The total phenolic compounds, flavonoids and antioxidant capacity (DPPH and FRAP methods) were determined according to the methodology previously described by Tomczyk et al. (2021) [61 (link)]. For the analysis, 20% solutions of honey in distilled water were used.
The protein content in honey was determined using the Bradford method according to Latimer (2016) [62 ]. Briefly, 1 mL of Bradford reagent (Biorad, Hercules, CA, USA) was added to 100 μL of each honey solution (20% in distilled water). Samples were incubated for 5 min at ambient temperature and the absorbance was read at 595 nm using a spectrophotometer (Biomate 3, Thermo Scientific, Waltham, MA, USA). The results were calculated on the basis of a calibration curve (y = 31.752x, r2 = 0.9919) prepared for bovine serum albumin in the range of 6.25–200 µg.
Diastase number was determined by spectrophotometric method with the Phadebas Honey Diastase test (Magle AB, Lund, Sweden) strictly according to the manufacturer′s instructions. The values of the diastase number (DN) were calculated using the following Equation (1):
The activity of three glycosidases: N-acetyl-β-glucosaminidase (NAG), β-galactosidase (β-GAL) and acid phosphatase (AP) was determined in tested honey samples according to the procedure described by Sidor et al. (2021) [44 (link)], using appropriate p-nitrophenolic substrate. The absorbance of released p-nitrophenol was measured using microplate reader (EPOCH2, BioTek, Winooski, VT, USA) at λ = 400 nm. Results were expressed as enzymatic units U (µmol/min/g).
The protein content in honey was determined using the Bradford method according to Latimer (2016) [62 ]. Briefly, 1 mL of Bradford reagent (Biorad, Hercules, CA, USA) was added to 100 μL of each honey solution (20% in distilled water). Samples were incubated for 5 min at ambient temperature and the absorbance was read at 595 nm using a spectrophotometer (Biomate 3, Thermo Scientific, Waltham, MA, USA). The results were calculated on the basis of a calibration curve (y = 31.752x, r2 = 0.9919) prepared for bovine serum albumin in the range of 6.25–200 µg.
Diastase number was determined by spectrophotometric method with the Phadebas Honey Diastase test (Magle AB, Lund, Sweden) strictly according to the manufacturer′s instructions. The values of the diastase number (DN) were calculated using the following Equation (1):
The activity of three glycosidases: N-acetyl-β-glucosaminidase (NAG), β-galactosidase (β-GAL) and acid phosphatase (AP) was determined in tested honey samples according to the procedure described by Sidor et al. (2021) [44 (link)], using appropriate p-nitrophenolic substrate. The absorbance of released p-nitrophenol was measured using microplate reader (EPOCH2, BioTek, Winooski, VT, USA) at λ = 400 nm. Results were expressed as enzymatic units U (µmol/min/g).
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