Development of LC-MS-ESI-TOF method for quantification of phytates in food using 13C-labelled maize as internal standard.

In this work, the LC-MS-ESI-TOF method for simultaneous determination of phytates (inositol mono-, bis-, tris-, tetrakis-, pentakis-, and hexakisphosphates, abbreviated to IP1, IP2, IP3, IP4, IP5, and IP6, respectively) in food samples was developed and validated. The suitability of U-13C-labelled maize as a source for labelled internal standards for quantification of phytates was elucidated. The effectiveness of liberating IP1, IP2, IP3, IP4, and IP5 from phytic acid extracted form U-13C-labelled maize was evaluated for a variety of hydrolysis conditions, including enzymatic and acid hydrolysis. Enzymatic degradation of phytic acid using phytase (PHYZYME XP 5000 L) was very effective; phytic acid was degraded to lower phytates, but their distribution was unequal. Chemical hydrolysis was conducted under acidic conditions using hydrochloric acid and elevated temperatures up to 140 °C. The highest yields of IP4, IP5, and IP6 and of IP1, IP2, and IP3 were achieved by chemical hydrolysis at 105 °C for 7 h and 24 h, respectively. Thus, a combination of these two chemical treatments was selected for internal standard production. The developed LC-MS-ESI-TOF method was tested and successfully validated using plant-based food samples with different distribution of phytates. With this method, different forms of phytates in foods were separated and quantified simultaneously within 20 min. The high accuracy and precision of the developed method were guaranteed using respective labelled internal standards derived from U-13C-labelled maize.

The Composition, Physicochemical Properties, Antioxidant Activity, and Sensory Properties of Estonian Honeys.

Thirty honey samples from different regions of Estonia were investigated to determine the chemical compositions, physicochemical properties, bioactive compounds, and sensory characteristics of typical honeys from a northern climate. The physicochemical parameters, such as electrical conductivity, moisture content, free acidity, hydroxymethylfurfural, diastase, and invertase activity were measured. The color was measured and expressed by L*-, a*-, and b*-coordinates. Sensory parameters were determined by using "fruity", "floral", "berry-like", "herbal", "woody", "spicy", "sweet", and "animal-like" as the main odor and flavor attributes. The total polyphenol and flavonoid contents were in the range of 26.2-88.7 mg gallic acid equivalents (GAE) per 100 g and 1.9-6.4 mg quercetin equivalents (QE) per 100 g, respectively. The identified polyphenols showed the highest intensities of caffeic acid, coumaric acid, and abscisic acid and its derivatives. The protocatechuic acid intensity was highest in honeys containing traces of honeydew elements and of cinnamic acid and myricetin in heather honey. The water-soluble antioxidant values were 37.8-311.2 mg ascorbic acid equivalents (AAE) per 100 g and the lipid soluble antioxidant values were 14.4-60.7 mg Trolox equivalents (TE) per 100 g. The major amino acid in the analyzed honeys was proline, with variable values depending on the honey's botanical source. Correlations were calculated based on the results obtained. It was revealed that the typical Estonian honey has floral, berry-like, sweet, and rather mild sensory characteristics. Most of the honeys lacked stronger spicy, woody, and animal-like attributes. The typical color of Estonian honey is quite light.

Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast.

Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells became tolerant to severe environmental stresses. Intracellular RNA concentration decreased, and massive downregulation of ribosomal biosynthesis genes occurred. We proved that the expression of new proteins during purine starvation is critical for cells to attain stress tolerance phenotype Msn2/4p targets are upregulated in purine-starved cells when compared to cells cultivated in purine-rich media. The overall transcriptomic response to purine starvation resembles that of stationary phase cells. Our results demonstrate that the induction of a strong stress resistance phenotype in budding yeast can be caused not only by natural starvation, but also starvation for metabolic intermediates, such as purines.

Impact of Fermentation and Phytase Treatment of Pea-Oat Protein Blend on Physicochemical, Sensory, and Nutritional Properties of Extruded Meat Analogs.

Plant materials that are used for the production of extruded meat analogs are often nutritionally incomplete and also contain antinutrients, thus there is a need to explore alternative plant proteins and pre-treatments. This study demonstrates application of phytase and fermentation to a pea-oat protein blend with a good essential amino acid profile and subsequent texturization using extrusion cooking. Enzymatic treatment reduced the content of antinutrient phytic acid by 32%. Extrusion also degraded phytic acid by up to 18%, but the effect depended on the material. Differences in physicochemical, sensorial, and textural properties between untreated and phytase-treated extruded meat analogs were small. In contrast, fermented material was more difficult to texturize due to degradation of macromolecules; physicochemical and textural properties of extrudates were markedly different; sensory analysis showed enhancement of flavor, but also detected an increase in some unwanted taste attributes (bitterness, cereal and off-taste). Phytic acid was not degraded by fermentation. Analysis of volatile compounds showed extrusion eliminated volatiles from the raw material but introduced Maillard reaction products. Overall, phytase treatment and fermentation demonstrated the potential for application in extruded meat analogs but also highlighted the necessity of optimization of process conditions.

Assessment of bioavailable B vitamin content in food using in vitro digestibility assay and LC-MS SIDA.

Standardized analytical methods, where each B vitamin is extracted from a given sample individually using separate procedures, typically ensure that the extraction conditions provide the maximum recovery of each vitamin. However, in the human gastrointestinal tract (GIT), the extraction conditions are the same for all vitamins. Here, we present an analytically feasible extraction protocol that simulates conditions in the GIT and provides a measure of the content of bioavailable vitamins using LC-MS stable isotope dilution assay. The results show that the activities of both human gastric and duodenal juices were insufficient to liberate absorbable vitamers (AV) from pure cofactors. The use of an intestinal brush border membrane (IBBM) fraction derived from the mucosal tissue of porcine small intestine ensured at least 70% AV recovery. The rate of AV liberation, however, was strongly dependent on the cofactor, e.g., in the case of NADH, it was magnitudes higher than in the case of thiamine diphosphate. For some vitamins in some food matrices, the use of the IBBM fraction assay resulted in lower values for the content of AV than conventional vitamin determination methods. Conventional methods likely overestimate the actual bioavailability of some vitamins in these cases. Graphical abstract Assessment of bioavailable B vitamin content in food.