ALTERNATIVE MICROBIAL SOURCES OF PECTINOLYTIC PREPARATIONS: APPLICATION IN ENOLOGY

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

Curitiba

Congreso; 4th International Congresson Bioprocess in Food Industries; 2010

CIETEP

Pectinases are multi-enzymatic complexes highly effective in pectin depolymerization. Addition of pectolytic enzymes is a common practice in winemaking. 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 at low temperatures (12-25ºC). The aim of the present work was to propose and evaluate new pectinolytic preparations obtained from alternative microbial sources to those commercially used in winemaking. Study of enzymatic activities included the partial characterization of two pectinolytic preparations produced from Bacillus sp. SC-Ch15 bacterial strain and Aureobasidium pullulans GM-R22 yeast-like strain. Both microorganisms have been isolated, selected and studied previously as producers of acidic- and low-temperature-active-pectinase enzymes. Main pectinolytic activities were determined on cell-free culture supernatants. Polymetilgalacturonase (PMG), polygalacturonase (PG), pectin lyase (PNL), pectate lyase (PAL) and pectinesterase (PE) activities were quantified by traditional methods. PMG and PG hydrolytic activities were assayed by quantification of reducing sugars released from substrates, using 3,5-dinitrosalicilic acid reagent. Lyase activities were carried out by recording the 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 -glucosidase activities 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 -glucosidase activity was shown. A. pullulans GM-R22 was the best pectinase producer, showing great levels of depolymerizing pectinolytic activities at low temperatures and acidic pH; besides its pectinases were constitutive. With respect to Bacillus sp. SC-Ch15, it was the first report of an acidic-pectinase-producer bacterium with activity at low temperature. Moreover, due to the type of microorganism involved, there is not the possibility of producing micotoxins and it is of rapid production, which is an advantage from the economic point of view. The obtained results at winemaking conditions were significant with respect to commercial enzymatic activities, which are lower in musts with low pH and manufactured at low temperatures. Thus, both microorganisms proved to be efficient sources of "cold" acidic pectinases for their potential use in vinification process.