COMPARISON OF METHODS FOR DETERMINING THE PROTEOLYTIC ACTIVITY OF A PLANT EXTRACT FROM ARTICHOKE FLOWER (Cynara scolymus L.)

Proteases extracted from artichoke flower (Cynara scolymus L.) can act as milk coagulants in the production of buttery cheeses. This study aimed to establish a rapid and reproducible method for quantifying the proteolytic activity of these extracts, adressing challenges related to pigmentation and the presence of phenolic compounds that interfere with colorimetric reactions and direct light absorption by samples at 280 nm. Extracts were clarified with activated charcoal, which reduced the standard deviation of replicates more than fivefold in preliminary tests. Three analytical methods were evaluated: (i) hydrolysis of azocasein with absorbance at 428 nm; (ii) hydrolysis of sodium caseinate with direct absorbance at 280 nm; and (iii) hydrolysis of sodium caseinate followed by reaction with Folin–Ciocalteu reagent and absorbance at 660 nm. In the azocasein method, the reaction lasted 40 minutes, was stopped with 10% TCA, centrifuged (17,000 g, 15 min, 25 °C), and neutralized with 5 M KOH. In the caseinate-based methods, 600 μL of extract and 400 μL of 0.5% substrate reacted for 60 minutes, followed by TCA addition and centrifugation (2,300 g, 15 min, 25 °C). For the Folin method, 0.1 mL of supernatant was mixed with 1.5 mL of 0.5 M NaOH and 1.5 mL of diluted Folin reagent (1:2), incubated in the dark for 20 minutes. Two extract concentrations (0.15 and 0.30 g/mL) and three temperatures (30, 33, and 36 °C) were tested in triplicate. Blanks were prepared by adding TCA before the enzymatic reaction. One unit of activity (U) was defined as the enzyme amount required to increase absorbance by 0.1 at the corresponding wavelength. Reproducibility was assessed by comparing coefficients of variation (CV), means, and medians. CVs for the Folin method ranged from 3.57 to 20.73 (mean = 9.27; median = 7.45); for the 280 nm method, from 2.13 to 23.72 (mean = 16.14; median = 18.71); and for azocasein, from 3.86 to 28.10 (mean = 14.99; median = 12.24). The Folin method showed the lowest mean and median CVs, indicating superior analytical precision and reproducibility. Additionally, it required less sample volume, making it advantageous when enzyme availability is limited. Furthermore, this method requires a smaller sample volume, making it particularly advantageous when enzyme material is limited, and therefore the most suitable option for quantifying the proteolytic activity of the evaluated plant extract.