A group of biologically active compounds with various chemical structures called folate is well known by its major role in the transport of one-carbon groups in numerous metabolic reactions in humans. It takes part in core intracellular cycles such as in the synthesis of nucleic acids, in production, methylation and regeneration of DNA, in remethylation of homocysteine and synthesis of various amino acids. Due to its crucial role in cell production, inadequate folate intake can lead to occurrence of various chronic diseases such as neural tube defects, anemia, certain types of cancer and cognitive degenerative diseases. As it is synthesized by plants and microorganisms, folate is widely present in nature. However, due to its minor concentrations in food, sensitivity and complexity of its chemical structure, and complexity of food matrices, folate analysis in food is challenging. Three main steps included in folate quantification are extraction, deconjugation and detection. Deconjugation or removal of polyglutamyl tail from the biologically active part of the molecule by the use of ɣ-glutamyl hydrolase (GGH) is a bottle-neck in folate analysis. Numerous studies have been performed in order to obtain reliable data on folate content, but there are many discrepancies in these data, depending on the natural variation of folate occurrence, but also on the method used for folate analysis. So far, a GGH of animal origin is mainly used for folate deconjugation. Microbiological assay as the only standardized method for folate detection in food is incapable of distinguishing among various folate forms, which have different stability and presumably bioavailability. Therefore, the main hypothesis in this PhD project is that the use of a recombinant plant origin GGH can be a starting point for the standardization of the deconjugation step by providing fast and effective deconjugation in various food matrices, with possibility of distinguishing among biologically active folate forms. The overall aim was to establish research based documentation for a new, accurate and precise chemical method for quantification of folate forms, and further investigate a new strategy to produce folate fortified food products.
Therefore, the main focus was on the development of a rapid, sensitive and reproducible method for folate quantification in various groups of foods. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection, was chosen as a method for analysis of various folate forms due to its selectivity and sensitivity. The developed LC-MS/MS method was validated for the quantification of folate forms such as tetrahydrofolate, formyl folate forms (10-formylfolic acid, 5,10-methenyltetrahydrofolate, 5-formyltetrahydrofolate), folic acid, 5-methyltetrahydrofolate and 10-methylfolic acid using three 13C5-labeled internal standards. A single-enzyme extraction provided satisfactory deglutamylation with enzyme activity being >95% after 1 hour of incubation.
Validation parameters of trueness at 80-120% in spiked samples and certified reference materials, and a precision <10% were met in all compounds of interest except tetrahydrofolate. In the future, quantification of tetrahydrofolate as the least stable folate form should be performed by the use of a corresponding isotopically labeled internal standard.
The single-laboratory validated method using recombinant GGH of plant origin was compared to microbiological assay and another LC-MS/MS method using the animal origin GGH in a round robin study on a large and diverse sample set. Food groups of fruits, vegetables, legumes, cereals, dairy products, meat and offal represented a sample set of 89 samples analyzed for the folate content. A significantly lower constant bias of 17% (p ≤ 0.05) was observed when the method using plant origin GGH was compared to microbiological assay. This indicates that folate daily intake calculated from the national food composition databases that normally contain data obtained by the use of microbiological assay, is overestimated. On the other side, comparison to another LCMS/MS method using animal origin GGH evidenced superiority of deconjugation activity of plant origin GGH in plant matrices. Especially in the groups of fruits, vegetables and cereals constant bias of 25% was shown (p ≤ 0.05). The reason for the difference is hypothesized to be due to inhibition of the GGH of animal origin in the plant food matrices. The superior activity of the GGH of plant origin over the commonly used GGH of animal origin encourage the incorporation of the new GGH for future standardization of an accurate, precise, and specific method for folate vitamers.
Food groups of offal, vegetables, legumes and certain fruits were shown to be sources rich in folate. 5-methyltetrahydrofolate was the most abundant folate form in the mentioned groups and the second most abundant in food groups of cereals, meat and dairy products after formyl forms. However, folate distribution varied within the food group and between different varieties of the same foodstuffs. Even though they were not considered as folate sources, food groups of cereals and dairy products contribute to the folate intake among Danes due to their wide consumption. Therefore, a fortification strategy to increase the folate content in products made from flour by fortification of flour with biologically active folate form 5-methyltetrahydrofolate was studied. Due to its instability, 5-methyltetrahydrofolate was incorporated into food system in the forms of nanocapsules, where it was protected by carbohydrate matrix produced by the electrospraying encapsulation. A significant improvement in the stability of 5-methyltetrahydrofolate was achieved via encapsulation, even though its incorporation into food system did not confirmed the need for encapsulation, indicating the importance of future investigation of this fortification strategy.