2.6. Zein Test

The irritant potential of the products (1% SCS with different concentrations of the extracts) was measured using the zein test [10–12]. 40 mL of the sample solution with 2 ± 0.05 g of zein from corn was shaken on a shaker with a water bath (60 min at 35°C). Then, the solutions were filtered on Whatman No. 1 filters and centrifuged at 6720g for 10 min. The nitrogen content in the solutions was determined by the Kjeldahl method. 1 mL of the centrifuged filtrate was mineralized in sulfuric acid (98%) containing copper sulfate pentahydrate and potassium sulfate. The mineralized solution was transferred (with 50 mL of Milli-Q water) into the flask of the Wagner–Parnas apparatus. 20 mL of 25 wt% sodium hydroxide was added. The released ammonia was distilled with steam. Ammonia was bound by sulfuric acid (5 mL of 0.1 N H₂SO₄) in the receiver of the Wagner–Parnas apparatus. The unbound sulfuric acid was titrated with 0.1 N sodium hydroxide. Tashiro solution was used as an indicator. The zein number (ZN) was calculated from the following equation:

where V1 is the volume (cm³) of sodium hydroxide used for titration of the sample. The final result was the arithmetic mean of five independent measurements.