Baths, Sand

Cell or tissue lipids were extracted by the procedures similar to the Folch method [4 (link)]. Chloroform/methanol (2:1, v/v) containing 0.005% butylated hydroxytoluene (as antioxidant) was added (usually 5 ml solvent added to 50–100 μl sample) and mixed vigorously for 1 min then left at 4°C overnight. One ml of 0.9% NaCl was added and mixed again. The chloroform phase containing lipids was collected. The remains were extracted with another 2 ml chloroform. The chloroform was pooled and dried under nitrogen and subjected to methylation. To monitor the recovery rate, the fatty acid C23:0 was added to the samples (usually 1 μg added to 2 mg tissue sample) as an internal standard.
Fatty acid methyl esters were prepared by methods similar to those described previously [5 (link),6 (link)] using BF₃/methanol reagent (14% Boron Trifluoride). Lipid sample was mixed with 1 ml hexane in 16 ml glass tubes with Teflon-lined caps. BF₃/MeOH reagent (1 ml) was added and the mixture was heated at 90–110°C in a metal block or a sand bath for 1 hour, cooled to room temperature and methyl esters extracted in the hexane phase after addition of 1 ml H₂O. Samples were allowed to stand for 20–30 min, and then the upper hexane layer was removed and concentrated under nitrogen.
Fatty acid methyl esters were analyzed by gas chromatography using a fully automated HP5890 system equipped with a flame-ionization detector, as described previously [7 (link)] The chromatography utilized an Omegawax 250 capillary column (30 m × 0.25 mm I.D.). Peaks were identified by comparison with fatty acid standards (Nu-chek-Prep, Elysian, MN), and area and its percentage for each resolved peak were analyzed using a Perkin-Elmer M1 integrator.

The technique used was previously described by Pérez Ruiz et al. [31 ], with some adaptation and modification described by Rodríguez et al. [32 ] and with some modifications and adaptations in our laboratory. All solutions were made with tridistilled H₂O and are only used for the assay and discarded. In brief, in new Corning sterile polypropylene centrifuge tubes, 250 μg of TAA homogenate protein and 500 μl of acid mixture (4 : 1 vol/vol of HNO₃+HCl) plus 500 μl of 10% H₂O₂ were added and incubated in a sand bath at 120°C for 4 hours. After incubation, 100 μl of tridistilled H₂O, 150 μl of 0.5 N NaOH, 200 μl of 30% formaldehyde, 200 μl of a mixture containing 0.5 N N₂S and 0.5 N Na₂SO₃, plus 250 μl of 0.01 M EDTA (pH 10.2), and 300 μl of 4 mM toluidine blue were added. Samples were incubated for 15 min at 25°C. At the end of the incubation, they were centrifuged at 448 rcf for 2 min and the absorbance was read at 600 nm. The calibration curve was determined with 100 ng/ml Na₂SeO₃ which was treated under conditions similar to the experimental samples.

Cells were grown in LB medium overnight and subcultured with 1 : 100 ratio into LB medium to an OD₆₀₀ of ~0.4. Cells were harvested, and levels of intracellular Fe were monitored by ICP-MS. All samples were washed once with chelex-treated phosphate-buffered saline (PBS) buffer containing 1 mM EDTA and then twice with chelex-treated PBS. Cell pellets were resuspended in 400 µl of buffer 2 (1× chelex-treated PBS buffer, 75 mM NaN₃, 1 % Triton X-100) and incubated at 37 °C for 90 min to lyse the cells. Lysed samples were spun down by centrifugation and the total protein content was quantified using a Bradford assay. Then, samples were mixed with 600 µl buffer 4 [5 % HNO₃, 0.1 % (v/v) Triton X-100] and heated in a 95 °C sand bath for 30 min. The debris was removed by centrifugation and the total metal ions in the diluted samples were analysed by Perkin-Elmer ELAN DRC II ICP-MS. Gallium was used as an internal standard. (mean±se; n=3).

The K, Ca, Mg, Na, Zn, Fe, Mn, and Cu elements contents of the wheat flour, optimal wheat–quinoa composite flour and bread were determined by flame atomic absorption spectrometry (FAAS) (AA-6300 Shimadzu, Kyoto, Japan). The analysis of the sample involved two stages: the mineralization of the sample and the metal dosage by spectrophotometry. During mineralization, the organic matter from the sample (5.00 ± 0.001 g) is destroyed by carbonization and combustion in a muffle furnace (Nabertherm, LE 2/11/R6, Bremen, Germany), with the temperature gradually increasing from 250 to 450 °C, up to 900 °C, for 8 h. 5 mL HCl 6 mol/L (STAS 13013/1-91) is added to the ash obtained, and then the acid is evaporated using a sand bath, and the residue is dissolved with 730 µL HNO3 69% and brought to the mark (50 mL) with deionized water. Deionized water was used as a control sample, following the same procedure. The spectrophotometric determination involved the following steps: activating the hollow cathode lamp corresponding to the elements (K, Ca, Mg, Na, Fe, Zn, Mn, Cu), adjusting the operational parameters (wavelength, sensitivity), activating and adjusting the flame, as well as establishing the curve standard by absorbing four working standard solutions of different concentrations. The calibration curve made for each element covers the range of 0.5–5.0 mg/L Ca, 0.5–2.5 mg/L Cu, 0.5–5.0 mg/L Fe, 0.05–0.30 mg/L Mg, 0.5–3.0 mg/L Mn, 0.05–0.60 mg/L Zn, 0.1–0.5 mg/L Na, and 0.2–1.0 mg/L K. The wavelengths taken into account when determining Ca, Cu, Fe, Mg, Mn, Zn, K and Na elements correspond to 422.7, 342.7, 248.3, 285.2, 279.5, 213.8, 589.0, and 766.5 nm. Air-acetylene as the flame type, a gas flow rate of 15.0 L/min, a pre-spray time of 10 s, an integration time of 5 s, and a response time of 1 s were also included as working conditions. The mineral elements are expressed as mg/100 g of flour and were calculated with Equation (1): $$\mathrm{E}=\frac{\mathrm{C}·\mathrm{F}·\mathrm{V}}{\mathrm{M}}$$
where: E—Mineral element concentration, mg/100 g; C—The concentration measured on the calibration curve, mg/L; F—Dilution factor; V—Sample volume, mL; M—Sample mass taken in the analysis, g.

Before use, clover flowers were ground to a fine powder using an Ultra Centrifugal Mill ZM 200 (Retsch, Haan, Germany). Grinding was performed at 4025 g (6000 rpm) using a 0.5 mm trapezoid hole sieve. The extraction was performed as in the previous study using excipient β-cyclodextrin (β-CD) [36 (link)].
Ultrasound-assisted extraction was performed using an ultrasound bath (38 kHz) (Grant Instruments™ XUB12 Digital, Cambridge, England). A sample of 0.3 ± 0.001 g of dried and milled flower heads was macerated in 10 mL of 50% ethanol. Additionally, 0.1 ± 0.001 g of β-CDs was added to the extraction mixture samples (10 mL) to prepare samples with CD concentrations of 1% (w/v). Ultrasound extraction time was 10 min, and the processing temperature was 40 ± 2 °C. After ultrasound processing, the samples were put in a round bottom flask and refluxed in a sand bath at 100 °C for 1 h. After that, the mixture was left to cool at a temperature of 25 ± 2 °C. The samples were centrifuged for 10 min at 3382 g (5500 rpm), followed by the decantation of the supernatant. The extract was filtered through the paper filter and used in the following research.