Helios zeta uv vis

Phytate content was determined using the method published by Haug and Lantzsch (1983 (link)) and adapted from Peng et al. (2010 (link)). This method is based on the precipitation of phytic acid using an acidic iron solution. The decrease of iron in the supernatant is proportional to the amount of phytic acid in the sample. Ferric solution (0.2 g of NH₄Fe (SO₄)₂·12H₂O in 100 ml HCl 2 M, with the volume raised to 1000 ml with distilled water) and bipyridine solution (1 g 2,2-bipyridine and 1 ml of thioglycolic acid, with the volume raised to 100 ml with distilled water) were prepared in advance. For the analysis, 50 mg of the sample was extracted with 10 ml HCl 2 M overnight at 4 °C. Then, the samples were vortexed, and 0.5 ml of the extract was added to a capped glass tube with 1 ml of ferric solution. The samples were then placed in a boiling water bath for 30 min. After cooling the samples to 25 °C, 2 ml of bipyridine solution was added, and the samples were vortexed and immediately measured by spectrophotometry at 519 nm (Helios Zeta UV/Vis, Thermo Scientific). For quantification, a calibration line was produced using phytic acid as standard (0–0.15 mg/ml). Results are expressed as milligrams of phytic acid per gram of dry sample.

Glucosamine content was used to estimate fungus growth, considering glucosamine, such as a product of the chitin hydrolysis (Aidoo et al. 1981 (link); Tomaselli Scotti et al. 2001 (link)). For fungal chitin hydrolysis into N-glucosamine, 100 mg of dried lentil and quinoa samples was incubated with 2.4 ml of 72% sulphuric acid (H₂SO₄) at 25 °C for 24 h. Then, samples were diluted with 55 ml of distilled water. The hydrolysis was carried out by sterilising the sample for 2 h at 121 °C. The hydrolysed products were neutralised to pH 7 using sodium hydroxide (NaOH) 10 M and 0.5 M. Next, 1 ml of hydrolysed product was added with 1 ml of acetylacetone reagent (1 ml of acetylacetone and 50 ml of sodium carbonate 0.5 M) in glass tubes and incubated in a boiling water bath for 20 min. After cooling the tubes, 6 ml of ethanol and 1 ml of Erhlick reagent (2.67 g p-dimethylamine benzaldehyde and ethanol:HCl solution 1:1 (v:v)) mixed into a 100-ml volumetric flask were added to the mixture. Then, the samples were incubated at 65 °C for 10 min, and absorbances were measured at 530 nm using a spectrophotometer (Thermo scientific, Helios Zeta UV/Vis). A calibration line taking glucosamine (0–0.5 mg/ml) as standard was used to quantify the fungus biomass. Results are expressed as milligrams of glucosamine per gram of dry basis.

Polyphenols were determined in samples using the Folin–Ciocalteu method following the indications of Espinosa-Páez et al. (2017 (link)) and Chang et al. (2006 (link)). An extraction with 80% methanol for 2 h in agitation (55 rpm, 25 °C, Intelli-Mixer RM-2) was performed to recover the hydrosoluble compounds from the samples. Methanol was added to the sample in a proportion of 1:20 (w:v). After agitation, samples were centrifuged (20 min, 14g-force, 20 °C), and the supernatant was used to quantify the polyphenols by visible spectrophotometry (Helios Zeta UV/Vis, Thermo Scientific). A gallic acid line was used to quantify the total polyphenols (0–200 mg/l). Results are expressed as milligrams of gallic acid per gram of dry basis.

Three methods were used to measure antioxidant activity in fermented samples following the indications of Thaipong et al. (2006 (link)) and Espinosa-Páez et al. (2017 (link)): (1) ABTS: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); (2) DPPH: 2,2-diphenyl-1-picrylhydrazyl and (3) FRAP: Ferric reducing antioxidant power. An extraction with 80% methanol was conducted to determine antioxidant activity. After centrifugation, supernatants were used for quantification using a spectrophotometer (Helios Zeta UV/Vis, Thermo Scientific). In all methods, a calibration line was required using Trolox as standard (0–200 mg/l). Results are expressed as milligrams of Trolox per gram of dry basis.

Antioxidant capacity (AC) was assessed using DPPH method following the methodology of Igual et al. [17 (link)]. UV-visible spectrophotometer (Thermo Electron Corporation, Waltham, MA, USA) was used for the absorbance at 515 nm. The final results were expressed as milligram trolox equivalents (TE) per 100 g (mg TE/100 g).
Total phenol content (PC) was carried out according to Agudelo et al. [12 (link)]. Absorbance was measured at 765 nm in a UV-visible spectrophotometer (Thermo Scientific, Helios Zeta UV-Vis, Loughborough, UK). The total phenolic content was expressed as mg of gallic acid equivalents (GAE) (Sigma-Aldrich, Steinheim, Germany) per 100 g of sample.

The total phenolics were determined spectrophotometrically according to Ribereau-Gayon method [32 ] in the modification by Guo et al. [33 (link)] using the addition of 0.5 mL of Folin-Ciocalteu reagent (Aktyn, Suchy Las, Poland) and 3 mL of 14% sodium carbonate (Stanlab, Poland) to tea infusions (0.04 mL) and measurement of the absorbance at a wavelength of 720 nm against the reference sample after 1 h (Thermo Scientific, Helios Zeta UV-VIS, Madison, WI, USA). The results were expressed as gallic acid equivalent with a reference curve plotted for this acid: $y=0.0223x-0.1916,R^2=0.990$

Antioxidant capacity was determined following the DPPH (2,2-diphenyl-1-picrylhydrazyl) method described by Kuskoski et al. [21 (link)] and Stratil et al. [22 (link)], with some modifications. A total of 0.1 mL of the extract, 0.9 mL of methanol, and 2 mL of the methanol–DPPH solution were mixed and absorbance was measured at 517 nm in a spectrophotometer (Thermo Scientific, Helios Zeta U/Vis). The results were expressed as milligrams of Trolox equivalent (TE) per gram of dry matter, using the Trolox calibration curve within a 0 to 500 mg/L concentration range.
The antioxidant activity was also evaluated following the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical method described by Re et al. [23 (link)]. A solution of 7 mM of ABTS and 2.45 mM of potassium persulfate was prepared and left to stand in the dark at room temperature for 16 h. ABTS was mixed with phosphate buffer to reach an absorbance of 0.70 ± 0.02, read at 734 nm. A 0.1 mL of extract was added to 2.9 mL of ABTS solution and absorbance was measured at 734 nm in a spectrophotometer (Thermo Scientific, Helios Zeta UV/Vis) after 0, 3 and 7 min of reaction time. The results were expressed as mg of Trolox equivalent (TE) per gram of dry matter.