2.3.2. Feeding of Aldehydes and Trapping of Carboxylic Acids

When feeding experiments were conducted by incubating 10 g of banana pulp slices with 50 μmol of exogenous C2–C6 straight chain or branched aldehydes, the incubation conditions were similar to Section 2.3.1 (Figure 2a,b). Due to the targeted products being carboxylic acids, specific procedures were applied as Figure 2b. Filter paper strips treated with NaOH (1 M) were dried before spotting exogenous substrate aldehydes to remove any possible acid impurities in the substrate aldehydes. A recovery flask (250-mL) was used for the incubation. After incubation, the pulp slices were well soaked with 1.5 mL of phosphoric acid (0.4 M) in the flask. Then, the flask was attached to a rotary evaporator in which a cotton ball was inserted at back-flow prevention glass wear. The cotton ball for the acid trap was previously treated with NaOH (0.1 M) and dried. After rotary evaporation (40 ◦C for 60 min), the cotton ball was put into a 17 mL vial and then mixed well with 5 mL of phosphoric acid (0.4 M). For ethanoic acid measurement, 5 mL of acidified ether was used instead of the phosphoric acid solution. For the phosphoric acid solution with carboxylic acids, 1 μL was injected into GC to determine the targeted carboxylic acids (Figure 2b). The GC conditions were: a glass column (3 m × 3 mm) of SP1200 (10%) + phosphoric acid (1%), and oven temperature 60–120 ◦C depending on molecular weight of the target acid. The recovery rate of this method was 53.3% ± 3.2 for ethanoic acid, 81.4% ± 4.7 for propanoic

acid, 92.6 ± 1.6 for 2-methylpropanoic acid, 95.6 ± 6.1 for butanoic acid, 97.2% ± 7.4 for 3-methylbutanoic acid, 94.9% ± 7.2 for pentanoic acid, and 97.6% ± 1.3 for hexanoic acid (n = 3).

**Figure 2.** Feeding experiments procedures. (**a**) Feeding alcohol or 2-keto-4-methylpentanoic acid feeding to banana pulps to produce alcohol/aldehyde/esters in air or nitrogen gas. (**b**) Incubating aldehyde vapor with banana pulps to produce corresponding carboxylic acid, and separation of the carboxylic acid from pulps.

The same method as mentioned above was used for the feeding of aldehydes and trapping of acids when pulp homogenate was used instead of pulp slices (Figure 2b). Ten grams of pulp slices were macerated together with 10 mL of phosphate buffer (0.2 M, pH 8.8), 0.8 g of Polyclar-VT, 1.0 mg of DTT, and 3 g of quartz sand with a mortar and pestle in an ice bath. The pH of the homogenate was adjusted to pH 8.8 with 0.1 M NaOH just after macerating.
