*2.9. Pathogenic Enteric Bacteria Used in the Study*

The pathogenic enteric bacteria that were used for the antimicrobial activity test included *E. coli* O157:H7 DMST 12743, *Shigella dysenteriae* DMST 1511 and *Salmonella* Typhi DMST 22842. *Escherichia coli* and *Vibrio cholera* were kindly obtained from the Microbiology Section, Department of Medical Technology, Faculty of Associated Medical Science, Chiang Mai University, Chiang Mai, Thailand. The bacterial strains were stored in glycerol stock at −20 ◦C and then grown on Mueller–Hinton (MH) agar (Difco™, Detroit, MI, USA) plates at 37 ◦C for 18–24 h.

## *2.10. Antimicrobial Activity of Kombucha Tea*

A single colony of the tested bacteria; *Escherichia coli*, *E. coli* O157:H7, *Shigella dysenteriae*, *Salmonella* Typhi, and *Vibrio cholera*, was transferred into Mueller–Hinton (MH) broth (Difco™, USA) and incubated at 37 ◦C for 18–24 h. The antimicrobial activity of kombucha tea after 15 days of fermentation was investigated using the agar well diffusion method. The turbidity of the bacterial culture was adjusted to McFarland standard no. 0.5 and swabbed on Mueller–Hinton (MH) agar (Difco™, USA). Wells of 10 mm in diameter were prepared on the agar plate with a sterile cork borer. Kombucha samples were sterilized by filtering them through a sterile microfilter (0.22 μm pore size), and they were then transferred into the wells in the agar plates. The plates were further incubated at 37 ◦C for 18–24 h. The zones of bacterial growth inhibition were then determined [17,18].

The antimicrobial activity of kombucha tea was compared to the non-fermented tea, and acetic acid was recorded at the same concentration of the kombucha tea after 15 days of fermentation. Moreover, kombucha tea was neutralized at pH 7.0 by adjusting the pH with 1 M HCl or 1 M NaOH. Heat denatured kombucha tea was prepared by treatment at 100 ◦C for 20 min and at 121.5 ◦C, at 15 pounds per square inch for 15 min by being autoclaved. After treatment, the kombucha tea was then sterilized by filtration and tested for its antimicrobial activity, as has been described previously.
