Development and Validation of Chromatographic Methods to Study Folate Derivatives Produced by Yeasts

Patring, J. 2007. Development and Validation of Chromatographic Methods to Study Folate Derivatives Produced by Yeasts. Doctoral thesis. ISSN 1652-6880. ISBN 91-576-7330-5. Folate, or folic acid, is an important water soluble B vitamin that exists in many different forms. Its presence is necessary for synthesis of DNA and RNA and methylation of homocysteine to methionine. Deficiency of folate is closely connected to increased risk of neural tube defects, a serious birth defect, and development of anaemia. Humans cannot synthesise folate by themselves and therefore depend on an adequate supply through food intake. However, the recommended daily intake of folate is not reached by many people, especially women in fertile ages. This thesis aimed to investigate possibilities for enhancing folate production from yeast for use as biofortificants in food to increase the natural folate content. To quantify folate, analytical methods were developed and validated. This involved optimisation of sample pre-treatment steps, HPLC methods and LC-MS methods. The main findings were that folate analysis of yeast could be facilitated by excluding an SPE step prior to HPLC analysis, that sample handling and choice of antioxidant greatly influenced folate stability, that choice of reversed-phase column considerably affected folate separation/retention and that LC-MS provided a powerful tool for yeast folate analysis. These developed analytical methods were used to investigate differences between yeast strains to produce folates. Furthermore, effect of cultivation conditions on folate content in yeast was studied. The main conclusions from these experiments were that careful selection of yeast strain may considerably increase folate content in food. It was also shown that folate concentration in yeast was significantly increased by optimising the cultivation procedure. The folate derivatives that were found to exist in yeast were tetrahydrofolate, 5methyltetrahydrofolate, 10-formylfolic acid and 5-formyltetrahydrofolate. The findings in this thesis show that there are great possibilities for increasing folate content in yeast-fermented foods, e.g. bread and dairy products, if a proper yeast strain is used under optimal growth conditions in appropriate culturing media.