Desenvolvimento de uma metodologia inovadora para a determinação e caracterização de aminas biogénicas em amostras de fluido ruminal
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2016
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No trato digestivo dos ruminantes, o alimento é extensivamente fermentado no rúmen pela população microbiana, produzindo maioritariamente ácidos gordos voláteis, metano, dióxido de carbono e amoníaco. Existem, ainda, outros compostos voláteis formados em menores quantidades como dimetilsulfureto, acetaldeído, aminas, álcoois, fenóis, aldeídos e cetonas. As aminas biogénicas resultam maioritariamente da descarboxilação de aminoácidos no rúmen. A influência destes compostos tem sido avaliada devido ao crescente interesse sobre a influência destes na saúde dos ruminantes.
Com este trabalho pretendeu-se estudar a presença de aminas biogénicas em fluido ruminal na tentativa de relacionar a sua composição com a dieta alimentar. Para isso foi desenvolvida uma metodologia de extração baseada na difusão gasosa que consiste na volatilização das aminas biogénicas, diretamente da amostra, que são retidas numa coluna de extração em fase sólida (SPE). As aminas são posteriormente derivatizadas na coluna de SPE com uma solução de o-ftalaldeído (OPA). De seguida é feita a eluição dos derivados com um solvente orgânico e análise por cromatografia líquida de alta eficiência com deteção fluorimétrica (HPLC-FLD).
A metodologia desenvolvida foi otimizada através do estudo de diversos parâmetros de extração e derivatização tais como o tempo e a temperatura de extração, o pH e a concentração OPA. A metodologia otimizada foi aplicada à identificação e quantificação de aminas biogénicas em amostras de fluido ruminal.
Foi também utilizada outra técnica de extração- microextração por difusão gasosa (GDME) na qual é possível realizar os processos de extração e derivatização em simultâneo.
As principais aminas detetadas nas amostras analisadas foram a metilamina, etilamina, tiramina e iso-pentilamina. No conteúdo ruminal do animal alimentado com feno-silagem verificou-se um maior teor destas aminas relativamente ao de palha.
In the digestive tract of ruminants, the feed is extensively fermented by the rumen microbial population, mainly producing volatile fatty acids, methane, carbon dioxide and ammonia. There are also other volatile compounds formed in minor amounts as dimethylsulfide, acetaldehyde, amines, alcohols, phenols, aldehydes and ketones. Biogenic amines result mainly from the decarboxylation of amino acids in the rumen. The influence of these compounds has been evaluated due to growing concern about their effect on the ruminants’ health. This work aims to study the presence of biogenic amines in ruminal fluid trying to relate its composition to the diet. For that an extraction methodology based on gas diffusion was developed based on the volatilization of biogenic amines directly from the sample, and their retention in a solid phase extraction (SPE) column. Subsequently the amines are derivatized in the SPE column with a o-phthaldehyde (OPA) solution. Then the elution of the derivatives is performed with an organic solvent and analyzed by high-performance liquid chromatography with fluorimetric detection (HPLC-FLD). The methodology was optimized by studying the various parameters of the extraction and derivatization such as the time and temperature of extraction, pH and concentration of OPA. The optimized method was then applied to the identification and quantification of biogenic amines in rumen fluid samples. It was also used another extraction technique, the gas-diffusion microextraction (GDME) in which the extraction and derivatization can be performed simultaneously. The main amines detected in the analyzed samples were methylamine, ethylamine, tyramine and iso-pentylamine. In the rumen content of animals fed hay silage the amines content was higher than those verified for the animals fed straw.
In the digestive tract of ruminants, the feed is extensively fermented by the rumen microbial population, mainly producing volatile fatty acids, methane, carbon dioxide and ammonia. There are also other volatile compounds formed in minor amounts as dimethylsulfide, acetaldehyde, amines, alcohols, phenols, aldehydes and ketones. Biogenic amines result mainly from the decarboxylation of amino acids in the rumen. The influence of these compounds has been evaluated due to growing concern about their effect on the ruminants’ health. This work aims to study the presence of biogenic amines in ruminal fluid trying to relate its composition to the diet. For that an extraction methodology based on gas diffusion was developed based on the volatilization of biogenic amines directly from the sample, and their retention in a solid phase extraction (SPE) column. Subsequently the amines are derivatized in the SPE column with a o-phthaldehyde (OPA) solution. Then the elution of the derivatives is performed with an organic solvent and analyzed by high-performance liquid chromatography with fluorimetric detection (HPLC-FLD). The methodology was optimized by studying the various parameters of the extraction and derivatization such as the time and temperature of extraction, pH and concentration of OPA. The optimized method was then applied to the identification and quantification of biogenic amines in rumen fluid samples. It was also used another extraction technique, the gas-diffusion microextraction (GDME) in which the extraction and derivatization can be performed simultaneously. The main amines detected in the analyzed samples were methylamine, ethylamine, tyramine and iso-pentylamine. In the rumen content of animals fed hay silage the amines content was higher than those verified for the animals fed straw.
Descrição
Dissertação de Mestrado em Bioquímica
Palavras-chave
Bioquímica , Aminas biogénicas , Composto volátil , Ruminante , Fluido ruminal