*2.3. Determination of Bitterness*

Bitterness was assessed by manual isooctane extraction as described in the American Society of Brewing Chemists (ASBC) Methods of Analysis with the following modifications [32]. A total of 5 mL of beer was acidified with hydrochloric acid (HCl; 0.5 mL, 3M) and isooctane (10 mL); subsequently, it was homogenized for 15 min using a mechanical shaker. The separation of organic and aqueous layers was performed by centrifugation at 400 *g* × 5 min. Finally, the isooctane phase (upper) was measured spectrophotometrically at 275 nm. A calculation of bitterness units (IBU) of beer was obtained, as shown in Equation (4).

$$\text{IBU} = \text{absorbrane}\_{275} \times 50\tag{4}$$
