its resolution. Thus, the microorganism Lactobacillus bulgaricus (LB-UFSC 001) was used in an experimental treatment to biodegrade ENA and DNMP by fermentating mortadella samples produced in pilot scale. There was a significant difference (p < 0.05) in ENA and DNMP concentrations in the mortadellas before and after treatment. The identification of the main metabolites contributed to the elucidation of the main metabolic pathways, demonstrating that LB-UFSC 001 uses nitro reductases and nitrile hydratases to modulate mutagenic compounds in the meat matrix. This thesis has shown that understanding the mechanisms involved in carcinogen formation and inhibition, as well as the chemical and microbiological composition of the matrix, can provide clearer evidence on the cancer risk ratings attributed to the consumption of processed meats worldwide

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.Carolina Turnes Pasini Deolindo acknowledges her master scholarship from CAPES (Project/process 88882.344959/2019-01). Cristina Bosch Orea acknowledges her scholarship Becas Iberoamérica Santander Investigación from Santander Group.Peer reviewe

Oxidative Stress Modulates DNA Methylation during Melanocyte Anchorage Blockade Associated with Malignant Transformation1

Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. lG-Nitro-l-arginine methyl esther (l-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-l-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both l-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism

Integrated analysis of marine biotoxins and contaminants of emerging concern in bivalve mollusks from Santa Catarina, Brazil

Santa Catarina is the main producer state of oysters and mussels in Brazil, reaching 98 % of national production. To assure the safety of bivalve mollusks production, control programs of marine biotoxins (MBs) have been continuously performed. Herein, the co-occurrence of MBs and contaminants of emerging concern (CECs) in oyster and mussels from the main production sites of Santa Catarina was reported, covering 178 compounds. Samples of wild and non-cultivated oysters and mussels were also assessed. Chemometric tools were used to evaluate and optimize several sample preparation techniques such as solid-liquid, ultrasound assisted, and pressurized liquid extraction. The optimized protocol was based on ultrasound assisted extraction followed by liquid chromatography coupled to tandem mass spectrometry. The results showed the incidence of several CECs and MBs. In the case of MBs, all results were below the regulatory limits for both cultivated and non-cultivated samples. Wild mollusks have shown a higher number of compounds. Regarding CECs, the more frequent compounds were caffeine, diclofenac, meloxicam, and sertraline. Domoic acid and okadaic acid were the main toxins detected. The results highlighted the need of monitoring for MBs and the potential of oyster and mussels as sentinel organisms to risk analysis of CECs in coastal regions. To the best of our knowledge, this is the first method to describe a simultaneous sample preparation and analysis of CECs and MBs in bivalve mollusks, as well as the first report of meloxicam and florfenicol in mussels and oysters.The authors would like to thank our colleague Mauricio Raimundo for the sampling of non-cultivated oysters from the São José bay area. Carolina Turnes Pasini Deolindo acknowledges her master scholarship from CAPES (Project/process 88882.344959/2019-01). Cristina Bosch-Orea acknowledges her scholarship Becas Iberoamérica Santander Investigación from Santander Group.Peer reviewe

A new analytical concept based on chemistry and toxicology for herbal extracts analysis: From phenolic composition to bioactivity

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