On yeasts from traditional fermented foods Characterization, phytate degradation, strain improvement and applications

Plant materials naturally contain minerals of iron, zinc and calcium [1]. However, plants also contain a compound called phytic acid, which can chelate the minerals and form insoluble complexes. Minerals from plant foods are unavailable for intestinal uptake when they are bound in phytate complexes [24]. Cereal-based diets low in meat, can thereby lead to micronutrient deficiencies of e.g. iron and zinc. Yeasts were isolated from food fermentations, and studied with the aim to find starter strains able to degrade phytate in cereal-based matrices. The overall phytate degrading ability of different strains was studied, followed by analysis of release of the phytase (phytate degrading) enzyme to the surrounding media by the two strains Pichia kudriavzevii TY13wt and TY1322, and of factors affecting the phytase release. Yeasts isolated from the traditionally fermented goat milk from the Yaghnob valley, Tajikistan, were genetically identified by ITS1-4 sequencing and restriction fragment length polymorphism. Phenotypic characterization was done by studying e.g. growth at various pH and temperatures, on different carbon sources, in presence of ox bile, ethanol or lactic acid, under osmotic and oxidative stress. Selected strains were used as starters for fermentation of some plant-based substrates. A prominent phytate degradation was observed from the strain P. kudriavzevii TY13wt, previously isolated from Tanzanian Togwa. Through mutagenesis by ultraviolet light exposure of strain TY13wt, mutant strain TY1322 was acquired, having improved phytate degrading ability. Strain TY1322 showed about eight times higher phytate degradation compared to wild type strain TY13wt under certain conditions. The phytase synthesis from strain TY1322 was not repressed by high phosphate levels (26mM), as opposed to the wild type strain. Both TY13wt and TY1322 were able to release phytase to the surrounding media, which was induced by yeast extract medium. Strains TY13wt and TY1322 could grow at pH 2, at 46°C, in presence of 2% ox bile or 6% ethanol, and under osmotic stress. The phytase produced showed two pH optima (3.5 and 5.5) and one temperature optimum (55°C). Yeast isolation from fermented goat milk of the Yaghnob valley resulted in 52 isolates belonging to Kluyveromyces marxianus (29), Pichia fermentans (12), Saccharomyces cerevisiae (10) and Kazachstania unispora (1). Characterization of the strains revealed i) two genetically different groups among the K. marxianus strains, ii) Yaghnob S. cerevisiae strains forming a separate cluster in a phylogenetic tree constructed of 350 previous S. cerevisiae isolates of various origins, iii) phenotypically intriguing traits of several strains, e.g. S. cerevisiae strains able to grow at elevated temperatures, 37°C (all ten strains), 40°C (9), 42°C (2), and 46°C (1). The phenotypic intra-species variations found among some of the Yaghnob strains may potentially indicate isolation of some new species. Finally, application of selected yeasts and lactic acid bacteria for soy milk and soy flour fermentations showed strong phytate degradation in fermentations containing strain P. kudriavzevii TY1322, and the phytate degrading effect of TY1322 was improved in co-cultures with Yaghnob strains K. marxianus AL2 or BL8. Traditional food fermentations constitute a valuable source of microbial strains. This work demonstrates the usefulness of phytase-active strains for phytate degradation in plant-based substrates, and new strain isolation revealed several strains able to grow under stress conditions that may occur during fermentation, or inside the gastrointestinal tract. This work can contribute to future strain selection for food and feed processes.