Microbial ingress procedure

For the microbial challenge test, 135 mL of tryptic soy broth (TSB; Becto, USA) was piped into each pouch before completely sealing it. A total of 210 pouches were tested. DH 5α Escherichia coli (E. coli) and KCCM 11335 Staphylococcus aureus (S. aureus) (obtained from the KCCM, Korean culture center of microorganisms) have been used in bioaerosol microbial package integrity testing, with a concentration of microorganisms in a solution of about 3.0 × 10⁸ CFU/mL [14]. Equal volumes of E. coli and S. aureus cell suspensions were combined to give approximately equal populations of each culture.

Defective pouches as described above were placed in a chamber (constructed of acrylic with approximate dimensions of 0.13 m³), which connected to the nebulizer (Pari LC SPIRINT 0123, Germany). Then, the microorganisms inoculum were nebulized into the chamber for 60 min, followed by waiting for 3 h to obtain a static condition. A 14-day incubation period was conducted at 37 °C, throughout which the pouches were visually inspected and periodically checked for the presence of microbial growth, which would indicate microbial ingress. A pouch with no defect was injected with 0.2 mL of a 10³ CFU/mL solution of the organisms to serve as a positive control. We applied this method to another type of packaging in our previous work [15].