Release of the bioactive compound, ferulic acid, from malt extracts.

During the brewing process, lipid oxidation can lead to undesirable flavours in beer [I]. Naturally occuring phenols in malt (germinated barley) have proven their effectiveness as antioxidants [2]. Ferulic acid (4-hydroxy-3methoxy cinnamic acid) is the most abundant phenolic acid in barley. It acccounts for 0.14% of dry matter in barley grains 131, and it is mainly esterified to arabinofuranosyl residues of heteroxylans in barley cell walls [4,5]. Athough ferulic acid is potentially a good antioxidant of beer, its action is limited due to the low concentration of free ferulic acid [6]. Any pathway, therefore, that involves hydrolysis of cell wall feruloylated polysaccharides into free ferulic acid during mashing and kilning, should be investigated and optimized in order to improve the natural antioxidant capacity of the wort and the beer obtained from it. Convesely, a very high concentration of ferulic acid can lead to off-flavours [7]. Ferulic acid esterases have been purified from different microbial sources (8-1 I]. These enzymes are able to release ferulic acid from feruloylated plant cell wall polysaccharides, with their action enhanced by the presence of other cell-wall degrading enzymes [8,9]. Preliminary experiments showed that an extract from malt released ferulic acid when incubated with methyl ferulate, a specific substrate for ferulic acid esterases. In this paper, we report the first step in the purification of a posible "ferulic acid esterase" from malt. Its activity was determined against several methyl esters of cinnamic acids, and a feruloylated oligosaccharide. In order to determine whether or not this "ferulic acid esterase" could release ferulic acid from malt cell walls, the semi-purified malt extract was incubated with spent grain, which is the brewing residue of malt and grain remaining in the mashkettle after the wort has been removed by filtration. Ground malt (500 mg) was suspended in 2.5 I of 100 mM MOPS buffer @H 6.0). stirred for 2 h at room temperature, and centrifuged. The supernatant was subjected to (NH,),SO, fractionation: ferulic acid esterase activity (against methyl ferulate) was found to be associated with the 4040% saturation fractions. The 40-6096 fraction pellet was resuspended and dialysed against water. The de-salted solution was concentrated by ultrafiltration to a final volume of 50 ml. This semi-purified malt extract (15.5 mg proteinlml) was active upon all the cinnamic acid methyl esters tested except methyl caffeate (Table I ) . It showed higher specificity for MFA and MpCA than for MSA. Activity on the feruloylated polysaccharide FAXX was slightly higher than on the corresponding methyl ester (MFA). The extract also exhibited acetyl esterase activity (Table I ) , but was free of xylanase activity. Microbial cinnamoyl esterases purified so far have showed different specificities for methyl esters [9,11], but much greater preference for FAXX against MFA [lo]. This indicates the presence of a complex mixture of xylandebranching enzymes, whose synergistic interactions influence the degradation of FAXX, or that malt contains a "ferulic acid esterase" with different properties to the esterases reported previously.