Pepsin powder

The nutmeg essential oil (Sigma-Aldrich, Singapore) from Myristica fragrans seeds was used as the core material in microcapsules. The essential oil is a limpid, light yellowish liquid and has a specific odor. Polysorbate 80 (Tween^® 80, Roth, Germany) and sucrose esters (Sisterna^® SP70, Sisterna, The Netherlands) were used as surfactants which stabilized the nutmeg essential oil emulsion. As the shell material, alginic acid sodium salt from brown algae (Sigma-Aldrich, Shanghai, China) was used. Calcium chloride (Farmalabor, Italy) salt was used to formulate microcapsules as a crosslinker which linked sodium alginate chains and formed a solid gel. Magnesium aluminometasilicate (Neusilin^® US2, Fuji Chemical Industries Co., Osaka, Japan) was used as an absorbent of volatile compounds. Ethanol 96% (Vilniaus degtinė, Vilnius, Lithuania) was used as a solvent for encapsulation efficiency determination. Distilled water was used throughout the experiment. Potassium dihydrogen phosphate (Supelco, Darmstadt, Germany), sodium hydroxide (Sigma-Aldrich, Munich, Germany), pancreas powder (Mezym 20000 U, Berlin-Chemie, Berlin, Germany), sodium chloride (Sigma-Aldrich, Munich, Germany), pepsin powder (Sigma-Aldrich, Soborg, Denmark), and hydrochloric acid (36% (Standard, Krakow, Poland) were used to produce simulated intestinal and gastric juices.

We studied the effect of gastric juices on seed viability by exposing both intact and scarified seeds to a solution mimicking the gastric juice of mallards Anas platyrhynchos [15 ]. We used demineralized water and 1M hydrochloric acid (HCl), with the addition of 1 mg/mL powdered pepsin (≥250 Pu/mg pepsin powder from porcine gastric mucosa, Sigma-Aldrich), to make a solution of approximately 250 Pu/mL pepsin and pH 2.5. Seeds of each species were immersed in 10 mL of simulated gastric juice (20 mL for the large Iris pseudacorus and Sparganium erectum seeds) in glass beakers in a stove for two hours (modal retention time in dabbling ducks [32 ]) at 42°C (mallard deep body temperature [33 ]). The seeds were put to germinate one to three days later and meanwhile stored in the dark at 4°C.