Metabolomic and transcriptomic analyses identify external conditions and key genes underlying high levels of toxic glycoalkaloids in tubers of stress-sensitive potato cultivars.

Introduction: High levels of toxic steroidal glycoalkaloids (SGAs) in potato tubers constitute a recognized food quality problem. Tuber SGA levels vary between potato cultivars and can increase after post-harvest stresses such as wounding and light exposure. A few cultivars, e.g., 'Magnum Bonum' and 'Lenape,' have been withdrawn from commercial sales due to excessive SGA levels during some cultivation years. However, these sudden SGA increases are diffucult to predict, and their causes are not understood. To identify external and genetic factors that underlie sudden SGA increases in certain potato cultivars, we have here in a 2-year study investigated 'Magnum Bonum' and five additional table potato cultivars for their SGA levels after wounding and light exposure. Results and methods: Results showed that 'Magnum Bonum' has an unusual strong SGA response to light exposure, but not to wounding, whereas 'Bintje' displayed an opposite regulation. Levels of calystegine alkaloids were not significantly altered by treatments, implicating independent metabolic regulation of SGA and calystegine levels also under conditions of high SGA accumulation. Metabolomic and transcriptomic analyses identified a small number of key genes whose expression correlated with SGA differences between cultivars. Overexpression of two key genes in transgenic low-SGA potato cultivars increased their leaf SGA levels significantly. Discussion: The results show that a strong response to light can underlie the SGA peaks that occasionally occur in certain potato cultivars and indicate that a between-cultivar variation in the expression of single SGA key genes can account for cultivar SGA differerences. We propose that current attempts to mitigate the SGA hazard will benefit from an increased consideration of cultivar-dependent SGA responses to post-harvest conditions, particularly light exposure. The identified key SGA genes can now be used as a molecular tool in this work.

Influence of Cultivation Conditions on the Sioxanthin Content and Antioxidative Protection Effect of a Crude Extract from the Vegetative Mycelium of Salinispora tropica.

Due to their bioavailability, glycosylated carotenoids may have interesting biological effects. Sioxanthin, as a representative of this type of carotenoid, has been identified in marine actinomycetes of the genus Salinispora. This study evaluates, for the first time, the effect of cultivation temperature (T) and light intensity (LI) on the total cellular carotenoid content (TC), antioxidant activity (AA) and sioxanthin content (SX) of a crude extract (CE) from Salinispora tropica biomass in its vegetative state. Treatment-related differences in TC and SX values were statistically significantly and positively affected by T and LI, while AA was most significantly affected by T. In the S. tropica CE, TC correlated well (R2 = 0.823) with SX and somewhat less with AA (R2 = 0.777). A correlation between AA and SX was found to be less significant (R2 = 0.731). The most significant protective effect against oxidative stress was identified in the CE extracted from S. tropica biomass grown at the highest T and LI (CE-C), as was demonstrated using LNCaP and KYSE-30 human cell lines. The CE showed no cytotoxicity against LNCaP and KYSE-30 cell lines.

Effect of Agroecological Conditions on Biologically Active Compounds and Metabolome in Carrot.

Carrot serves as a source of health-beneficial phytochemicals for human diet whose content is affected by agroecological conditions. The effect of conventional, integrated and organic farming on ascorbic acid (AA) and α,ß-carotene levels of new carrot cultivars Cortina F1 and Afalon F1 was investigated and their metabolomic profiles were measured by direct analysis in real time ion source coupled with a high-resolution mass spectrometer (DART-HRMS). Cortina and Afalon exhibited high levels of AA and total carotenes under all agroecological conditions tested that fluctuated in broad ranges of 215-539 and 173-456 mg AA.kg-1 dry biomass and 1069-2165 and 1683-2165 mg carotene.kg-1 dry biomass, respectively. The ratio of ß- to α-carotene in both cultivars was about 1.3. The most important variable for the PCA and the partial least squares discriminant analysis (PLS-DA) models for ethyl acetate extracts measured in positive and negative ionization mode was 6-methoxymellein (6-MM). Total carotene content and 6-MM levels were higher in the organic carrot compared to the conventional one and were correlated with a higher level of spontaneous infection. Other important compounds identified were sitosterol, hexose and various organic acids including antioxidant ferulic and coumaric acids. The findings allow comparison of metabolomic profiles and the AA and carotene contents of both cultivars with those of other commercially used carrots.

Feed Composition Differences Resulting from Organic and Conventional Farming Practices Affect Physiological Parameters in Wistar Rats-Results from a Factorial, Two-Generation Dietary Intervention Trial.

Recent human cohort studies reported positive associations between organic food consumption and a lower incidence of obesity, cancer, and several other diseases. However, there are very few animal and human dietary intervention studies that provide supporting evidence or a mechanistic understanding of these associations. Here we report results from a two-generation, dietary intervention study with male Wistar rats to identify the effects of feeds made from organic and conventional crops on growth, hormonal, and immune system parameters that are known to affect the risk of a number of chronic, non-communicable diseases in animals and humans. A 2 × 2 factorial design was used to separate the effects of contrasting crop protection methods (use or non-use of synthetic chemical pesticides) and fertilizers (mineral nitrogen, phosphorus and potassium (NPK) fertilizers vs. manure use) applied in conventional and organic crop production. Conventional, pesticide-based crop protection resulted in significantly lower fiber, polyphenol, flavonoid, and lutein, but higher lipid, aldicarb, and diquat concentrations in animal feeds. Conventional, mineral NPK-based fertilization resulted in significantly lower polyphenol, but higher cadmium and protein concentrations in feeds. Feed composition differences resulting from the use of pesticides and/or mineral NPK-fertilizer had a significant effect on feed intake, weight gain, plasma hormone, and immunoglobulin concentrations, and lymphocyte proliferation in both generations of rats and in the second generation also on the body weight at weaning. Results suggest that relatively small changes in dietary intakes of (a) protein, lipids, and fiber, (b) toxic and/or endocrine-disrupting pesticides and metals, and (c) polyphenols and other antioxidants (resulting from pesticide and/or mineral NPK-fertilizer use) had complex and often interactive effects on endocrine, immune systems and growth parameters in rats. However, the physiological responses to contrasting feed composition/intake profiles differed substantially between the first and second generations of rats. This may indicate epigenetic programming and/or the generation of "adaptive" phenotypes and should be investigated further.

Influence of Harvest Date and Postharvest Treatment on Carotenoid and Flavonoid Composition in French Marigold Flowers.

The aim of this study was to characterize carotenoids and flavonoids present in French marigold flowers (Tagetes patula "Durango Red") as well as to assess the effects of harvest dates and postharvest treatments on these compounds. Carotenoids and flavonoids were quantified using their respective standards or semiquantified using relevant related standards. Lutein and its derivatives largely dominated the flower carotenoids, while the flavonoids were diverse with patuletin leading the list. Of the different postharvest treatments, ensilage leads to the highest content of carotenoids (from 5.0 to 7.8 g kg-1 dw) and flavonoids (from 19 to 50 g kg-1 dw). The composition of individual flavonoids was greatly influenced by different postharvest treatments, while the influence of harvest dates was secondary. Ensilage and drying induced separate metabolic pathways leading to degradation of high-molecular glycosidic compounds, converting the compounds either to their aglycones or into the intermediate flavonoid glycosides. We conclude that according to the intended industrial application, different postharvest techniques can be employed to acquire desired flavonoids on a large scale.

Effect of resveratrol and Regrapex-R-forte on Trichosporon cutaneum biofilm.

Microorganisms that cause chronic infections exist predominantly as surface-attached stable communities known as biofilms. Microbial cells in biofilms are highly resistant to conventional antibiotics and other forms of antimicrobial treatment; therefore, modern medicine tries to develop new drugs that exhibit anti-biofilm activity. We investigated the influence of a plant polyphenolic compound resveratrol (representative of the stilbene family) on the opportunistic pathogen Trichosporon cutaneum. Besides the influence on the planktonic cells of T. cutaneum, the ability to inhibit biofilm formation and to eradicate mature biofilm was studied. We have tested resveratrol as pure compound, as well as resveratrol in complex plant extract-the commercially available dietary supplement Regrapex-R-forte, which contains the extract of Vitis vinifera grape and extract of Polygonum cuspidatum root. Regrapex-R-forte is rich in stilbenes and other biologically active substances. Light microscopy imaging, confocal microscopy, and crystal violet staining were used to quantify and visualize the biofilm. The metabolic activity of biofilm-forming cells was studied by the tetrazolium salt assay. Amphotericin B had higher activity against planktonic cells; however, resveratrol and Regrapex-R-forte showed anti-biofilm effects, both in inhibition of biofilm formation and in the eradication of mature biofilm. The minimum biofilm eradicating concentration (MBEC80) for Regrapex-R-forte was found to be 2222 mg/L (in which resveratrol concentration is 200 mg/L). These methods demonstrated that Regrapex-R-forte can be employed as an anti-biofilm agent, as it has similar effect as amphotericin B (MBEC80 = 700 mg/L), which is routinely used in clinical practice.

Ascorbic Acid and Glucosinolate Levels in New Czech Cabbage Cultivars: Effect of Production System and Fungal Infection.

Nutritional value and disease-preventive effects of cabbage are well-known. Levels of the antioxidant compounds ascorbic acid (AA) and glucosinolates (GSL) in new Czech cabbage cultivars were determined in the context of different production systems. The contents of AA and GSLs in cabbage biomass were determined by HPLC. Individual GSLs were identified according to their exact masses with sinigrin used as the external standard. Artificial infection with A. brassicicola generally raised the AA levels. The major GSLs (≥10 mg kg-1) were glucobrassicin, sinigrin, and glucoiberin. Indole and aliphatic GSLs were present, but no aromatic ones were detected. Ecological growth conditions and the artificial fungal infection increased the total content of GSLs and, also, of the methoxylated indole GSLs. Sulforaphane, iberin, indole-3-carbinol, and ascorbigen resulting from the hydrolysis of GSLs were found in both cultivars. The amounts and profiles of GSLs present in the two Czech cultivars demonstrated their good nutritional value. The decomposition products sulforaphane, iberin, indole-3-carbinol, and ascorbigen detected improve its health-promoting qualities and represent a suitable component of the human diet.

Assessment of rosehips based on the content of their biologically active compounds.

In this study, an in-depth analysis of the unique set of rosehip samples from 71 Rosa genotypes was conducted with the aim to identify the most suitable ones for applications in the food and pharmaceutical industries based on the content of biologically active compounds. In the first part of our experiments, the antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl assay and the genotypes with the highest values were selected for the follow-up analysis. In the second part of experiments, the major classes of biologically active compounds in rosehips such as carotenoids, tocopherols, flavonoids, and triterpenoic acids were further quantified using liquid chromatography-based techniques. Large variation was observed among all the analyzed compounds with intraspecific variation often hiding interspecific or intersectional differences. The compounds studied herein thus do not provide a sharp tool for chemotaxonomic resolution of the genus Rosa. High intraspecific variation indicates the necessity to screen and utilize individual rose genotypes rather than representatives of the species when searching for sources of biologically active compounds. In the final stage of the study, 10 genotypes were selected for further cultivation and use, based on the highest concentrations of the analyzed biologically active compounds.

Analytical strategies for controlling polysorbate-based nanomicelles in fruit juice.

This study focused on the detection and quantification of organic micelle-type nanoparticles (NPs) with polysorbate components (polysorbate 20 and polysorbate 80) in their micelle shells that could be used to load biologically active compounds into fruit juice. Several advanced analytical techniques were applied in the stepwise method development strategy used. In the first phase, a system consisting of ultrahigh-performance liquid chromatography employing a size exclusion column coupled with an evaporative light scattering detector (UHPLC-SEC-ELSD) was used for the fractionation of micelle assemblies from other, lower molecular weight sample components. The limit of detection (LoD) of these polysorbate micelles in spiked apple juice was 500 µg mL(-1). After this screening step, mass spectrometric (MS) detection was utilized to confirm the presence of polysorbates in the detected micelles. Two alternative MS techniques were tested: (i) ambient high-resolution mass spectrometry employing a direct analysis in real time ion source coupled with an Orbitrap MS analyzer (DART-Orbitrap MS) enabled fast and simple detection of the polysorbates present in the samples, with a lowest calibration level (LCL) of 1000 µg mL(-1); (ii) ultrahigh-performance reversed-phase liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-HRTOF-MS) provided highly selective and sensitive detection and quantification of polysorbates with an LCL of 0.5 µg mL(-1).

Status quo and future research challenges on organic food quality determination with focus on laboratory methods.

Organic food quality determination needs multi-dimensional evaluation tools. The main focus is on the authentication as an analytical verification of the certification process. New fingerprinting approaches such as ultra-performance liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, direct analysis in real time-high-resolution mass spectrometry as well as crystallization with and without the presence of additives seem to be promising methods in terms of time of analysis and detecting organic system-related parameters. For further methodological development, a system approach is recommended, which also takes into account food structure aspects. Furthermore, the authentication of processed organic samples needs more consciousness, hence most of organic food is complex and processed.

Glycoalkaloid and calystegine levels in table potato cultivars subjected to wounding, light, and heat treatments.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (α-chaconine and α-solanine) and calystegines (A3, B2, and B4) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Effect of crop protection and fertilization regimes used in organic and conventional production systems on feed composition and physiological parameters in rats.

Very little is known about the effects of an organic or conventional diet on animal physiology and health. Here, we report the effect of contrasting crop protection (with or without chemosynthetic pesticides) and fertilization (manure or mineral fertilizers) regimes on feed composition and growth and the physiological parameters of rats. The use of manure instead of mineral fertilizers in feed production resulted in lower concentrations of protein (18.8 vs 20.6%) and cadmium (3.33 vs 4.92 µg/100 g) but higher concentrations of polyphenols (1.46 vs 0.89 g/100 g) in feeds and higher body protein (22.0 vs 21.5%), body ash (3.59 vs 3.51%), white blood cell count (10.86 vs 8.19 × 10³/mm³), plasma glucose (7.23 vs 6.22 mmol/L), leptin (3.56 vs 2.78 ng/mL), insulin-like growth factor 1 (1.87 vs 1.28 µg/mL), corticosterone (247 vs 209 ng/mL), and spontaneous lymphocyte proliferation (11.14 vs 5.03 × 10³ cpm) but lower plasma testosterone (1.07 vs 1.97 ng/mL) and mitogen stimulated proliferation of lymphocytes (182 vs 278 × 10³ cpm) in rats. There were no main effects of crop protection, but a range of significant interactions between fertilization and crop protection occurred.

Furanocoumarins in celeriac from different farming systems: a 3-year study.

BACKGROUND: The aim of this 3-year study was to investigate the effect of different celeriac cultivation strategies on the content of naturally occurring toxicants furanocoumarins, represented by psoralen, bergapten, xanthotoxin and isopimpinellin. The products from organic farming in which anaerobically fermented pig slurry was used were compared against those obtained from other treatment systems: mineral, combined and non-fertilised. RESULTS: The average levels of furanocoumarins for all 3 years (determined by gas chromatography-mass spectrometry) in varieties Albin and Kompakt were 2.6 mg kg⁻¹ and 10.2 mg kg⁻¹, respectively. In all crop years higher levels were found in variety Kompakt. By using linear discriminant analysis it was possible to separate the whole data set according to variety from 85.7%, in individual crop years the recognition ability was more than 90%. According to the crop year, it was possible to separate tested samples from 70.8%, for individual variety the separation was 100%. CONCLUSIONS: The method of fertilisation did not have a significant effect on the levels of plant secondary metabolites, furanocoumarins. The climatic conditions, in particular the growing periods and the celeriac variety, had an important role in the occurrence of furanocoumarins.

Analysis of isoflavones in soybeans employing direct analysis in real-time ionization-high-resolution mass spectrometry.

A direct analysis in real-time (DART) ion source coupled to a high-resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC-orbitrap MS method was achieved, in the case of the analysis of non-hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC-orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the "free" genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95-102%) and repeatabilities (RSD: 7-15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.