On Folates in Yeasts -Exploring Yeasts as Vehicles for Biofortification of Folates in Food

High intake of the B vitamin folate during pregnancy is known to decrease the risk for development of neural tube defects. In addition, a good folate status may prevent the progression of several diseases such as megaloblastic anaemia, cardiovascular disease, cancer and Alzheimer’s disease. However, reaching sufficient amounts via the diet only is complicated and new strategies must be developed to increase the mean folate intake. The aim of this thesis is to explore the use of yeasts for biofortification of folates in food. Yeasts synthesise folates de novo and may constitute potent vehicles for natural folates. Current work includes investigations of folate content and composition in yeasts as well as studies of the impact of growth rate and chemical environments on folate levels in Saccharomyces cerevisiae (Baker’s yeast). The applicability of the results was then investigated in two food model systems (bread and a cereal-based fermented porridge called togwa) to demonstrate the practical relevance. In a screening of 52 yeast strains, the folate content ranged from 40 to 145 \ub5g per gram dry matter of yeast, showing that inherent variations in yeast folate levels exist. Several S. cerevisiae strains showed at least a two-fold higher folate content than a commercial Baker’s strain, indicating possibilities to increase folates in fermented foods simply by choosing the proper strain. With regard to cultivation conditions, folates in S. cerevisiae were demonstrated to increase linearly with growth rate in a defined medium, showing that high growth rate, i.e. respiro-fermentative metabolism, is most favourable for high folate content. Cultivation in a rich medium yielded cells containing lower amounts of folate compared to cells grown in a poor chemical environment even though the specific growth rate was higher in the rich medium. The results obtained show that the growth rate per se could not predict intracellular folate concentrations and that the chemical environment has a substantial impact on intracellular folates. By applying strain selection and cultivation at optimal conditions prior to baking we were able to increase folates five-fold in white wheat bread compared to bread leavened with commercial Baker’s yeast. In addition, folates increased at most 23-fold during yeast fermentations in a cereal-based porridge (togwa) compared to togwa raw material. Final folate levels were shown to be highly dependent on strain and cultivation time. The results demonstrate that yeasts constitute flexible and controllable vehicles for biofortification of folates in fermented food

On Folates in Yeasts -Exploring Yeasts as Vehicles for Biofortification of Folates in Food

High intake of the B vitamin folate during pregnancy is known to decrease the risk for development of neural tube defects. In addition, a good folate status may prevent the progression of several diseases such as megaloblastic anaemia, cardiovascular disease, cancer and Alzheimer’s disease. However, reaching sufficient amounts via the diet only is complicated and new strategies must be developed to increase the mean folate intake. The aim of this thesis is to explore the use of yeasts for biofortification of folates in food. Yeasts synthesise folates de novo and may constitute potent vehicles for natural folates. Current work includes investigations of folate content and composition in yeasts as well as studies of the impact of growth rate and chemical environments on folate levels in Saccharomyces cerevisiae (Baker’s yeast). The applicability of the results was then investigated in two food model systems (bread and a cereal-based fermented porridge called togwa) to demonstrate the practical relevance. In a screening of 52 yeast strains, the folate content ranged from 40 to 145 \ub5g per gram dry matter of yeast, showing that inherent variations in yeast folate levels exist. Several S. cerevisiae strains showed at least a two-fold higher folate content than a commercial Baker’s strain, indicating possibilities to increase folates in fermented foods simply by choosing the proper strain. With regard to cultivation conditions, folates in S. cerevisiae were demonstrated to increase linearly with growth rate in a defined medium, showing that high growth rate, i.e. respiro-fermentative metabolism, is most favourable for high folate content. Cultivation in a rich medium yielded cells containing lower amounts of folate compared to cells grown in a poor chemical environment even though the specific growth rate was higher in the rich medium. The results obtained show that the growth rate per se could not predict intracellular folate concentrations and that the chemical environment has a substantial impact on intracellular folates. By applying strain selection and cultivation at optimal conditions prior to baking we were able to increase folates five-fold in white wheat bread compared to bread leavened with commercial Baker’s yeast. In addition, folates increased at most 23-fold during yeast fermentations in a cereal-based porridge (togwa) compared to togwa raw material. Final folate levels were shown to be highly dependent on strain and cultivation time. The results demonstrate that yeasts constitute flexible and controllable vehicles for biofortification of folates in fermented food

Growth rate and medium composition strongly affect folate content in Saccharomyces cerevisiae

Folate content in a Saccharomyces cerevisiae strain was monitored during aerobic batch fermentation in synthetic growth medium, yeast peptone dextrose medium, and a molasses based medium. During growth in the synthetic medium large differences in intracellular folate content was observed at different phases. Specific folate levels, expressed per unit biomass, were highest during respiro-fermentative growth (120 mu g/g) and decreased during the respiratory and stationary phases. Thus, the physiological state of the cells clearly affects the folate content. This was confirmed in chemostat Cultures where total intracellular folate content increased linearly with increasing growth rate (r(2) = 0.998), indicating high growth rate i.e. respiro-fermentative growth to be most favourable to obtain high specific folate content. In complex media however, much lower folate content (15-40 mu g/g) was found throughout the batch growth. Only minor growth-phase related differences were detected. This shows the impact of cultivation medium on folate content in yeast. To further investigate which components that influence folate content, batch experiments in synthetic medium with addition of specific components were performed. Adding a raw mixture of peptides and amino acids (peptone) decreased folate levels extensively (90%) whereas adding amino acids one-by-one only had minor effects on the intracellular folate content. Furthermore, supplementing synthetic medium with pABA, folate or nucleotides did not change the intracellular folate content. This work constitutes the first steps towards an optimised process for production of natural folates for fortification Purposes, as well as an effort to gain fundamental understanding of folate requirements in yeast in relation to environmental conditions

Biofortification of folates in white wheat bread by selection of yeast strain and process

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 mu g/100 dry matter), compared to white wheat bread leavened with commercial Baker\u27s yeast (27-43 mu g/100 g). The commercial Baker\u27s yeast strain had been industrially produced, using a fedbatch process. thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products

Inherent biodiversity of folate content and composition in yeasts

We have studied folate content and composition in 44 yeast strains cultivated in a synthetic medium at standardised conditions. Folates were measured by a validated HPLC method. The total folate content ranged from 4000 to 14,500 mu g/100g dry matter showing a large biodiversity among studied yeasts. The forms found were 5-CH3-H(4)folate and H(4)folate, also varying extensively in relative amounts between different strains. Several strains showed a showed two-fold or higher folate content as compared to the control strain-a commercial strain of Baker's yeast. This indicates that folate content in yeast-fermented foods may be more than doubled merely by choosing a proper strain

Develpoment of a Simplified Method for the Determination of Folates in Baker\ub4s Yeastby HPLC with Ultraviolet and Fluorescence Detection

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C-18, specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker\u27s yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 mu g/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains