Alternative Microbial Sources of Pectinolytic Preparations: Application in Enology

MARTÍN, M. C; .MERÍN, M.G.; CASTRO, G.R.; MORATA, V.I.

Curitiba

Congreso; 4th International Congress on Bioprocess in Food Industries, CIETEP; 2010

Pectinases are multi-enzymatic complexes highly effective in pectin depolymerization, and commonly used in winemaking practice. Commercial pectinases are acidic and of fungal origin, specially obtained from Aspergillus niger strains. A recent technique applied to vinification process is the use of low temperatures to obtain wines of high added value. Hence, it demands the utilization of enzymes with good activity in the low temperature range (12-25ºC). The aim of the present work was to evaluate new pectinolytic preparations obtained from native microbial sources to those commercially used in argentine winemaking. Studies of enzymatic activities including the partial characterization of two pectinolytic preparations produced from Bacillus sp. SC-Ch15 and Aureobasidium pullulans GM-R22 (a yeast-like strain) are reported. Both wild-type microorganisms have been isolated, selected as producers of acidic- and low-temperature-active-pectinolytic enzymes. Pectinolytic activities determined on cell-free culture supernatants were polymetilgalacturonase (PMG), polygalacturonase (PG), pectin lyase (PNL), pectate lyase (PAL) and pectinesterase (PE). PMG and PG hydrolytic activities were assayed by reducing sugars using 3,5-dinitrosalicilic acid reagent. Lyase activities were carried out by recording changes on absorbance at 235 nm. These activities were determined on citrus pectin (60% DE) and polygalacturonic acid, under vinification conditions (pH 3.6 and 20ºC) and at higher pH-values and temperatures. Besides, the PE activity was quantified by titration with 0.01N NaOH. In addition, xylanase, cellulase and activities -glucosidase were measured on corresponding substrates. CM-Ch15 y GM-R22 pectinolytic preparations exhibited a specific pectinase activity at pH 3.6 and 20ºC of 0.647 and 17.182 UE/mg, respectively. Hydrolytic enzymes under winemaking conditions showed good activities (PMG: 0.062±0.004 and 0.567±0.075 UE/mL for CM-Ch15 and GM-R22, respectively). PG activity gave greater values at pH 6 (0.165±0.023 and 1.143±0.078 UE/mL) than at 3.6; however, the relative activity at the latter pH represented a 35% and 71% respectively for both enzymes. These preparations also revealed important lyase activities; PNL maintained, under vinification conditions, a 41% and 58% of their maximum activities at pH 6-50ºC, respectively; and PAL at these last conditions showed high values (1.827±0.117 and 2.121±0.041 UE/mL). PE was 0.200±0.060 UE/mL for CM-Ch15, whereas for GM-R22 it was not detected. Furthermore, the preparations exhibited cellulase and xylanase activities, and no alpha-glucosidase activity was shown. A. pullulans GM-R22 was selected because is showing higher levels of depolymerizing pectinolytic activities at low temperatures and acidic pH than Bacillus sp. SC-Ch15; besides its pectinases were constitutive. To our knowledge, this is the first report of an acidic-pectinase-producer microorganism displaying activities at low temperature. Moreover, other advantages are the lack of mycotoxin synthesis by the microorganism, and the fast enzyme production during the exponential growth phase. Also, the pectinoltic activites detected in our selected strains were higher compared to commercial ones in musts under winemaking conditions at low pHs and temperatures. Thus, both microorganisms proved to be efficient sources of "cold" acidic pectinases for their potential use in vinification process at industrial scale