Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA) and using the yeast α-acetolactate synthase gene (ILV2) as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely.Uvođenjem ilv2:GLA isječka iz pMGI6, kao nosioca gena za glukoamilazu (GLA) i uporabom rekombinacijske sekvencije gena za α-acetolaktat sintazu (ILV2), konstruiran je rekombinantni soj pivskoga kvasca Saccharomyces pastorianus. Utvrđeno je da soj može koristiti škrob kao jedini izvor ugljika, da aktivnost glukoamilaze iznosi 6,3 U/mL, a da je aktivnost α-acetolaktat sintaze smanjena za 33,3 %. Uvedeni gen GLA, integriran u gen primatelja ILV2, raskinuo je jednu kopiju gena ILV2, dok je druga kopija ostala nepromijenjena. Prvi test fermentacije sladovine potvrdio je da je fermentacijom s pomoću rekombinantnog soja koncentracija diacetila smanjena za 65,6 %, a koncentracija ostatka šećera za 34,2 % u usporedbi sa sojem primatelja. U industrijskim uvjetima zrenje piva fermentiranog s pomoću rekombinantnog soja smanjeno je sa 7 na 4 dana, pri čemu nema značajne razlike u izgledu i okusu, te je ono zadovoljilo visoke zahtjeve. Stoga se ovaj soj može sigurno upotrijebiti u proizvodnji piva
