Materials and methods
Isolation of thermotolerant yeast strains
Yeasts were isolated from soils and traditional
fermented foods collected from various areas in
Thailand. Isolation was carried out at 40 C by an
enrichment technique using yeast extract peptone
dextrose (YPD) broth containing 1 % yeast extract,
2 % peptone, and 2 % glucose, supplemented with
4 % (v/v) ethanol, 0.025 % sodium propionate, and
0.02 % chloramphenicol. Two grams of soil or
fermented food were aseptically placed in 250-ml
Erlenmeyer flasks containing 50 ml enrichment broth
and incubated at 40 C on a rotary shaker (Bio-Shaker
BR-300LF; Taitec, Japan) at 150 rpm for 2 days. A
loopful of the enriched culture was streaked on an agar
plate containing the same medium and incubated at
40 C until yeast colonies appeared. Yeast colonies
were picked up and purified by cross-streaking on
YPD agar. Purified yeast strains were suspended in
YPD broth, supplemented with 10 % glycerol and
maintained at -80 C.