Jelena Jastrebova

HPLC determination of folates in raw and processed beetroots

Abstract A sensitive HPLC method with fluorescence detection and gradient elution has been developed for the determination of folates in vegetables. The method involved extraction of folates from food matrix by heat treatment, deconjugation of folate polyglutamates to monoglutamates by incubation with hog kidney conjugase and purification of food extracts by solid-phase extraction with strong-anion exchange cartridges. The chromatographic separation of folates was achieved on Zorbax SB C 8 column, which was found to be superior over conventional C 18 column in terms of selectivity and sensitivity. Validation of the method included linearity tests, the addition of standard folates for the determination of recovery, repeatability and stability tests. The method developed was applied to analysis of raw and processed beetroots; 5-methyltetrahydrofolate was found to be the main folate form in beetroots. Cultivar differences and growing conditions were found to have a pronounced effect on the folate content in beetroots. Processing resulted in considerable losses of folates, whereas losses during storage appeared to be moderate.

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 microg/100 dry matter), compared to white wheat bread leavened with commercial Bakers yeast (27-43 microg/100 g). The commercial Bakers yeast strain had been industrially produced, using a fed-batch 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.

Occurrence, stability, and determination of formyl folates in foods.

The B-vitamin folate has specific tasks as a one-carbon (C1) group supplier in the building and repair of DNA and RNA as well as in the methylation of homocysteine to methionine. Folate occurs in all living cells as a dynamic pool of several interconvertible forms carrying different C1 groups. Along the food chain, this dynamic pool of folates constantly changes due to either enzymatic or chemical interconversions during food processing and storage. These interconversions make it difficult to determine individual folate forms in foods. The formyl folates, the second most predominant forms of food folates, after 5-methyltetrahydrofolate, are particularly prone to interconvert at low pH. Today, this knowledge is often neglected, leading to risks for analytical underestimation of formyl folates. The purpose of the review is to explore the stability and interconversions of formyl folates in foods as well as to analyze the pitfalls in the determination of formyl folates.

Challenges in the Determination of Unsubstituted Food Folates: Impact of Stabilities and Conversions on Analytical Results

Tetrahydrofolate is the parent molecule of the folate coenzymes required for one carbon metabolism. Together with other unsubstituted folates such as dihydrofolate and folic acid, tetrahydrofolate represents the third pool of dietary folates following 5-methyltetrahydrofolate and formyl folates. Low intake of dietary folates and poor folate status are common problems in many countries. There is a critical need for reliable methods to determine folate in foods to accurately estimate folate intakes in populations. However, current values for folates in foods in databanks are often underestimated due to the high instability of several folate forms, especially tetrahydrofolate. The present review highlights the occurrence of unsubstituted folates in foods and their oxidation mechanisms and chemical behavior as well as interconversion reaction between tetrahydrofolate and 5,10-methylenetetrahydrofolate. The review shows also the important role of antioxidants in protecting folates during analysis and describes strategies to stabilize unsubstituted folates throughout all steps of the analytical procedure.

Antioxidant Hydroquinones Substituted by β-1,6-Linked Oligosaccharides in Wheat Germ

Seven new compounds that demonstrate antioxidant properties, 4-hydroxy-3-methoxyphenyl beta-d-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1), 4-hydroxyphenyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (2), 4-hydroxy-3-methoxyphenyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)beta-D-glucopyranoside (3), 4-hydroxy-3-methoxyphenyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (4), 4-hydroxy-3,5-dimethoxyphenyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (5), 4-hydroxy-3,5-dimethoxyphenyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (6), and 4-hydroxy-2-methoxyphenyl beta-d-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (7), were isolated from wheat germ. The structures were determined by spectroscopic and chemical methods. Compound 1 was the most abundant, approximately 2 mg isolated from each gram of wheat germ. The antioxidant activity of compounds 1-7 was determined by the Trolox equivalent antioxidant capacity assay, and 2 and 7 showed higher values than the other compounds. Compounds 1 and 3-6 reacted with the radical cation reagent within a few seconds, whereas 2 and 7 required several minutes for complete reaction. Compound 1 was shown to protect plasmid DNA from oxidative stress damage caused by hydrogen peroxide; this effect was concentration-dependent.

Novel Fortification Strategies for Staple Gluten-Free Products

Gluten is the major storage protein in cereals such as wheat, rye and barley, or their crossbreds. In the wheat flour the gluten proteins contribute 80–85 % of the total protein content. These proteins contain peptides with high glutamine/proline content which are resistant to digestion by human proteases and may trigger damage to the small intestines. Gluten intolerance is a lifelong intolerance to gluten proteins [1]. A couple of decades ago, gluten intolerance was considered an uncommon disorder in the world, with prevalence rates of 1 in 1,000 or lower [2]. However, the development of novel sensitive and specific screening methods for gluten intolerance improved considerably diagnosis rates and resulted in an epidemiologic shift. Recent population studies have reported a much higher prevalence of gluten intolerance and it is now estimated to be 1:100–1:200 [1, 3].

Folate and Bifidobacteria

Folate is the generic term for a group of chemically related and biologically active forms of vitamin B9, needed by all organisms. Folic acid is the fully oxidized synthetic form of the vitamin. The chapter starts with nomenclature and chemical structure of folate forms followed by methodologies for extraction and chemical analysis of bacterial folate. Folates are unstable and susceptible to interconversion reactions so the special care needed to correctly determine folates in bacteria is discussed. Next follows folate biosynthesis, which happens commonly in plants and many microbes, including most bifidobacteria, but is absent in mammals, such as humans. Relevant genes, enzymes, and intermediate metabolites are described. Thereafter, the chapter continues to review folate metabolism, which describes cycling of different one-carbon units, using folate as vehicle, necessary for, for example, nucleic acid biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), and methylation of macromolecules, such as proteins and DNA, which includes epigenetic maintenance. Next follows a section on how biotechnology can be applied to produce high levels of microbial folate, to be used either as a food additive or as folate-producing probiotics. Finally, the main concerns and health implications from insufficient folate intake are described.